RESEARCH ARTICLE Open Access

lncRNA TINCR sponges miR-214-5p toupregulate ROCK1 in hepatocellularcarcinomaMin Hu1* , Yaowu Han1, Ying Zhang2, Yuanfeng Zhou2 and Lin Ye3

Abstract

Background: Our preliminary bioinformatics analysis showed that lncRNA TINCR may absorb miR-214-5p by servingis sponge, while miR-214-5p targets ROCK1. This study aimed to investigate the interactions among these 3 factorsin hepatocellular carcinoma (HCC).

Methods: Expression of TINCR, ROCK1 and miR-214-5p in HCC and non-tumor tissues was detected by performingqPCR. The correlations among TINCR, ROCK1 and miR-214-5p in HCC tissues were analyzed by performing linearregression. Overexpression experiments were performed to analyze gene interactions. Cell proliferation wasanalyzed by CCK-8 assay.

Results: We found that TINCR and ROCK1 were upregulated, while miR-214-5p was downregulated in HCC. TINCRand ROCK1 were positively correlated, while TINCR and miR-214-5p were not significantly correlated. In HCC cells,TINCR overexpression is followed by ROCK1 overexpression, while miR-214-5p overexpression induced thedownregulation of ROCK1. In addition, TINCR and miR-214-5p did not affect the expression of each other. TINCRand ROCK1 overexpression led to increased rate of cancer cell proliferation, while miR-214-5p played an oppositerole and reduced the effects of TINCR overexpression. Therefore, TINCR sponges miR-214-5p to upregulate ROCK1in HCC, thereby promoting cancer cell invasion and migration.

Keywords: Hepatocellular carcinoma, lncRNA TINCR, miR-214-5p, ROCK1

BackgroundIncidence of hepatocellular carcinoma (HCC) ranks topplaces all over the world [1]. With the popularization ofrisk factor screening program and the application of pre-vention regimens, incidence of HCC has dropped signifi-cantly in many regions, such as parts of China and Japan[2]. However, an increasing trend in both incidence andmortality rates of HCC has been observed in many Euro-pean countries and North America owing to the highprevalence of hepatitis B virus (HBV) and hepatitis C virus(HCV) infections [3]. In spite of the considerable numberof risk factors identified for HCC, pathogenesis of this dis-ease remains poorly understood [4, 5], which is a big chal-lenge in the development of novel therapeutic targets.

ROCK1, or Rho-associated protein kinase 1, acts at thedownstream of small GTPase RhoA to mediate the forma-tion of contractile force [6]. Oncology studies have shownthat ROCK1 is usually overexpressed during cancer devel-opment and ROCK1 overexpression promotes cancer de-velopment by regulating cancer cell behaviors, such asproliferation, invasion and migration [7, 8]. In effect, inhib-ition of ROCK1 is novel therapeutic choice for cancer [7,8]. It is known that, the expression of ROCK1 can be regu-lated by certain miRNAs [9, 10], and the function of miR-NAs can be attenuated by lncRNA sponges [11]. In arecent study, Zhang et al. reported that miR-214-5p inhibitsosteosarcoma cell proliferation by directly targeting ROCK1[12]. TINCR has been reported to be an oncogenic lncRNAin many types of cancers including HCC [13]. Our bioinfor-matics analysis showed that TINCR may sponge miR-214-5p. This study was performed to investigate the interactionsamong TINCR, miR-214-5p and ROCK1 in HCC.

© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: pdmnzwhj4623501@126.com1Department of Pathology, Anhui University of Chinese Medicine, Hefei, 1Qianjiang Road, Hefei 230012, Anhui province, ChinaFull list of author information is available at the end of the article

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MethodsPatients and specimensForm December 2016 to December 2018 a total of 137HCC patients were admitted by Anhui University of Chin-ese Medicine. From those patients our study selected 60cases (gender: 37 males and 23 females; age: 33 to 67years; mean: 49.9 ± 6.3 years) to serve as research subjects.Inclusion criteria: 1) No therapies for any clinical disor-ders were performed within 100 days; 2) new HCC cases.Exclusion criteria: 1) patients with other clinical disorders;2) patients with recurrent HCC; 3) patients with previoushistory of malignancies. The HCC patients were educatedwith the experimental principle and all of them involvedin this study provided signed informed consent. All pa-tients were diagnosed by histopathological biopsy. Duringthe operation of biopsy, non-tumor and HCC tissues werecollected from each patient. All tissue specimens wereconfirmed by at least 3 experienced pathologists. Thisstudy has been approved by Ethics Committee of AnhuiUniversity of Chinese Medicine.

HCC cells and transient transfectionsIn vitro experiments in this study were performed usingHCC cell lines H1581 (Cat# ATCC® CRL-5878™) andSNU-475 (ATCC® CRL-2236™) from ATCC (USA). Cellswere cultivated at 37 °C with 5% CO2 in RPMI-1640medium (10% FBS) before use. PcDNA3.1 vectors ex-pressing TINCR and ROCK1 were constructed by RIBO-BIO (Guangzhou, China). MiR-214-5p mimic andnegative control miRNA were also from RIBOBIO.H1581 cells were harvested and were counted, followedby transfection of 10 nM pcDNA3.1 vector expressingTINCR or ROCK2, or 10 nM empty pcDNA3.1 vector(negative control, NC), or 30 nM miR-214-5p mimic, or30 nM negative control miRNA (NC) into 106 cellsthrough lipofectamine 2000 (Invitrogen, USA)-mediatedmethod. Cells with no transfections were also includedto serve was control (C) group. The interval betweensubsequent experiments and transfection was 24 h.

RNA extractions and qPCRNon-tumor and HCC tissues were ground. H1581 cellswere harvested and counted. To extract total RNAs,0.015 g tissue or 106 cells were mixed with 1 ml Trizolreagent (Invitrogen, USA). To retain miRNA, 80% etha-nol was used to participate and wash RNAs. MMLV Re-verse Transcriptase (Lucigen, USA) and QuantiTectSYBR Green PCR Kit (Qiagen, Shanghai, China) wereused to perform total RNA reverse transfections andprepare qPCR mixtures to detect the expression ofTINCR and ROCK2 with GAPDH as endogenous con-trol. miScript II RT Kit (QIAGEN) and miScript SYBRGreen PCR Kit (QIAGEN) were used to perform miRNAreverse transcriptions and prepare qPCR mixtures to

detect the expression of miR-214-5p with U6 as en-dogenous control. All experiments were performed intriplicate manner and expression levels were normalizedusing 2-ΔΔCT method.

Western blot analysisH1581 cells were collected and mixed with RIPA solu-tion (RIBOBIO) with a ratio of 105 cells per 1 ml solu-tion. Total proteins were extracted following theprotocol provided by RIBOBIO. Protein samples werequantified using BCA kit (RIBOBIO), followed by de-naturing in boiled water for 5 min. Following 12% SDS-PAGE gel electrophoresis, gel transfer (PVDF mem-brane) and blocking (2 h in 5% non-fat milk at roomtemperature) were performed. Following that, primaryantibodies of rabbit GAPDH (1: 1500, ab37168, Abcam)and RCOK1 (1: 1200, ab66320, Abcam) were used toblot the membranes at 4 °C for 15 h. After that, HRPgoat anti-rabbit (IgG) secondary antibody (1:1400;ab6721; Abcam) was used to further blot the membranesfor 2 h at 24 °C. All signal developments were performedusing ECL solution (Sangon, Shanghai, China). Image Jv1.46 software was used to normalize all gray values.

RNA-RNA interaction predictionThe interaction between TINCR and miR-214-5p was per-formed using online program IntaRNA (http://rna.informa-tik.uni-freiburg.de/IntaRNA/Input.jsp). Sequence of TINCRwas used as the long sequence and sequence of miR-214-5p was used as the short sequence. All other parameterswere default.

Cell proliferation analysisH1581 and SNU-475 cells were harvested and counted. 1ml RPMI-1640 medium (10% FBS) was mixed with 5 ×104 cells to prepare single cell suspensions. The cells werecultivated in a 96-well plate (0.1ml per well) at 37 °C in a5% CO2 incubator. Three replicate wells were set for eachexperiment. To monitor cell proliferation, 10ul CCK-8 so-lution (Sigma-Aldrich) was added at 2 h before the end ofcell culture. Cell collection was performed every 24 h for atotal of 4 days. After the termination of cell culture ODvalues at 450 nm were measured. OD value of Controlgroup was set to “100”, all other time pointes and othergroups were normalized to this value.

Statistical analysisData from 3 biological replicates were used to calculatemean values, which were used in all data analyses. Cor-relations were analyzed by linear regression. Differencesin gene expression levels and cell proliferation ratesamong aforementioned different cell transfection groupswere analyzed by ANOVA (one-way) and post hocTukey test. Differences in levels of gene expression

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between non-tumor and HCC tissues were exploredusing paired t test. With the median expression level ofTINCR in HCC as cutoff value, the 60 patients were di-vided into high and low TINCR level group (n = 30).Chi-squared test was performed to analyze the relation-ship between patients’ clinical data and levels of TINCRexpression in HCC. p < 0.05 was statistically significant.

ResultsExpression of TINCR, ROCK1 and miR-214-5p was alteredin HCCExpression of TINCR, ROCK1 and miR-214-5p in HCCand non-tumor tissues was detected by performing qPCR.Expression levels were compared between non-tumor andHC tissues by paired t test. Compared to non-tumor tissues,expression levels of TINCR (Fig. 1a) and ROCK1 (Fig. 1b)were significantly higher, whereas expression level of miR-214-5p was significantly lower (Fig. 1c) in HCC (p < 0.05).Chi-squared test showed that levels of TINCR in HCC tis-sues were significantly correlated with tumor size and TNMstage (p < 0.05), but not age, gender, histological differenti-ation, HBV or HCV infections and liver cirrhosis (p < 0.05,Table 1).

The correlations among TINCR, ROCK1 and miR-214-5p inHCCThe correlations among TINCR, ROCK1 and miR-214-5pin HCC tissues were analyzed by performing linear regres-sion. In HCC tissues, TINCR and ROCK1 were positivelycorrelated (Fig. 2a), while TINCR and miR-214-5p were notsignificantly correlated (Fig. 2b). In addition, miR-214-5pand ROCK1 were also not significantly correlated (Fig. 2c).

TINCR may sponge miR-214-5pThe interaction between TINCR and miR-214-5p werepredicted using the aforementioned methods. It can beobserved that TINCR may form base pairing with miR-214-5p from position 258 to position 276 (Fig. 3). The

hybridization energy is − 22.7 kcal/mol. Therefore,TINCR can be a sponge of miR-214-5p.

The interactions among TINCR, ROCK1 and miR-214-5p inH1581 cellsTINCR and ROCK1 expression vectors as well as miR-214-5p mimic were transfected into H1581 cells. At 24 hpost-transfection, expression levels of TINCR, ROCK1 andmiR-214-5p were significantly upregulated Compared toNC and C groups (Fig. 4a, p < 0.05). In addition, TINCR

Fig. 1 Expression of TINCR, ROCK1 and miR-214-5p was altered in HCC. Expression of TINCR (a), ROCK1 (b) and miR-214-5p (c) in HCC and non-tumor tissues was detected by performing qPCR. All qPCR reactions were performed 3 times. Expression levels were compared between non-tumor and HC tissues by paired t test (*, p < 0.05)

Table 1 Correlation between levels of TINCR expression levelsin HCC and patients’ clinical dataVariables n High (n =

30)Low (n =30)

Chisquare

p

Gender

Male 37 17 20 0.63 0.43

Female 23 13 10

Age (years)

> 50 28 13 15 0.27 0.60

< =50 32 17 15

Histological differentiation

Well 14 5 9 1.55 0.46

Moderate 21 11 10

Poor 25 14 11

HBV or HCV infections

Yes 48 22 26 1.67 0.20

No 12 8 4

Liver cirrhosis

Absence 11 5 6 0.11 0.74

Presence 49 25 24

Tumor size

> 5 cm 33 10 23 11.38 0.0007

< = 5 cm 27 20 7

TNM stage

I + II 31 11 20 5.41 0.02

III + IV 29 19 10

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overexpression is followed by ROCK1 overexpression, whilemiR-214-5p overexpression induced the downregulation ofROCK1 and attenuated the effects of ROCK1 overexpres-sion (Fig. 4b, p < 0.05). In addition, TINCR and miR-214-5pdid not affect the expression of each other (Fig. 4c).

TINCR promoted H1581 and SNU-475 cell proliferationthrough ROCK1 and miR-214-5pCell proliferation data were analyzed. Compared to Cand NC groups, TINCR and ROCK1 overexpression ledto increased rate of cancer cell proliferation, while miR-214-5p played an opposite role and reduced the effectsof TINCR overexpression (Fig. 5, p < 0.05).

DiscussionThis study mainly investigated the interactions amongTINCR, ROCK1 and miR-214-5p in HCC. We found thatTINCR can upregulate ROCK1 possibly by sponging miR-214-5p, which can directly target ROCK1.Previous studies have identified numerous miRNAs that

can directly target ROCK1 [9, 10, 12]. In HCC, miR-148btargets ROCK1 to inhibit cancer cell proliferation, invasion

and migration [14]. In a recent study, miR-214-5p was re-ported to directly target ROCK1 to inhibit cancer cell behav-iors [12]. In the present study we also observed the reducedexpression levels of ROCK1 in HCC cells after miR-214-5pexpression. Therefore, miR-214-5p may also target ROCK1to regulate its expression in HCC. Those data also suggestthat ROCK1 can be targeted by multiple miRNAs.In this study we observed the upregulated ROCK1

and downregulated miR-214-5p in HCC tissues.Interestingly, we did not observe a significant correl-ation between miR-214-5p targets ROCK1 in HCCtissues. This observation suggests the existence of asponge of miR-214-5p. MiRNA sponges only absorbmiRNAs to attenuate their functions but may notaffect their expression levels [15]. Therefore, if asponge exists, the downregulation of miR-214-5p ex-pression may not be significantly correlated with theupregulated ROCK1. The sponge may serve buffer tofinely regulate the levels of functional miRNAs.TINCR is a well-characterized oncogenic lncRNA

in many types of cancer [13, 16, 17]. In some cases,TINCR may sponge miRNAs to promote cancer

Fig. 2 The correlations among TINCR, ROCK1 and miR-214-5p in HCC. The correlations between TINCR/ROCK1 (a), TINCR/miR-214-5p (b) and miR-214-5p/ROCK1 (c) were analyzed by linear regression

Fig. 3 TINCR may sponge miR-214-5p. The interaction between TINCR and miR-214-5p was performed using online program IntaRNA. Sequenceof TINCR was used as the long sequence and sequence of miR-214-5p was used as the short sequence. All other parameters were default

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Fig. 4 The interactions among TINCR, ROCK1 and miR-214-5p in HCC cells. TINCR and ROCK1 expression vectors as well as miR-214-5p mimicwere transfected into H1581 cells. At 24 h post-transfection, overexpression of TINCR, ROCK1 and miR-214-5p was confirmed by qPCR (a). Theeffects of TINCR and miR-214-5p on ROCK1 overexpression were analyzed by qPCR and western blot (b). The interaction between TINCR andmiR-214-5p was analyzed by qPCR (c). All experiments were repeated 3 times and mean values were presents. NC1, empty vector transfection;NC2, negative control miRNA transfection (*, p < 0.05)

Fig. 5 TINCR promoted HCC cell proliferation through ROCK1 and miR-214-5p. The effects of TINCR, ROCK1 and miR-214-5p on HCC cellproliferation were analyzed by cell proliferation assay. All experiments were repeated 3 times and mean values were presents. NC1, empty vectortransfection; NC2, negative control miRNA transfection (*, p < 0.05)

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progression. For instance, TINCR sponges miR-375to regulate PDK1, thereby promoting gastric cancer[16]. In colorectal cancer, TINCR sponges miR-7-5pto aggregate disease conditions [17]. In this study weshowed that TINCR may sponge miR-214-5p to up-regulated ROCK1 in HCC, and the upregulation ofROCK1 by TINCR is involved in the regulation ofHCC cell proliferation. However, other lncRNAs mayalso sponge miR-214-5p to participate in thisprocess. Our future studies will explore thispossibility.

ConclusionsIn conclusion, TINCR played an oncogenic role in HCCby sponging miR-214-5p to upregulate ROCK1.

AbbreviationsFBS: Fetal bovine serum; GAPDH: Glyceraldehyde 3-phosphate dehydrogen-ase; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; HCV: Hepatitis Cvirus; ROCK1: Rho Associated Coiled-Coil Containing Protein Kinase 1;TINCR: TINCR ubiquitin domain containing

AcknowledgementsNot applicable.

Authors’ contributionsMH supervised the whole study, designed the concept, analyzed the data,and edited the final manuscript. YWH, YZ, YFZ and LY collected andanalyzed the data, prepared the manuscript. All authors read and approvedthe final manuscript.

FundingWe thank for the financial support from the Anhui Province ReturnedOverseas Students Innovation Project; China Postdoctoral Science FundProject(2016 M592037) and 2016 Anhui Postdoctoral Fund Project (2016B096)for the funding to support our purchase of experimental equipment andmaterials.The funding bodies played no role in the design of the study and collection,analysis, and interpretation of data and in writing of the manuscript.

Availability of data and materialsThe analyzed data sets generated during the study are available from thecorresponding author on reasonable request.

Ethics approval and consent to participateThe present study was approved by the Ethics Committee of the Maternityand Child Care Center of Liuzhou. The research has been carried out inaccordance with the World Medical Association Declaration of Helsinki. Allpatients and healthy volunteers provided written informed consent prior totheir inclusion within the study.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Pathology, Anhui University of Chinese Medicine, Hefei, 1Qianjiang Road, Hefei 230012, Anhui province, China. 2Graduate School,Anhui University of Chinese Medicine, Hefei 230038, Anhui, China.3Department of Psychology, Anhui University of Chinese Medicine, Hefei230012, Anhui, China.

Received: 14 October 2019 Accepted: 23 December 2019

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