dysregulated sp1/mir-130b-3p/hoxa5 axis contributes to ... · 3 45 microvessel densities. we...

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1 Dysregulated Sp1/miR-130b-3p/HOXA5 axis contributes to tumor 1 angiogenesis and progression of hepatocellular carcinoma 2 3 Yadi Liao 1* , Chenwei Wang 1,2* , Zhiwen Yang 1,2 , Wenwu Liu 1 , Yichuan Yuan 1,2 , Kai 4 Li 1,2 , Yuanping Zhang 1,2 , Yongjin Wang 1,2 ,Yunxing Shi 1,2 , Yuxiong Qiu 1,2 , Dinglan 5 Zuo 1 , Wei He 1,2 ,Jiliang Qiu 1,2 , Xinyuan Guan 1,3 , Yunfei Yuan 1,2 , Binkui Li 1,2 6 7 1 State Key Laboratory of Oncology in South China, Collaborative Innovation 8 Center for Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou 9 510060, P. R. China 10 2 Department of Liver Surgery, Sun Yat-Sen University Cancer Center, 11 Guangzhou 510060, P. R. China 12 3 Department of Clinical Oncology, The University of Hong Kong, 852 Hong Kong, 13 P. R. China 14 15 *Yadi Liao and Chenwei Wang contributed equally to this work. 16 Corresponding authors: 17 Dr. Binkui Li. Tel: 86-020-87343951, E-mail: [email protected]; 18 Dr. Yunfei Yuan. Tel: 86-020-87343118, E-mail: [email protected]. 19 20 21

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Page 1: Dysregulated Sp1/miR-130b-3p/HOXA5 axis contributes to ... · 3 45 microvessel densities. We further demonstrated that miR-130b-3p could be 46 directly transcriptionally regulated

1

Dysregulated Sp1/miR-130b-3p/HOXA5 axis contributes to tumor 1

angiogenesis and progression of hepatocellular carcinoma 2

3

Yadi Liao1*, Chenwei Wang1,2*, Zhiwen Yang1,2, Wenwu Liu1, Yichuan Yuan1,2, Kai 4

Li1,2, Yuanping Zhang1,2, Yongjin Wang1,2,Yunxing Shi1,2, Yuxiong Qiu1,2, Dinglan 5

Zuo1, Wei He1,2,Jiliang Qiu1,2, Xinyuan Guan1,3, Yunfei Yuan1,2, Binkui Li1,2 6

7

1State Key Laboratory of Oncology in South China, Collaborative Innovation 8

Center for Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou 9

510060, P. R. China 10

2Department of Liver Surgery, Sun Yat-Sen University Cancer Center, 11

Guangzhou 510060, P. R. China 12

3Department of Clinical Oncology, The University of Hong Kong, 852 Hong Kong, 13

P. R. China 14

15

*Yadi Liao and Chenwei Wang contributed equally to this work. 16

Corresponding authors: 17

Dr. Binkui Li. Tel: 86-020-87343951, E-mail: [email protected]; 18

Dr. Yunfei Yuan. Tel: 86-020-87343118, E-mail: [email protected]. 19

20

21

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2

Abstract 22

Angiogenesis, one of the hallmarks of cancer, is essential for both tumor growth 23

and metastasis. However, its molecular mechanisms in hepatocellular carcinoma 24

(HCC) are largely unknown. Here, we report the role of HOXA5 in tumor 25

angiogenesis of HCC. 26

Methods: The expression of miR-130b-3p and HOXA5 was determined by qRT-27

PCR and immunohistochemistry, respectively. Capillary tube formation assay, 28

chicken chorioallantoic membrane assay, and subcutaneous xenograft 29

experiments were performed to investigate the role of miR-130-3p and HOXA5. 30

Luciferase reporter assay and chromatin immunoprecipitation assay were 31

performed to evaluate the interaction between Sp1, miR-130b-3p and HOXA5. 32

Results: miR-130b-3p was found up-regulated in HCC and correlated with a 33

poor prognosis. miR-130b-3p promoted HCC angiogenesis both in vitro and in 34

vivo. Mechanistically, HOXA5 was validated as a direct target of miR-130b-3p. 35

Furthermore, we demonstrated that HOXA5 was down-regulated in HCC and its 36

down-regulation was associated with larger tumor size, shorter overall survival, 37

and higher recurrence probability. Moreover, HOXA5 was significantly associated 38

with angiogenesis biomarkers such as CD31 and CD34. Functional studies 39

revealed that the knockdown of HOXA5 also significantly promoted HCC 40

angiogenesis both in vitro and in vivo. Knocking-down HOXA5 significantly 41

provoked HCC cells to induce the capillary tube formation, migration and 42

proliferation of endothelial cells. In xenograft animal models, we found that a 43

decrease of HOXA5 effectively enhanced tumor growth and increased 44

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3

microvessel densities. We further demonstrated that miR-130b-3p could be 45

directly transcriptionally regulated by Sp1. 46

Conclusions: This study showed that a dysregulation in the Sp1/miR-130b-47

3p/HOXA5 axis contributed to HCC progression and angiogenesis, and that 48

HOXA5 can be considered as a promising therapeutic target for treating HCC. 49

50

Keywords: Hepatocellular carcinoma, Angiogenesis, miR-130b-3p, HOXA5 51

52

Graphical Abstract: 53

54

55

Introduction 56

Angiogenesis is recognized as one of the hallmarks of cancer and essential for 57

both tumor growth and metastasis [1, 2]. Hepatocellular carcinoma (HCC) is a 58

hypervascular tumor with frequent intrahepatic and extrahepatic metastasis, 59

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4

which is responsible for the high recurrence rate and poor prognosis of HCC [3-5]. 60

Therefore, anti-angiogenesis has been considered as an attractive therapeutic 61

strategy for HCC. 62

The involvement of proangiogenic molecules, such as VEGF, VEGF-C/VEGFR3, 63

Ang2/Tie2, in cancer development have been previously described [6]. However, 64

the molecular mechanisms of tumor angiogenesis in HCC is yet to be fully 65

elucidated. Currently, targeted agents including sorafenib, regofenib and 66

lenvatinib, are being used for HCC treatment [7]. Of the multiple effects on 67

tumors, the anti-angiogenic effect is fundamental to the clinical benefits of 68

targeted therapies. Moreover, the combination of anti-angiogenic agents and 69

immunotherapies could significantly improve tumor response rates [8]. Thus, 70

anti-angiogenic treatment is indispensable for HCC patients. The molecular 71

mechanisms of HCC angiogenesis are under urgent need to be investigated. 72

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs and 73

are involved in nearly every biological process by targeting mRNAs for cleavage 74

or translational repression. Emerging evidence indicates that the dysregulation of 75

miRNA is directly implicated in the process of angiogenesis. For example, miR-76

93, miR-135b, and miR-205 exert pro-angiogenesis effects through the 77

suppression of integrin-β8 [9], HIF-1 [10], and PTEN [11], respectively. On the 78

other hand, miR-145, miR-15a/16, and miR-4306 exert an anti-angiogenesis 79

activity by suppressing the expression of p70S6K1 [12], VEGFA [13], and 80

SIX1/Cdc42/VEGFA [14], respectively. Our previous study revealed that miR-81

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130b-3p was up-regulated in HCC [15], however, its role in the regulation of 82

tumor angiogenesis for HCC is still unclear. 83

HOXA5, a member of the HOX family, contains a conserved DNA binding 84

domain, known as the homeobox-containing domain. The HOX family contains 85

39 HOX genes that are classified into four clusters namely A to D [16-18]. 86

Transcription factors encoded by the homeobox genes have a pivotal role in 87

governing the process of tumor angiogenesis. Previous studies have indicated 88

that HOXB9 promotes tumor cell proliferation and angiogenesis in breast cancer 89

and is associated with poor clinical outcomes [19, 20]. Overexpression of 90

HOXC10 promotes angiogenesis in human glioma by interacting with PRMT5 91

[21]. The overexpression of HOXB13 was shown to be correlated with 92

angiogenesis and poor prognosis in pancreatic carcinoma [22]. However, the 93

function of HOXA5 in HCC is still unclarified. 94

In this study, we demonstrated that HOXA5 was an HCC tumor suppressor. Its 95

dysregulation was critical to enhance the angiogenic phenotype of HCC. 96

Furthermore, HOXA5 was identified as an important prognostic predictor and 97

could be a potential therapeutic target for treating HCC. 98

99

Methods 100

Human tissue specimens. 101

All patients included in the present study underwent an initial surgical treatment 102

at the Sun Yat-sen University Cancer Center (SYSUCC; Guangzhou, China), 103

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none of them received any local or systemic anticancer therapies before surgical 104

resection, and no postoperative anticancer treatments were administered before 105

any identified relapse. The diagnosis of all cases was confirmed pathologically 106

based on the terminology criteria established by the International Working Party. 107

The studied patients were classified into 2 cohorts. Cohort 1 comprised of 108

archived paraffin-embedded pathological specimens from 107 consecutive HCC 109

patients who received treatment between July 2003 and July 2007. Cohort 2 110

comprised of a separate set of 449 HCC patients who were treated between 111

December 2003 and May 2015. All patients’ data were marked as anonymous 112

prior to analysis. 113

Clinicalpathological characteristics are presented in Table S1 and Table S3, 114

respectively. Written informed consent was obtained from each patient. The 115

study was approved by the Institute Research Ethics Committee at the Cancer 116

Center. 117

Preparation of tumor cell-conditioned medium (TCM). 118

Stably-transfected tumor cells (3×106) were seeded in a 10 cm dish and 119

incubated for 48 h, after which the medium was removed and the cells were 120

washed thrice with 1 × phosphate –buffered saline (PBS). All cells were then 121

cultured in 10 mL serum-free medium (SFM) for 24 h. Following the incubation 122

period, the TCM was collected and centrifuged at 3000 rpm (4 °C, 5 min) to 123

remove detached cells and was then filtered through a 0.45 μm membrane 124

(Millipore) to discard cell debris. The TCM supernatant was concentrated to 200 125

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7

μL by ultrafiltration with the use of a Millipore 3 kDa Centricon column. The TCM 126

was stored in aliquots at -80 °C until use. The TCM loading volume was adjusted 127

according to the number of living cells in each sample. 128

Capillary tube formation assay. 129

HUVECs (2.4×104) were grown in the TCM for 12 hours at 37 °C in a 48-well 130

plate coated with Matrigel (354230, Corning). The formation of capillary-like 131

structures was captured by a light microscope. The branch points of the formed 132

tubes, which represented the ability of angiogenesis, were counted in three low-133

power fields (100×). 134

Chicken chorioallantoic membrane (CAM) assay. 135

CAM assay was performed according to a protocol previously described [23]. 136

Briefly, a 1-cm-diameter window was opened in the shell of each egg with a 7-137

day-old chicken embryo (Yueqin Breeding Co.). The surface of the dermic sheet 138

on the floor of the air sac was removed to expose the CAM. First, a 0.5-cm-139

diameter filter paper was placed on top of the CAM, and 200 μL of TCM was 140

added onto the center of the paper. After the window was closed with sterile 141

adhesive tape, the eggs were incubated at 37 °C under 80% relative humidity for 142

48 h. Following fixation with a stationary solution (methanol/acetone, 1:1) for 15 143

min, the CAMs were cut and mounted on slides. Gross photos of each CAM were 144

taken under a digital camera. The effect of TCM was evaluated by counting the 145

number of second-and third-order vessels. 146

In vitro cell migration assay. 147

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In vitro cell migration assays were performed in Transwell chambers (8 µm pore 148

size; Costar) according to the manufacturer’s instructions. 2×104 cells were 149

placed into the top chamber of each insert (BD Biosciences) and were incubated 150

at 37 °C for 8 h. 151

Cell proliferation assay. 152

Two experiments were performed to analyze the proliferation of HUVECs. In the 153

CCK-8 assay, HUVECs cells were seeded at 500 cells per well in 96-well 154

microplates for 12 h and then, the complete medium was replaced with TCM and 155

cultured for the indicated hours. Cell proliferation was measured using the Cell 156

Counting Kit-8 (CCK-8) assay kit (Dojindo Corp.) according to the manufacturer’s 157

instructions. In another proliferation assay, HUVECs (6×105) were grown in 158

complete medium for 12 h at 37 °C in a 6-well plate, and then replace the 159

complete medium with TCM and cultured for another 24 h. The cells were 160

trypsinized and counted using the ScepterTM Handheld Automated Cell Counter 161

(Millipore). Three independent experiments were performed for this step. 162

Animal studies. 163

Five-week-old male nude mice were used for subcutaneous xenograft model. 164

Hepatoma cells were suspended in 50 µL of Dulbecco’s Modified Eagle Medium 165

(DMEM) and Matrigel (1:1), and were then injected subcutaneously into either 166

side of the posterior flank of the same male BALB/c athymic nude mice. Eight 167

nude mice were included and their tumor growth was examined during the 168

following 30 days. The mice were sacrificed and the tumors were dissected, fixed 169

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in formalin, and embedded in paraffin. All experimental procedures involving 170

animals were performed according to the Guide for the Care and Use of 171

Laboratory Animals (NIH publications Nos. 80-23, revised 1996), and in 172

accordance with the institutional ethical guidelines for animal experiments. 173

Luciferase reporter assay. 174

To explore the effect of miR-130b-3p on the HOXA5 3’-UTR, HEK293T cells in a 175

6-well plate were co-transfected with 10nM of miR-130b-3p or NC duplex, 1 µg 176

pEZX-HOXA5-3’-UTR-WT or pEZX-HOXA5-3’-UTR-MUT and cultured for 24 h. 177

The cells were then trypsinized and transferred to a 96-well plate. After 24 hours, 178

a luciferase reporter assay was performed following the manufacturer’s 179

instructions. Renilla luciferase activity was normalized to firefly luciferase activity. 180

To dissect the promoter region of miR-130b-3p, Huh7 and MHCC-97H cells were 181

transfected with firefly luciferase reporter plasmids in a 48-well plate. To explore 182

the effect of Sp1 knockdown on the miR-130b-3p promoter activity, Huh7 and 183

MHCC-97H grown in a 48-well plate were co-transfected with siSp1 duplex and 184

firefly luciferase reporter. Cell lysates were collected 48 h after the transfection 185

and were subjected to luciferase activity assays using the luciferase reporter 186

system (Genecopoeia). The data are presented as the mean ± standard error of 187

the mean (SEM) from at least three independent experiments. 188

RNA isolation, quantitative real-time PCR, and western blot. 189

Total RNA was extracted from the HCC tissues and cell lines using Trizol reagent 190

kit (Life Technologies, Carlsbad, CA). After treatment with DNase I (TaKaRa, 191

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Dalian, China), 2 µg of total RNA was used for cDNA synthesis with random 192

hexamers and Superscript III (Invitrogen). The cDNA templates were subjected 193

to PCR amplification. 194

qRT-PCR analysis of miR-130b-3p was performed on an ABI PRISM 7900 195

Sequence Detector using an SYBR Green PCR Kit (Applied Biosystems, 196

Carlsbad, CA). The following primers were used for detection: HmiRQP0159 for 197

miR-130b-3p, HmiRQP9001 for U6 (Genecopoeia, Guangzhou, China). All 198

reactions were run in triplicate. The cycle threshold (Ct) values should not differ 199

more than 0.5 among triplicates. The miR-130b-3p level was normalized to 200

RNU6B, which yielded a 2- ΔΔCt value. 201

Equal amounts of cell protein lysates were separated in 10% SDS-202

polyacrylamide gels and electrophoretically transferred to polyvinylidene 203

difluoride membranes (Millipore), then detected with rabbit polyclonal antibody 204

specific for HOXA5 (Sigma-Aldrich) and a commercial ECL kit (Pierce). Protein 205

loading was estimated using a mouse anti-β-actin monoclonal antibody (Sigma-206

Aldrich). 207

Immunohistochemical staining. 208

Paraffin-embedded tissue sections from nude mice or HCC patients were applied 209

to IHC using a rabbit Ab against human CD31 (cat. ab28364, Abcam), CD34 (cat. 210

ab81289, Abcam), VEGF (cat. ab46154, Abcam) or HOXA5 (cat. HPA029319, 211

Sigma-Aldrich), respectively. Immunoreactivity for VEGF and HOXA5 proteins 212

was scored using a semi-quantitative method by evaluating the number of 213

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positive tumor cells over the total number of tumor cells. Scores were assigned 214

by using 5% increments (0%, 5%, 10% till 100%), as described in a previous 215

study [24]. Any discrete cluster or single cell stained for CD31 or CD34 was 216

considered as one microvessel. Five representative fields were counted, and the 217

average number of microvessels per field (×200) is presented [25]. 218

Chromatin immunoprecipitation (ChIP) assay. 219

ChIP was performed using the SimpleChIP Enzymatic Chromatin IP kit (Cell 220

Signaling Technology). HCC cells were crosslinked with formaldehyde, lysed with 221

SDS buffer followed by ultrasonication, then, incubated with specific antibodies or 222

normal mouse IgG. After washing with high salt and low salt, DNA was eluted 223

and de-crosslinked, and enrichment was examined using PCR. 224

Statistical analysis. 225

Survival curves were computed using the Kaplan-Meier method and analyzed by 226

the log-rank test. Significant prognostic factors found by univariate analysis (p < 227

0.05) were entered into a multivariate analysis using the Cox proportional 228

hazards model. The Fisher test was used to analyze the association of HOXA5 229

expression with the investigated patients’ clinicopathological factors. The Student 230

t test or the Mann-Whiney U test was used to compare the values between 231

subgroups. Data were expressed as mean ± SD. The program Statistical 232

Package for Social Science version 22 (SPSS Inc., Chicago, IL, USA) and R 233

statistical package (R software version 3.4.1; R Foundation for Statistical 234

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Computing, Vienna, Austria) was used for all analyses. A p value < 0.05 was 235

considered statistically significant. 236

237

Results 238

miR-130b-3p is upregulated in HCC and predicts poor prognosis. 239

Using miRNA microarray, we found that miR-130b-3p was up-regulated in HCC 240

[15]. To validate the expression of miR-130b-3p, we quantified the level of miR-241

130b-3p in 107 paired HCC and adjacent non-tumor liver tissues (Cohort 1). 242

Notably, the expression of miR-130b-3p in HCC was markedly increased, 243

compared with adjacent non-tumor liver tissues (p < 0.001) (Figure 1A). 244

Moreover, the expression of miR-130b-3p in patients with recurrence was higher 245

than those without recurrence (p < 0.001) (Figure 1B). 246

Next, we investigated whether the overexpression of miR-130b-3p in HCC tissue 247

correlated with the clinicopathological features and prognosis of the HCC 248

patients. An association between the increased miR-130b-3p expression and 249

increased tumor size was observed (Table S1). The Kaplan-Meier analyses 250

revealed that higher miR-130b-3p level was associated with both shorter overall 251

survival and recurrence-free survival of the HCC patients (p = 0.037 and 0.001, 252

respectively) (Figure 1C-D), and was also confirmed using the TCGA cohort and 253

GEO data set (GSE116182) (p = 0.018 and 0.004, respectively) (Figure 1E-F). 254

Notably, multivariate analyses confirmed that overexpressed miR-130b-3p level 255

was an independent predictor for both shorter RFS and OS in HCC patients (p 256

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<0.001 and p = 0.047, respectively) (Table S2). Collectively, these data suggest 257

that an overexpression of miR-130b-3p may contribute to the progression of HCC 258

and predict poor prognosis of HCC patients. 259

260

miR-130b-3p enhances the capacity of liver cancer cells to promote 261

angiogenesis. 262

To evaluate the role of miR-130b-3p in the angiogenesis of HCC, in vitro capillary 263

tube formation assay and chicken chorioallantoic membranes (CAMs) assay 264

were performed. Huh7 and BEL-7402 with low endogenous miR-130b-3p 265

expression were stably transfected with miR-130b-3p (Figure S1A-B). The 266

conditioned medium of stable cell lines was collected and supplied to the culture 267

medium for HUVECs. We observed that HUVECs cultured with the conditioned 268

medium derived from the miR-130b-3p overexpressed cell lines developed more 269

capillary-like structures and branch points; implying the pro-angiogenesis 270

function of miR-130b-3p in HCC (Figure 2A). Besides, the CAMs assay also 271

revealed that ectopic expression of miR-130b-3p strongly facilitated the formation 272

of second- and third-order micro-vessels (Figure 2B). Moreover, we observed 273

that the TCM of miR-130b-3p- transfectants could promote HUVECs proliferation 274

(Figure 2C). Finally, the angiogenesis related genes, including MMP9, FGF2, 275

VEGFA, VEGFC, PDGFA and PDGFC, were found to be upregulated in miR-276

130b-3p overexpressed HCC cells (Figure 2D). On the other hand, HUVECs 277

treated with the TCM of anti-miR-130b-3p- transfectants had less capacity for 278

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angiogenesis (Figure S2). In summary, these findings suggest that miR-130b-3p 279

could enhance the pro-angiogenesis capacity of HCC cells in vitro. 280

To further assess the in vivo effects of miR-130b-3p overexpression on 281

angiogenesis, miR-130b-3p-BEL-7402 cells and control cells were 282

subcutaneously implanted into nude mice. At 9 days post injection, the mean 283

volumes of xenograft tumors generated from the miR-130b-3p-BEL-7402 cells 284

were markedly larger than those originating from the control cells (n = 8 animals 285

per group, p < 0.01) (Figure 2F, left panel). At 30 days post injection, the mice 286

were sacrificed and the tumors were weighted. The mean tumor weight of the 287

miR-130b-3p-BEL-7402 group was greater than the control group (p = 0.017) 288

(Figure 2F, right panel). 289

Next, the microvessel density of the tumors was analyzed by 290

immunohistochemical staining using CD31 and CD34 antibodies. Both of them 291

showed a significant increase of microvessel density in tumors with miR-130b-3p 292

overexpressed as compared to the controls (p < 0.001). Besides, the expression 293

of VEGF was also found to be upregulated in miR-130b-3p overexpressed 294

tumors. These results clearly showed that miR-130b-3p was involved in 295

promoting tumor angiogenesis in HCC (Figure 2G-H). 296

297

HOXA5 is a direct target gene of miR-130b-3p. 298

The potential targets of miR-130b-3p were predicted by the following five 299

bioinformatics algorithms, targetScan, picTar, RNA22, PITA and miRanda. The 300

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intersections were retrieved by starbase V2.0 [26] and thirty-one genes were 301

found to be potential targets (Table S6). The top 5 genes, JARID2, DCBLD2, 302

E2F7, HOXA5, and PHF3, were screened by qRT-PCR. Among them, HOXA5 303

was found to be the most significantly downregulated target in HCC cells 304

transfected with miR-130b-3p (Figure S3). As transcription factors encoded by 305

the homeobox genes have a pivotal role in governing the process of tumor 306

angiogenesis, we decided to explore the function of HOXA5. 307

Two conserved binding sites for miR-130b-3p were identified in 3’UTR region of 308

the HOXA5 gene. To elucidate whether HOXA5 was directly regulated by miR-309

130b-3p, we constructed a luciferase reporter plasmid containing the 3’UTR of 310

HOXA5. As shown in Figure 3B, the relative luciferase activity of the reporter 311

containing the wild-type 3’UTR was significantly decreased when co-transfected 312

with miR-130b-3p mimics. On the contrary, the luciferase activity of the mutant-313

type 3’UTR was similar between the miR-130b-3p mimics and control mimics. As 314

shown in Figure 3C, the protein expression of HOXA5 was significantly 315

decreased after the overexpression of miR-130b-3p. These indicated that 316

HOXA5 was directly regulated by miR-130b-3p. 317

To explore the correlation between miR-130b-3p and HOXA5, the expression of 318

miR-130b-3p and HOXA5 in HCC tissues were detected by qRT-PCR and 319

immunohistochemistry, respectively. After normalization, the expression of miR-320

130b-3p and HOXA5 were analyzed by Pearson’s correlation coefficient analysis. 321

Interestingly, the HOXA5 protein level was inversely correlated with miR-130b-3p 322

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expression level (Figure 3D). This implied that endogenous HOXA5 in HCC were 323

negatively regulated by miR-130b-3p. 324

Furthermore, we evaluated the role of HOXA5 in miR-130b-3p-mediated 325

phenotypes. We found that the overexpression of HOXA5 in miR-130b-3p-326

transfectants attenuated the pro-angiogenic effect of miR-130b-3p (Figure 3E). 327

These data suggested that HOXA5 acted as a direct functional target gene of 328

miR-130b-3p. 329

330

Down-regulation of HOXA5 promotes tumorigenicity and angiogenesis in 331

HCC. 332

To explore the anti-oncogenic role of HOXA5 in HCC, two shRNAs (shHOXA5 333

#33 and shHOXA5 #34) specially targeting HOXA5 were transfected into Huh7 334

and MHCC-97H, respectively. The silencing effect was validated by western 335

blotting analysis, and the results revealed that both shRNAs could specifically 336

down-regulate the expression of HOXA5 (Figure 4A). Since VEGF is a pivotal 337

activator of angiogenesis-related pathways, we examined the VEGF level in the 338

TCM. ELISA assay revealed that the VEGF levels were significantly higher in the 339

shHOXA5 groups (Figure 4B). To explore the biological significance of HOXA5 in 340

tumor angiogenesis, in vitro tube formation assays were performed. TCMs from 341

tumor cells were supplied to the culture medium for HUVECs. We observed that 342

the HUVECs treated with TCM derived from the shHOXA5-transfectants 343

developed more capillary-like structures compared to cells treated with TCM 344

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derived from scramble shRNAs-transfectants (Figure 4C). In addition the TCM 345

from shHOXA5-transfectants significantly promoted the proliferation and 346

migration of HUVECs, compared to the corresponding controls (Figure 4D-E). 347

Moreover, the angiogenesis-related genes were upregulated in HCC cells 348

transfected with shHOXA5 (Figure S4E-F). However, the ectopic expression of 349

HOXA5 had little impact on tumor angiogenesis (Figure S4C-D, and Figure S5). 350

To further verify the in vivo anti-oncogenic role of HOXA5, we conducted 351

subcutaneous injection of HCC cells into nude mice, which were then sacrificed 352

after four weeks for collecting the tumoral tissues. As shown in Figure 5A-C, both 353

the tumor size and weight were significantly higher in the Huh7-shHOXA5 group 354

than in Huh7-shCtrl group. IHC staining of the tumoral tissues showed markedly 355

lower HOXA5 level and higher CD31, CD34 and VEGF levels in the Huh7-356

shHOXA5 group than in the Huh7-shCtrl group (Figure 5D-E). Collectively, these 357

data proved that HOXA5 may suppress angiogenesis in HCC. 358

359

Down-regulation of HOXA5 predicts poor prognosis and negatively 360

correlates with angiogenesis markers. 361

The protein level of HOXA5 in 449 human HCC samples and matched adjacent 362

nontumor liver tissues were detected by immunohistochemistry (IHC). The 363

results revealed that HOXA5 was significantly down-regulated in HCC tissues 364

(Figure 6A-B). Clinicopathological analyses revealed that HOXA5 was negatively 365

correlated with tumor size and AFP (Table S3). Further survival analysis showed 366

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that the low HOXA5 group had shorter overall survival and recurrence-free 367

survival than the high HOXA5 group (Figure 6C). Besides, the prognostic value 368

of HOXA5 in HCC was also confirmed in the GEO data set (Figure S6A-B). 369

Multivariate Cox regression analysis further demonstrated that low HOXA5 370

expression was an independent predictive indicator for overall survival (hazard 371

ratio [HR] 1.758, 95% confidence interval [CI], 1.295-2.386; p < 0.001) and 372

recurrence-free survival (hazard ratio [HR] 1.625, 95% confidence interval [CI], 373

1.239-2.132; p < 0.001) in HCC (Table S4). Moreover, the expression levels of 374

CD31 and CD34 in tumor specimens from 314 HCC patients were also detected 375

by IHC staining, to investigate the correlation between HOXA5 and MVD. These 376

patients were divided into two groups, namely the HOXA5_high group (n = 170) 377

and the HOXA5_low group (n = 144) based on the HOXA5 expression. 378

We found that the rate of strong CD31 staining was higher in the HOXA5_low 379

group, compared to that in the HOXA5_high group (82/144, 56.9% vs. 70/170, 380

41.2%, p < 0.05) (Figure 6E, left panel). Consistently, the rate of strong CD34 381

staining was also higher in the HOXA5_low group (84/144, 58.3%) than in the 382

HOXA5_high group (61/170, 35.9%, p < 0.001) (Figure 6E, right panel). We also 383

found significant negative correlations between HOXA5 and CD31 (R = -0.25, p < 384

0.001), as well as CD34 in HCC tissues (R = -0.31, p < 0.001) (Figure 6F). 385

Consistently, the correlations between HOXA5 and CD31/CD34 were also 386

validated in the GEO data set (Figure S6C-D). 387

388

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Sp1 regulates miR-130b-3p expression through miR-130b-3p promoter 389

binding. 390

To determine the mechanism of miR-130b-3p up-regulation in HCC, the 391

AliBaba2.1 (http://www.gene-regulation.com/pub/programs/alibaba2/) software 392

was used to search for predicted transcription binding sites. Sixty three 393

transcription factors which may bind to the promoter of miR-130b-3p were 394

predicted (Table S7). The top 3 transcription factors, which had the most 395

potential binding sites, were Sp1 (97 sites), C/EBPalpha (11 sites) and NF-1 (10 396

sites). A previous study reported that Sp1 may bind to the putative miR-130b-3p 397

promoter sequence, as predicted by TSSG promoter prediction program[27]. 398

Moreover, since Sp1 is considered as a basal transcription factor and regulates 399

angiogenesis in multiple tumors [28-30], we thereby chose Sp1 for further 400

analysis. 401

The miR-130b-3p upstream region (-1 to -2000kb) was analyzed by JASPAR 402

(http:// http://jaspar.genereg.net/), and six binding sites of Sp1 were predicted in 403

the putative promoter region (Figure 7A), using a cut-off score of 10 and a 404

relative profile score threshold of 80% (Table S8). We used RNA interference to 405

silence the Sp1 expression (Figure 7H). Our findings showed that silencing Sp1 406

resulted in a significant decrease in the promoter activity of p-(-2.0k) (Figure 7B) 407

as well as the endogenous miR-130b-3p level (Figure 7C). To further confirm the 408

functional Sp1 binding sites within the putative promoter region, a 5’ deletion 409

analysis was conducted (Figure 7A). Compared with the promoter activity of p-(-410

2.0k), no significant change in p-(-0.7k), p-(-0.5k) or p-(-0.3k) was detected and a 411

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remarkably decreased activity in the promoter activity of p-(-0.1k) was observed 412

(Figure 7D). These results indicated that the -0.3 to -0.1kb region was crucial for 413

miR-130b-3p transcription. Between the -0.3 to -0.1kb region, there was only one 414

Sp1 potential binding site (Figure 7A). Sp1 silencing dramatically reduced the 415

activity of p-(-0.3k) (Figure 7E) but had no effect on that of p-del without Sp1 416

binding site within the -0.3 to -0.1kb region (Figure 7F). In addition, ectopic 417

expression of Sp1 did not change the promoter activity of p-(-2.0 k) (Figure S7B). 418

Silencing of Sp1 expression reduced the promoter activity of p-(ΔA) and p-(ΔB) 419

(Figure S7C-D). To determine whether Sp1 bounded directly to the miR-130b-3p 420

promoter, we performed chromatin immunoprecipitation (ChIP) assay with anti-421

Sp1 antibody, and the result demonstrated an interaction between Sp1 and the 422

miR-130b-3p promoter (Figure 7G). Furthermore, the expression of Sp1 was 423

found to be positively correlated with miR-130b-3p in HCC specimens (Figure 7I). 424

These data collectively demonstrated that Sp1 transcriptionally regulated miR-425

130b-3p expression in HCC cells. 426

427

Down-regulation of HOXA5 promotes angiogenesis in HCC via the 428

PI3K/AKT/mTOR signaling pathway. 429

To further explore the molecular mechanism of the anti-angiogenesis function of 430

HOXA5, transcriptome profiling by RNA-Seq was performed. Kyoto Encyclopedia 431

of Genes and Genomes (KEGG) pathway enrichment analysis showed that the 432

PI3K/AKT/mTOR signaling pathway was significantly altered upon knocking-433

down HOXA5 (Figure 8A). Furthermore, the phosphorylation of the mammal 434

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21

target of rapamycin (mTOR) and other proteins, such as protein kinase B (AKT) 435

and p70 S6 kinase, involved in the PI3K/AKT/mTOR signaling pathway were 436

upregulated in cells transfected with shHOXA5 (Figure 8B). Additionally, HOXA5 437

has been reported to be involved in several pathways, including NF-kb, P53, 438

STAT3 and MMP2. However, no positive relationship between HOXA5 and the 439

above reported pathways were detected in this present study. (Figure S8). 440

441

Discussion 442

HOXA5 has been reported to be deregulated in several malignant tumors, 443

including colorectal cancer, lung cancer, cervical cancer, and gastric cancer [31-444

35]. However, the biological role and underlying mechanism of HOXA5 in HCC 445

are still unclear. In the present study, we investigated the biological function and 446

molecular mechanism of HOXA5 in HCC. Our results demonstrated that HOXA5 447

was down-regulated in HCC tissues and its expression was closely correlated 448

with tumor size. Survival analyses indicated that a low HOXA5 expression 449

predicted unfavorable OS and high recurrence probability in HCC patients. 450

Functional experiments showed that HOXA5 significantly suppressed HCC 451

angiogenesis. 452

As a member of the HOX family, HOXA5 plays an important role in tumor 453

development and progression. Previous studies indicated that HOXA5 could up-454

regulated linc00312 expression, and inhibit proliferation and promote apoptosis in 455

non-small cell lung cancer [34]. HOXA5 has also been reported to be down-456

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regulated by miR-429, and promote colorectal cancer progression and 457

metastasis [35]. Moreover, HOXA5 was observed to inhibit proliferation and 458

induce apoptosis in cervical cancer cells via regulation of protein kinase B and 459

p27 [32]. In this present study, we attempted to investigate the biological 460

function and molecular mechanism of HOXA5 in HCC progression. Our results 461

demonstrated that HOXA5 was down-regulated in HCC tissues and its 462

expression closely correlated with tumor size. Survival analyses indicated that a 463

low HOXA5 expression predicted unfavorable OS and high recurrence probability 464

in HCC patients. It is worth noting that previous studies considered HOXA5 as a 465

tumor-suppressor mainly due to its capacity to inhibit tumor proliferation and 466

metastasis but not angiogenesis. The hypervascular nature underlines the 467

importance of angiogenesis in most HCC tumors [36].Based on our research, we 468

may be the first to discover that both the in vitro and in vivo down-regulation of 469

HOXA5 promoted angiogenesis, and HOXA5 was regulated by the Sp1/miR-470

130b-3p axis and inhibited angiogenesis in HCC via the PI3K/AKT/mTOR 471

signaling pathway. 472

According to the in silico target prediction algorithms, miR-130b-3p has a 473

complementary binding sequence in the 3’-UTR of HOXA5 mRNA. Previous 474

studies have shown that the miR-130b-3p expression was significantly 475

upregulated in HCC and it emerged as an oncogene [37, 38]. However, a recent 476

report demonstrated that miR-130b-3p inhibited, rather than promoted, cell 477

motility in hepatoma cell lines and may function as a tumor suppressor [39]. In 478

this current study, we found that miR-130b-3p promoted angiogenesis in HCC by 479

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23

directly targeting HOXA5. In addition, high miR-130b-3p expression levels were 480

significantly associated with aggressive characteristics and poor prognosis in 481

HCC patients. Based on the previous works from other groups and this study 482

findings, we confirmed miR-130b-3p as an oncogene in HCC. 483

Sp1 is considered as a basal transcription factor and has been found to regulate 484

angiogenesis in multiple tumors. Chen et al reported that JWA suppresses tumor 485

angiogenesis in gastric cancer by inhibiting Sp1 activity and downregulating the 486

proangiogenic MMP-2 expression [40]. However, the specific role of Sp1 in HCC 487

angiogenesis has not been clarified. Here, we demonstrated that Sp1 regulated 488

HCC angiogenesis by transcriptional activation of miR-130b-3p based on the 489

following evidences. First, the knockdown of Sp1 dramatically decreased the 490

miR-130b-3p promoter activity and the cellular miR-130b-3p expression. Second, 491

mutation of the potential Sp1 binding site significantly reduced the miR-130b-3p 492

promoter activity. Third, ChIP assays revealed that Sp1 interacted with the miR-493

130b-3p promoter sequence in vivo. 494

The PI3K/AKT/mTOR pathway is activated in majority of human cancers and 495

plays a key role in tumor angiogenesis [41, 42]. Although anti-angiogenesis 496

drugs such as sorafenib and regorafenib, have been approved for advanced 497

HCC, the prognoses of these patients are still unsatisfactory. It implies that the 498

molecular mechanism of abnormal hypervascular in HCC needs to be more 499

intensely investigated. Although our present study indicated that the down-500

regulation of HOXA5 activated the PI3K/AKT/mTOR pathway, however, the detail 501

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24

mechanism of how HOXA5 regulates the PI3K/AKT/mTOR pathway needs to be 502

further clarified. 503

504

Conclusions 505

In summary, we demonstrated that miR-130b-3p was frequently up-regulated in 506

HCC and was correlated with poor prognosis. HOXA5 was identified as a direct 507

target of miR-130b-3p. Furthermore, we demonstrated that the down-regulation 508

of HOXA5 played a crucial role in angiogenesis for HCC. In addition, miR-130b-509

3p was found to be transcriptionally regulated by Sp1. Our findings highlight the 510

importance of Sp1/miR-130b-3p/HOXA5 axis in tumor angiogenesis and 511

recurrence, and implicate HOXA5 as a potential target for HCC treatment. 512

513

Abbreviations 514

CAM: Chicken chorioallantoic membrane; ChIP: Chromatin immunoprecipitation; 515

Ct: cycle threshold; DMEM: Dulbecco’s Modified Eagle Medium; FBS: fetal 516

bovine serum; HCC: hepatocellular carcinoma; HUVEC: Human umbilical vein 517

endothelial cell; IHC: immunohistochemistry; miRNA: microRNA; mTOR: 518

mammal target of rapamycin; PBS: phosphate –buffered saline; qRT-PCR: Real-519

time quantitative polymerase chain reaction; SFM: serum free medium; siRNA: 520

small interference RNA; TCM: tumor cell-conditioned medium 521

522

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25

Acknowledgments 523

This work was supported by grants from the National Natural Science Foundation 524

of China (Nos. 81101863, 81772598 and 81772625), the Guangdong Provincial 525

Natural Science Foundation of China (No. 2017A030311006), the Guangzhou 526

Science and Technology Program of China (No. 201804020093), and the Sun 527

Yat-sen University Clinical Research 5010 Program (No. 2012010). We thank 528

Shi-Mei Zhuang, Mu-Yan Cai and Li-sheng Zheng for revising this manuscript. 529

530

Author Contributions 531

Y.-D.L and C.-W.W performed experiments, analyzed the data and wrote the 532

manuscript. Z.-W.Y, W.-W.L, D.-L.Z and Y.-C.Y assisted with the 533

immunohistochemistry assays and animal experiments. K.L., Y.-P.Z, Y.-J.W, Y.-534

X.S, Y.-X.Q, J.-L.Q and W.H collected the clinical samples. Y.-F.Y, X.-Y.G and 535

B.-K.L designed the study and revised the manuscript. 536

537

Competing interests 538

The authors declare that they have no competing interests. 539

540

541

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References 542

543

1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011; 144:646-544

674. 545

2. Qian CN, Pezzella F. Tumor vasculature: a sally port for inhibiting cancer cell spreading. 546

Cancer Commun (Lond). 2018; 38:52. 547

3. Heimbach JK, Kulik LM, Finn RS, Sirlin CB, Abecassis MM, Roberts LR, et al. AASLD 548

guidelines for the treatment of hepatocellular carcinoma. Hepatology. 2018; 67:358-380. 549

4. He W, Zheng Y, Zou R, Shen J, Yang J, Qiu J, et al. Long- versus short-interval follow-up 550

after resection of hepatocellular carcinoma: a retrospective cohort study. Cancer 551

Commun (Lond). 2018; 38:26. 552

5. Pan YX, Chen JC, Fang AP, Wang XH, Chen JB, Wang JC, et al. A nomogram predicting the 553

recurrence of hepatocellular carcinoma in patients after laparoscopic hepatectomy. 554

Cancer Commun (Lond). 2019; 39:55. 555

6. Welti J, Loges S, Dimmeler S, Carmeliet P. Recent molecular discoveries in angiogenesis 556

and antiangiogenic therapies in cancer. J Clin Invest. 2013; 123:3190-3200. 557

7. Villanueva A. Hepatocellular Carcinoma. N Engl J Med. 2019; 380:1450-1462. 558

8. Khan KA, Kerbel RS. Improving immunotherapy outcomes with anti-angiogenic 559

treatments and vice versa. Nature Reviews Clinical Oncology. 2018; 15:310. 560

9. Fang L, Deng Z, Shatseva T, Yang J, Peng C, Du WW, et al. MicroRNA miR-93 promotes 561

tumor growth and angiogenesis by targeting integrin-beta8. Oncogene. 2011; 30:806-562

821. 563

10. Umezu T, Tadokoro H, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH. Exosomal miR-564

135b shed from hypoxic multiple myeloma cells enhances angiogenesis by targeting 565

factor-inhibiting HIF-1. Blood. 2014; 124:3748-3757. 566

11. He L, Zhu W, Chen Q, Yuan Y, Wang Y, Wang J, et al. Ovarian cancer cell-secreted 567

exosomal miR-205 promotes metastasis by inducing angiogenesis. Theranostics. 2019; 568

9:8206-8220. 569

12. Xu Q, Liu LZ, Qian X, Chen Q, Jiang Y, Li D, et al. MiR-145 directly targets p70S6K1 in 570

cancer cells to inhibit tumor growth and angiogenesis. Nucleic Acids Res. 2012; 40:761-571

774. 572

13. Sun CY, She XM, Qin Y, Chu ZB, Chen L, Ai LS, et al. miR-15a and miR-16 affect the 573

angiogenesis of multiple myeloma by targeting VEGF. Carcinogenesis. 2013; 34:426-435. 574

14. Zhao Z, Li L, Du P, Ma L, Zhang W, Zheng L, et al. Transcriptional Downregulation of miR-575

4306 serves as a New Therapeutic Target for Triple Negative Breast Cancer. Theranostics. 576

2019; 9:1401-1416. 577

15. Wei R, Huang GL, Zhang MY, Li BK, Zhang HZ, Shi M, et al. Clinical significance and 578

prognostic value of microRNA expression signatures in hepatocellular carcinoma. Clin 579

Cancer Res. 2013; 19:4780-4791. 580

Page 27: Dysregulated Sp1/miR-130b-3p/HOXA5 axis contributes to ... · 3 45 microvessel densities. We further demonstrated that miR-130b-3p could be 46 directly transcriptionally regulated

27

16. McGinnis W, Krumlauf R. Homeobox genes and axial patterning. Cell. 1992; 68:283-302. 581

17. Scott MP. Vertebrate homeobox gene nomenclature. Cell. 1992; 71:551-553. 582

18. Krumlauf R. Hox genes in vertebrate development. Cell. 1994; 78:191-201. 583

19. Li H, Zhang Y, Chen SW, Li FJ, Zhuang SM, Wang LP, et al. Prognostic significance of 584

Flotillin1 expression in clinically N0 tongue squamous cell cancer. Int J Clin Exp Pathol. 585

2014; 7:996-1003. 586

20. Hou J, Zhou Y, Zheng Y, Fan J, Zhou W, Ng IO, et al. Hepatic RIG-I predicts survival and 587

interferon-alpha therapeutic response in hepatocellular carcinoma. Cancer Cell. 2014; 588

25:49-63. 589

21. Tan Z, Chen K, Wu W, Zhou Y, Zhu J, Wu G, et al. Overexpression of HOXC10 promotes 590

angiogenesis in human glioma via interaction with PRMT5 and upregulation of VEGFA 591

expression. Theranostics. 2018; 8:5143-5158. 592

22. Zhuang SM, Zhang GH, Chen WK, Chen SW, Wang LP, Li H, et al. Survival study and 593

clinicopathological evaluation of trichilemmal carcinoma. Mol Clin Oncol. 2013; 1:499-594

502. 595

23. Jiang L, Lin C, Song L, Wu J, Chen B, Ying Z, et al. MicroRNA-30e* promotes human 596

glioma cell invasiveness in an orthotopic xenotransplantation model by disrupting the 597

NF-kappaB/IkappaBalpha negative feedback loop. J Clin Invest. 2012; 122:33-47. 598

24. Cai MY, Tong ZT, Zheng F, Liao YJ, Wang Y, Rao HL, et al. EZH2 protein: a promising 599

immunomarker for the detection of hepatocellular carcinomas in liver needle biopsies. 600

Gut. 2011; 60:967-976. 601

25. Lin XJ, Fang JH, Yang XJ, Zhang C, Yuan Y, Zheng L, et al. Hepatocellular Carcinoma Cell-602

Secreted Exosomal MicroRNA-210 Promotes Angiogenesis In Vitro and In Vivo. Mol Ther 603

Nucleic Acids. 2018; 11:243-252. 604

26. Li JH, Liu S, Zhou H, Qu LH, Yang JH. starBase v2.0: decoding miRNA-ceRNA, miRNA-605

ncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data. Nucleic 606

Acids Res. 2014; 42:D92-97. 607

27. Yeung ML, Yasunaga J, Bennasser Y, Dusetti N, Harris D, Ahmad N, et al. Roles for 608

microRNAs, miR-93 and miR-130b, and tumor protein 53-induced nuclear protein 1 609

tumor suppressor in cell growth dysregulation by human T-cell lymphotrophic virus 1. 610

Cancer Res. 2008; 68:8976-8985. 611

28. Liu N, Ding D, Hao W, Yang F, Wu X, Wang M, et al. hTERT promotes tumor angiogenesis 612

by activating VEGF via interactions with the Sp1 transcription factor. Nucleic Acids Res. 613

2016; 44:8693-8703. 614

29. Chen Y, Huang Y, Huang Y, Xia X, Zhang J, Zhou Y, et al. JWA suppresses tumor 615

angiogenesis via Sp1-activated matrix metalloproteinase-2 and its prognostic 616

significance in human gastric cancer. Carcinogenesis. 2014; 35:442-451. 617

30. Cho SG, Yi Z, Pang X, Yi T, Wang Y, Luo J, et al. Kisspeptin-10, a KISS1-derived 618

decapeptide, inhibits tumor angiogenesis by suppressing Sp1-mediated VEGF expression 619

and FAK/Rho GTPase activation. Cancer Res. 2009; 69:7062-7070. 620

Page 28: Dysregulated Sp1/miR-130b-3p/HOXA5 axis contributes to ... · 3 45 microvessel densities. We further demonstrated that miR-130b-3p could be 46 directly transcriptionally regulated

28

31. Hu LL, Wang XX, Chen X, Chang J, Li C, Zhang Y, et al. BCRP gene polymorphisms are 621

associated with susceptibility and survival of diffuse large B-cell lymphoma. 622

Carcinogenesis. 2007; 28:1740-1744. 623

32. Cheng J, Huo DH, Kuang DM, Yang J, Zheng L, Zhuang SM. Human macrophages promote 624

the motility and invasiveness of osteopontin-knockdown tumor cells. Cancer Res. 2007; 625

67:5141-5147. 626

33. Xu T, Zhu Y, Xiong Y, Ge YY, Yun JP, Zhuang SM. MicroRNA-195 suppresses 627

tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells. 628

Hepatology. 2009; 50:113-121. 629

34. Zhu Q, Lv T, Wu Y, Shi X, Liu H, Song Y. Long non-coding RNA 00312 regulated by HOXA5 630

inhibits tumour proliferation and promotes apoptosis in Non-small cell lung cancer. J 631

Cell Mol Med. 2017; 21:2184-2198. 632

35. Han Y, Zhao Q, Zhou J, Shi R. miR-429 mediates tumor growth and metastasis in 633

colorectal cancer. Am J Cancer Res. 2017; 7:218-233. 634

36. Morse MA, Sun W, Kim R, He AR, Abada PB, Mynderse M, et al. The Role of Angiogenesis 635

in Hepatocellular Carcinoma. Clin Cancer Res. 2019; 25:912-920. 636

37. Ma S, Tang KH, Chan YP, Lee TK, Kwan PS, Castilho A, et al. miR-130b Promotes CD133(+) 637

liver tumor-initiating cell growth and self-renewal via tumor protein 53-induced nuclear 638

protein 1. Cell Stem Cell. 2010; 7:694-707. 639

38. Tu K, Zheng X, Dou C, Li C, Yang W, Yao Y, et al. MicroRNA-130b promotes cell 640

aggressiveness by inhibiting peroxisome proliferator-activated receptor gamma in 641

human hepatocellular carcinoma. Int J Mol Sci. 2014; 15:20486-20499. 642

39. Lin YH, Wu MH, Liao CJ, Huang YH, Chi HC, Wu SM, et al. Repression of microRNA-130b 643

by thyroid hormone enhances cell motility. J Hepatol. 2015; 62:1328-1340. 644

40. Liu JJ, Lin XJ, Yang XJ, Zhou L, He S, Zhuang SM, et al. A novel AP-1/miR-101 regulatory 645

feedback loop and its implication in the migration and invasion of hepatoma cells. 646

Nucleic Acids Res. 2014; 42:12041-12051. 647

41. Karar J, Maity A. PI3K/AKT/mTOR pathway in angiogenesis. Frontiers in molecular 648

neuroscience. 2011; 4:51. 649

42. Zhao P, Chen H, Wen D, Mou S, Zhang F, Zheng S. Personalized treatment based on mini 650

patient-derived xenografts and WES/RNA sequencing in a patient with metastatic 651

duodenal adenocarcinoma. Cancer Commun (Lond). 2018; 38:54. 652

653

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29

Figure 1 654

655

Figure 1. miR-130b-3p is upregulated in HCC and predicts poor prognosis. 656

(A) The expression of miR-130b-3p in HCC is markedly upregulated, compared 657

with adjacent non-tumor liver tissues. 658

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30

(B) The expression of miR-130b-3p is higher in tumor samples of recurrent HCC 659

patients. 660

(C) The Kaplan-Meier method reveals that higher miR-130b-3p level is 661

associated with shorter overall survival of HCC patients. 662

(D) The Kaplan-Meier method reveals that higher miR-130b-3p level is 663

associated with shorter recurrence-free survival of HCC patients. 664

(E) Kaplan–Meier analyses of the correlation between miR-130b-3p levels and 665

the overall survival in the TCGA HCC cohort. 666

(F) Kaplan–Meier analyses of the correlation between miR-130b-3p levels and 667

the overall survival in GEO database (GSE116182). 668

669

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31

Figure 2 670

671

Figure 2. miR-130b-3p promotes tumor angiogenesis in vitro and in vivo. 672

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32

(A) TCM from miR-130b-3p transfected HCC cells induced tube formation of 673

HUVECs. Representative images of capillary-like structures and the number of 674

branch points of HUVECs are shown. 675

(B) Effect of miR-130b-3p on vascularization in the CAM angiogenesis model. 676

Filter discs soaked with TCM were loaded on the CAMs of Day 8 chick embryos. 677

After incubation for 5 days, the area under and surround the filter was fixed and 678

photographed. Representative images of neovascularization and the number of 679

new blood vessels are shown. 680

(C) HUVECs proliferation was promoted by the TCM from miR-130b-3p 681

transfected HCC cells. HUVEC cells were seeded on 6-well plate with a density 682

of 3 × 105 cells per well and were cultured with SFM supplemented with 20% 683

FBS and 0.3% EGF for 6 hours and shifted to TCM from indicated cells and 684

cultured for additional 24 h. The number of HUVEC cells was counted using the 685

ScepterTM Handheld Automated Cell Counter. Results were based on six 686

independent experiments. 687

(D) The mRNA level of angiogenesis relevant genes was upregulated by miR-688

130b-3p. The expression of MMP9, FGF2, VEGFA, VEGFC, PDGFA, PDGFC in 689

Huh7 cells transfected with miR-130b-3p or vector were determined by qRT-PCR. 690

Results were based on at least three independent experiments. Data are 691

presented as their mean ± SD. 692

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33

(E) miR-130b-3p overexpressed and control BEL-7402 cells were 693

subcutaneously injected into nude mice (n = 8). The nude mice were sacrificed 694

on day 30 after inoculation and the tumor were harvested. 695

(F) The tumor weight on day-30 after inoculation are presented (left). The tumor 696

growth curve was measured every 3 days for 30 days after inoculation. The 697

mean tumor volume was significantly increased in the miR-130b-3p 698

overexpression group (right). 699

(G) Representive immunohistochemical images of endothelial markers CD31, 700

CD34 and VEGF on serial sections of tumor samples are shown. Scale bar, 100 701

µm. 702

(H) The staining intensity of CD31, CD34, and VEGF from tumor samples are 703

shown. Data are presented as their mean ± SD. Statistical analysis was 704

performed using the Student’s t-test. 705

* p < 0.05, **p < 0.01, ***p < 0.001. 706

707

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34

Figure 3 708

709

Figure 3. HOXA5 is a direct target of miR-130b-3p. 710

(A) Luciferase reporter plasmids were inserted with wild-type or mutant HOXA5 711

3’-UTR immediately downstream of the luciferase gene. The sequences of two 712

predicted miR-130b-3p binding sites within the HOXA5 3’-UTR, including wild-713

types and mutants (underlined) are shown. 714

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35

(B) Relative luciferase activity was analyzed in HEK-293T cells transfected with 715

indicated reporter plasmids and miR-130b-3p mimic or control. 716

(C) miR-130b-3p overexpression downregulated endogenous HOXA5 protein 717

level in Huh7 and BEL-7402 cells. 718

(D) HOXA5 protein level negatively correlated with miR-130b-3p in HCC. Left, 719

representative immunohistochemical images of HOXA5 in HCC are shown 720

(magnification ×200). Right, the correlation between miR-130b-3p and HOXA5 721

protein level in HCC tissues are shown (n = 20). miR-130b-3p was detected by 722

qRT-PCR and normalized to U6 expression. Statistical analysis was performed 723

using the Spearman’s correlation coefficient. 724

(E) HOXA5 partially impaired the miR-130b-3p induced angiogenesis phenotype. 725

HCC cells were co-transfected with miR-130b-3p mimic or control mimic and 726

pEZ-HOXA5 or vector and the TCMs were collected 24 h after transfection. 727

HUVECs were seeded above the Matrigel basement membrane matrix (growth 728

factor reduced) and cultured with TCM for 8 h. Representative images of 729

capillary-like structures (Left) are shown. Quantification of tube formation (Right) 730

was performed basing on the number of branches. Results were based on at 731

least three independent experiments.Data are presented as their mean ± SD. 732

Statistical significance was determined by Student’s t-test. * p < 0.05, **p < 0.01, 733

***p < 0.001. 734

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36

Figure 4 735

736

Figure 4.The down-regulation of HOXA5 promotes angiogenesis in HCC in vitro. 737

(A) The knockdown efficiency of shHOXA5 in HCC cells by Western Blot (Huh7 738

and MHCC-97H). 739

(B) The down-regulation of HOXA5 increased the levels of secreted VEGF in 740

HCC cells. VEGF was measured by ELISA in the supernatants of indicated HCC 741

cells. 742

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37

(C) The down-regulation of HOXA5 enhanced HCC cell-induced tube formation 743

of HUVECs. HUVECs were cultured in the presence of 75% TCM from Huh7 or 744

MHCC-97H cells transfected with indicated plasmids. Representative images of 745

tube formation and the number of branch points of HUVECs are presented. 746

(D) Endothelial recruitment assay revealed an enhanced effect of HOXA5 down-747

regulation on HCC cell-induced HUVECs migration . HUVECs were seeded in 748

the upper transwell chambers with TCM in the lower compartments and 749

incubated for 12 h. 750

(E) The down-regulation of HOXA5 promoted HCC cell-induced HUVECs 751

proliferation. HUVECs were grown in complete medium for 12 h at 37 °C in a 96-752

well plate and then replace the complete medium with TCM and cultured for an 753

indicated period of time. Cell viability was measured using the Cell Counting Kit-8 754

(CCK-8) assay kit (Dojindo Corp.) according to the manufacturer’s instructions. 755

Three independent experiments were performed. 756

*p < 0.05; **p < 0.01. 757

758

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38

Figure 5 759

760

Figure 5. The down-regulation of HOXA5 promotes HCC tumorigenicity and 761

angiogenesis in vivo. 762

(A) HOXA5 down-regulation promoted the growth of Huh7 xenograft tumors. 763

HOXA5 down-regulated and control Huh7 cells were subcutaneously injected 764

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39

into nude mice (n = 8). The nude mice were sacrificed on day-28 after inoculation 765

and the tumor were harvested. 766

(B) The tumor weight on day-28 after inoculation are presented. 767

(C) The tumor growth curve was measured every week after inoculation. The 768

mean tumor volume was significantly increased in the HOXA5-downregulated 769

group. 770

(D) HOXA5 down-regulation elevated the expression of angiogenesis markers. 771

Immunohistochemical expression of angiogenesis markers CD31, CD34 and 772

VEGF were detected on serial sections of tumor samples. Scale bar, 100 µm 773

(100x), 25 µm (400x). 774

(E) The staining intensity of CD31, CD34, and VEGF from tumor samples were 775

shown.Data are presented as their mean ± SD. Statistical analysis was 776

performed with Student’s t-test. 777

*p < 0.05; ***p < 0.001. 778

779

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40

Figure 6 780

781

Figure 6. The down-regulation of HOXA5 predicts poor prognosis and negatively 782

correlates with angiogenesis in HCC. 783

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41

(A) Representative IHC staining images of HOXA5 in HCC and adjacent non-784

tumor liver tissues. Scale bar, 500 μm. 785

(B) HOXA5 expression was significantly decreased in HCC tissues. 786

Quantification of HOXA5 levels according to IHC scores. 787

(C) Kaplan-Meier plot revealed shorter overall and recurrence-free survival in the 788

lower HOXA5 group. Based on the minimum p-value approach, the 49th 789

percentile of the HOXA5 level in 449 HCC tissues was chosen as the cut-off 790

value for dividing the HOXA5-low level group (n = 220) from the HOXA5-high 791

level group (n = 229). 792

(D) (E) and (F) HOXA5 was negatively correlated with angiogenesis markers 793

CD31, CD34 in HCC. (D) Representative staining images of HOXA5 and CD31, 794

CD34 are shown. (E) The expression of CD31 and CD34 in HOXA5-low and 795

HOXA5-high group, respectively. The median IHC scores were chosen as the 796

cut-off value for separating the low and high groups (n = 314). 797

(F) Correlations between HOXA5 and CD31, CD34 in HCC were assessed using 798

the Pearson correlation and linear regression analytics (n = 314). 799

* p < 0.05, *** p < 0.001. 800

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42

Figure 7 801

802

Figure 7. Sp1 is essential for miR-130b-3p transcription. 803

(A) Schematic diagram of firefly luciferase reporter constructs containing the 804

indicated genomic segments upstream of miR-130b-3p gene. Putative Sp1 805

binding sites are depicted as short vertical lines. Deletion of the Sp1 binding site 806

is depicted as triangle (Δ). 807

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43

(B) Silencing of Sp1 decreased the miR-130b-3p promoter activity. HCC cells 808

were reversely transfected with NC or siSp1 for 24 h, followed by transfection 809

with p-(-2.0 k) for another 48 h before luciferase activity analysis. 810

(C) Silencing of Sp1 reduced miR-130b-3p level. The indicated HCC cells were 811

transfected with NC or siSp1 for 48 h before qRT-PCR analysis. 812

(D) Effect of fragment deletions on miR-130b-3p promoter activity. Huh7 cells 813

were transfected with the indicated luciferase reporter for 48 h before luciferase 814

activity analysis. The mean value of pEZX-PG04.1-transfectant from multiple 815

independent experiments was set as control. 816

(E) Silencing of Sp1 reduced the promoter activity of p-(-0.3 k). 817

(F) Silencing of Sp1 did not change the activity of p-del. 818

In (E) and (F), Huh7 cells were reversely transfected with negative control (NC) 819

or siSp1 duplex for 24 h and then transfected with the indicated luciferase 820

reporter for another 48 h. The mean value of NC-transfectant from multiple 821

independent experiments was set as control. 822

(G) Sp1 interacted with the miR-130b-3p promoter in HCC cells . Scheme of the 823

amplicon is shown on top. The putative Sp1 binding site located at the -0.3 to -824

0.1-kb region is illustrated as a black diamond. The solid arrows represent 825

primers used for semi-quantitive PCR. ChIP assays detected a specific band of 826

the expected size in the input DNA and the Sp1 antibody (Anti-Sp1)-precipitated 827

DNA. Genomic region upstream of GAPDH was used as a negative control. 828

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44

(H) Certification of knockdown efficiency of siSp1 in HCC cells (Huh7 and 829

MHCC-97H). 830

(I) The mRNA level of Sp1 positively correlated with miR-130b-3p in HCC 831

specimens (n = 62). Sp1 and miR-130b-3p was detected by qRT-PCR, and 832

normalized to actin and U6, respectively. Statistical analysis was performed 833

using Pearson correlation coefficient. 834

ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. 835

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45

Figure 8 836

837

Figure 8. The down-regulation of HOXA5 in HCC activates the PI3K/AKT/mTOR 838

signaling pathway. 839

(A) Top 20 statistics of KEGG pathway enrichment based on the differentially 840

expressed genes (DEGs) after HOXA5 knockdown in Huh7 cells. In the scatter 841

plot, RichFactor was the ratio of DEG numbers noted in this pathway term, to all 842

gene numbers noted in this pathway term, indicating intensiveness. P value 843

ranges from 0 to 1, and a lower P-value represents greater intensity. The 844

PI3K/AKT/mTOR signaling pathway was one of the most regulated biofunctions 845

upon HOXA5 knockdown. 846

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46

(B) The down-regulation of HOXA5 increased the phosphorylation of the 847

PI3K/AKT/mTOR signaling pathway-related proteins. 848

(C) Schematic regulatory network of the Sp1/miR-130b-3p/HOXA5 axis that 849

regulates angiogenesis in HCC. 850