mir-190a-3p promotes proliferation and migration in glioma

Research ArticlemiR-190a-3p Promotes Proliferation and Migration in GliomaCells via YOD1

Lili Zhou ,1 Lingzhi Li ,2 Yan Chen ,3 Chun Chen ,1 Zhongwen Zhi ,1 Luxia Yan ,1

Yuqian Wang ,1 Bing Liu ,3 and Qijin Zhai 1

1Department of Neurology, The Affiliated Huai’an Hospital of Xuzhou Medical University, Huai’an, 223002 Jiangsu, China2Department of ICU in Emergency Center, The Affiliated Huai’an Hospital of Xuzhou Medical University, Huai’an,223002 Jiangsu, China3Department of Neurology, Siyang Hospital of Traditional Chinese Medicine, Siyang, 223700 Jiangsu, China

Correspondence should be addressed to Bing Liu; [email protected] and Qijin Zhai; [email protected]

Received 23 May 2021; Revised 11 August 2021; Accepted 13 August 2021; Published 6 September 2021

Academic Editor: Lei Chen

Copyright © 2021 Lili Zhou et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction. To investigate the function of miR-190a-3p on the proliferation and migration of glioma. Methods. Twenty gliomasamples and 6 normal brain tissue samples were collected. Normal human glial cell line HEB and glioma cell lines were used forthe experiments. We then used TargetScan to predict the target genes of miR-190a-3p. Dual-luciferase reporter assay was alsoused to validate. Results. Combined with dual-luciferase reporter experiment, we finally verified that YOD1 was the aim, and itwas low-expressed in glioma. Besides, a series of mechanism experiments then proved that miR-190a-3p negatively regulatesYOD1 expression. Conclusions. Our research was the first to demonstrate the promoting function of miR-190a-3p in theproliferation and migration of glioma and provided new views for the treatment of glioma. miR-190a-3p was expected to be anew target for molecular therapy of glioma.

1. Introduction

Glioma is one of the most usual primary intracranial tumors,accounting for nearly 50% of all primary intracranial tumors[1]. It is characterized by high morbidity, mortality, and poorprognosis [2]. According to the degree of malignancy, gliomacan be divided into astrocytoma, oligodendroglioma, stromaloligodendroglioma, and glioblastoma. Patients with highlymalignant glioblastoma multiforme have a median survivaltime less than one year. Glioma is highly aggressive whichshows invasive growth and has an unclear boundary withhealthy brain tissue [3]. At present, the therapy of gliomamainly rely on the surgery-assisted radiotherapy and chemo-therapy, and the patients who suffer from these treatmentsoften have poor prognosis and are very prone to relapse [4].The occurrence and development of glioma are a compre-hensive process involving multiple factors, genes, and mole-cules. Based on the current status of glioma, the treatmentand survival of patients, researches on the pathogenesis of

glioma are urgently needed to lay the foundation for the sub-sequent study of glioma and to provide new points for thetherapy of glioma.

miRNAs are series of 20 to 22 nt RNAs without protein-coding function. miRNAs regulate the mRNA expression viacombining with the target mRNA [5]. They participate in aseries of biological processes, for example, cell propagationand differentiation, stress, and apoptosis, and therefore haveimportant significance in tumor research [6]. Ciafrè et al.analyzed 245 miRNAs in 9 pairs of human glioma tissuesand 10 glioma cell lines. They found that 20 miRNAs areexpressed differently in glioma, of which 9 miRNA areupregulated; 11 miRNA are downregulated [7]. Liu et al.analyzed medulloblastoma tissue through high-throughputmicroarray technology and found that miR-113, miR-99a,miR-102, and miR-109b are significantly increased, whereasmiR-216, miR-29a, miR-128a, miR-29c, and miR-128-5p aresignificantly reduced [8]. With the progress of miRNAresearch, more and more scholars have discovered that

HindawiComputational and Mathematical Methods in MedicineVolume 2021, Article ID 3957738, 12 pageshttps://doi.org/10.1155/2021/3957738

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https://doi.org/10.1155/2021/3957738

miRNAs may be involved in regulating malignant progres-sion, angiogenesis, growth, recurrence, and migration of gli-oma [9–12].

miR-190 has two primary mature forms, including miR-190a-5p and miR-190a-3p [13]. It is well known that miR-190a-5p plays an essential part in various diseases, especiallyin malignant tumors. According to the reports, miR-190a-5pacts a dual role in different cancers, for instance, miR-190a-5p is increased in bladder cancer and gastric cancer, while itis decreased in rectal cancer and liver cancer [14–16]. How-ever, the function of miR-190a-3p in various diseases is stillpoorly understood.

YOD1 is a well-known deubiquitinating enzyme, and itis highly conserved that can cleave ubiquitin residues of ubi-quitinated proteins, but its function has not been determinedin mammalian cells yet [17]. Ubiquitination and deubiquiti-nation of proteins control many biological processes, such ascell cycle, transcriptional activation, and signal transduction,and are closely associated with the tumorigenesis [18]. Deu-biquitination is known to stabilize tumor suppressor genesand thus inhibit the occurrence and progression of tumors[19]. In the current research, we confirmed that miR-190a-3p promoted the proliferation and migration of glioma byinhibiting the expression of YOD1, which provided newideas for the therapy of glioma.

2. Materials and Methods

2.1. Clinical Specimens. 20 glioma samples and 6 normalbrain tissue samples were collected from The AffiliatedHuai’an Hospital of Xuzhou Medical University during2019-2020. The 20 glioma tissue samples were gradedaccording to the WHO glioma grading standard. Tissueswere frozen in liquid nitrogen until further RNA extraction.This study had been ratified by the Ethics Committee of TheAffiliated Huai’an Hospital of Xuzhou Medical University.All patients or their relatives had signed informed consentsin advance.

2.2. Cell Culture. Normal human glial cell line HEB and gli-oma cell lines T98G, U87, SHG44, and U251 were boughtfrom Tongpai Biotechnology (Shanghai, China). DMEMwith high glucose and sodium glutamate (Hyclone, GE,Marlborough, USA), added with 10% FBS (Gibco, Invitro-gen, California, USA) and 1% antibiotic mixture (Sigma-Aldrich, St. Louis, USA), was applied to culture cells. Allcells were cultivated in a 37°C incubator (Panasonic, Osaka,Japan) containing 5% CO2.

2.3. Cell Transfection. The miR-190a-3p inhibitor expressionplasmid (pGCMV-EGFP-miR-190a-3p inhibitor), shYOD1expression plasmid (pGPU6-Neo-shYOD1), and their corre-sponding negative control plasmid were constructed byGenePharma (Shanghai, China). The logarithmic phaseU87 and U521 cells were inoculated in plates overnight to80% confluence, then change to serum-free DMEM. Themixture of Lipo3000 transfection regents (Sangon Biotech,Shanghai, China) and miR-190a-3p inhibitor expressionplasmid, shYOD1 expression plasmid, and NC plasmid

was prepared by serum-free DMEM and then added intothe wells. 4 h later, the normal DMEM was changed andthe subsequent experiments were carried out after 24 h ofcultivation. Transfection efficiency of miR-190a-3p inhibitorwas detected by the GFP under the microscope. Cell linesstably transfected with YOD1 shRNA were screened byneomycin.

2.4. Real-Time Quantitative PCR. Total RNA was extractedby using a commercial extraction kit (Biomarker, Beijing,China) that followed the instructions in the product manual.The SYBR Green RT-qPCR kit was obtained from Biosharp(Hefei, China), and the SLAN-96S Real-time PCR System(HongShi, China) was used to examine the level of miR-190a-3p and YOD1. U6 and β-actin were internal refer-ences. A 2−ΔΔCt method was applied for relative quantitativeanalysis. All primers used are listed in Table 1.

2.5. Cell Proliferation. U87 and U251 cells were first inocu-lated in cell culture plates and grown to 80% confluence,respectively, then added 10μl CCK-8 reagent (Sangon Bio-tech, Shanghai, China) per well, and continued to cultivatecells for another 4 h. Finally, OD450 was measured using amicroplate reader (Synergy H1, USA) to determine the cellviability.

2.6. Colony Formation Assay. The logarithmic growthphased U87 and U251 cells were digested into single cellsand inoculated in a Petri dish with 200 cells per well, thencultured cells for 2-3 weeks until visible colonies appeared.The supernatant was discarded, washed with PBS twice, thentreated with fixative for 15min, stained with Giemsa for30min, and finally, rinsed with PBS for at least five times.The number of clones was calculated under the microscope.

2.7. Wound Healing Assay. Glioma cells introduced withmiR-190a-3p or NC inhibitor were inoculated in a 6-wellplate until grown to 90% confluence. Use a sterile tip tocause a wound line in each well, then record the wound heal-ing under the microscope at 0 h and 48 h after the operation.The wound healing degree was calculated by software.

2.8. Transwell Migration and Invasion Assay. Cells treatedwith miR-190a-3p or NC inhibitor were digested and seededin the Transwell chamber without Matrigel (Millipore,USA). After 24 h, cells in the lower chamber were immobi-lized with aldehyde fixative for 30min and then stained with0.1% crystal violet, and photos were shot with the micro-scope. The procedure for the invasion experiment is thesame as above, except that the Transwell chamber used forthe invasion experiment was covered with Matrigel on theupper side.

2.9. Western Blot. Preparing cell lysates with RIPA Buffer(Solarbio, China) for the first, the protein content was ana-lyzed with the BCA Kit (Solarbio, China). SDS-PAGE wasconducted and then transferred the separated protein tothe PVDF film. Blocked with BSA, the film was treated withthe primary antibody and secondary antibody in sequenceand finally developed with enhanced chemiluminescence

2 Computational and Mathematical Methods in Medicine

reagent ECL (PE0010, Solarbio, China). All antibodies used inthis article were purchased from Abcam (Cambridge, MA).

2.10. TUNEL Assay. Cells were first inoculated in 24-wellplates and grown until 80% confluence. Discarding theupper medium, the cells were fixed in turn with fixativeand permeated with operating instructions of the TUNELreaction kit (Beyotime, China). Then, add DAPI dye forstaining cells and recording with a fluorescence microscopeat a wavelength of 490nm; the apoptotic cells representedgreen fluorescence. Using software to calculate the apoptosisrate.

2.11. Dual-Luciferase Reporter Assay. YOD1 was screened tobe the target of miR-190a-3p that related to glioma by usingTargetScan 7.2 software. The wild-type and mutant 3′UTRof YOD1 were synthesized and inserted into the pmirGLOdual-luciferase reporter vector (Biovector, China). miR-190a-3p/NC mimics and the vector were cotransfected toglioma cell lines with Lipo3000 Reagent (Invitrogen, CA,USA). Luciferase activity of cells was detected after 48 h.

2.12. Statistical Analysis. The data were expressed as themean ± standard deviation (SD) of three parallel experi-ments. Using SPSS 23.0 for data statistics, GraphPad wasalso used for survival curves. p < 0:05 was considered statis-tically significant.

3. Results

3.1. miR-190a-3p Is High-Expressed in Glioma. Firstly, wechecked the level of miR-190a-3p in both glioma tissuesand cell lines to explore the relationship between the miR-190a-3p and the glioma. As seen in Figure 1(a), the level ofmiR-190a-3p in glioma tissues was much higher. Further-more, miR-190a-3p was also higher in grade III+IV gliomathan that in grade I+II (Figure 1(b)). By analyzing themiR-190a-3p level and the overall survival rate of includedclinical samples, we found that the survival rate was lowerin samples with the high-level miR-190a-3p, as shown inFigure 1(c). Next, we tested the miR-190a-3p level in a seriesof glioma cell lines. It was obvious that miR-190a-3p waslow-expressed in HEB cells but was highly expressed inT98G, U87, SHG44, and U251 cell lines (Figure 1(d)).

Besides, we designed and transfected miR-190a-3p inhibitorinto U87 and U251 cell lines to further seek the effect ofmiR-190a-3p in glioma (Figure 1(f)). Herein, GFP was usedto verify the transfection efficiency of miR-190a-3p inhibitor(Figure 1(e)).

3.2. Suppression of miR-190a-3p Inhibits the Proliferation ofGlioma and Promotes Its Apoptosis. Through CCK-8 andcolony formation experiment, we found that the cell viabilityof U87 and U251 decreased significantly after adding themiR-190a-3p inhibitor (Figure 2(a)), and the number of col-onies was also considerably reduced (Figure 2(b)), whichindicated that suppression of miR-190a-3p restrains thepropagation of glioma cells. Next, we studied the apoptosisof glioma cells after introducing miR-190a-3p. The resultsof TUNEL showed that the apoptosis of cells was increasedafter the addition of miR-190a-3p inhibitor; at least 50% ofcells had undergone apoptosis (Figure 2(c)). Moreover, theexpression of a few proteins that were related to apoptosiswas examined. It was shown in Figure 2(d) that the level ofBax and cleaved caspase-3/9 was increased, whereas Bcl-2was decreased.

3.3. Suppression of miR-190a-3p Inhibits the Progression ofGlioma. In order to investigate the effect of suppression ofmiR-190a-3p on the progression of glioma, wound healingand Transwell assays were conducted. It could be seen thatthe wounds were caused after 48 h, and the wounds of U87and U251 cells gradually healed (Figure 3(a)). Moreover, asshown in Figures 3(b) and 3(c), introducing miR-190a-3pinhibitor obviously impeded the migration and invasion ofcells. Furthermore, after miR-190a-3p was inhibited,changes of two essential proteins ICAM-1 and VCAM-1were examined, which were closely related to migrationand invasion. The results showed that ICAM-1 andVCAM-1 were visibly decreased after inhibiting miR-190a-3p (Figure 3(d)).

3.4. miR-190a-3p Negatively Regulates Its Target YOD1. Inorder to illustrate the mechanism of miR-190a-3p’s effecton glioma, we first used TargetScan (V7.2) to predict the tar-gets that interacted with miR-190a-3p. Among the candidategenes, YOD1 was selected for further study. The predictedinteraction site of 3′UTR of target YOD1 to miR-190a-3pis shown in Figure 4(a). Meanwhile, according to the pre-dicted interaction site of the 3′UTR of YOD1 with miR-190a-3p, dual-luciferase reporter plasmids were synthesizedand cointroduced with miR-190a-3p mimics into gliomacells. The results are shown in Figure 4(b), indicating thatYOD1 was indeed the target of miR-190a-3p. Next, weexamined the level of YOD1 in glioma; it was shown thatYOD1 was low-expressed in both glioma tissues and cells(Figure 4(c)). To investigate the relationship between thelevel of YOD1 and miR-190a-3p, we analyzed the includedclinical samples and found that the level of YOD1 wasnegatively related to miR-190a-3p (Figure 4(d)). In addi-tion, increased YOD1 expression was detected in gliomacells after the introduction of miR-190a-3p inhibitor(Figures 4(e) and 4(f)).

Table 1: RT-qPCR primer sequences.

Gene Primer sequences (5′-3′)

miR-190a-3pForward 5′-CTAGGAGCTTCAGTAACGTTA-3′Reverse 5′-GAATTCGAATCGGTAACAGCT-5′

YOD1Forward 5′-CTTTGAAGCCAAGTAGCTTC-3′Reverse 5′-AAGTCAGTTGVATGAGCTGA-3′

U6 snRNAForward 5′-CTGATCAGTGAGTAAGATG-3′Reverse 5′-ATCGAATATGCAGTGTCAG-3′

β-ActinForward 5′-ACTGTTATGATAGCATCGA-3′Reverse 5′-CATACTGCATCCTATCGAAT-3′

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Figure 1: miR-190a-3p is high-expressed in glioma. (a) miR-190a-3p in normal and cancer tissues. (b) miR-190a-3p in glioma tissues ofgrade I+II and III+IV. (c) Comparison of overall survival rate of clinical samples with high and low expression of miR-190a-3p. (d)Relative level of miR-190a-3p in HEB and four different glioma cell lines. (e, f) The miR-190a-3p inhibitor was introduced into U87 andU251 cell lines, and GFP was used to verify the transfection efficiency. ∗∗p < 0:01. Scale bar = 100μm for (e).


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Figure 2: Low-expressed miR-190a-3p can suppress the proliferation of glioma and promote its apoptosis. (a, b) The influence of inhibitingmiR-190a-3p on the growth of U87 and U251 cell lines was evaluated by CCK-8 and colony formation assays. (c, d) TUNEL assay (c) andWestern blot (d) were conducted to evaluate the cell apoptosis after transfection of miR-190a-3p inhibitor. ∗∗p < 0:01. Scale bar = 50μm for (c).


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Figure 4: miR-190a-3p negatively regulates its target YOD1. (a) The predicted binding site of YOD 3′UTR and miR-190a-3p. (b) Theluciferase activity significantly decreased after introducing miR-190a-3p inhibitor and WT 3′UTR of YOD1, while the MUT YOD1 3′UTR group had almost no change. (c) Relative expression of YOD1 in glioma cell lines and tissues. (d) Relative expression of miR-190a-3p and YOD1 in clinical sample tissues was negatively correlated. (e, f) After introducing miR-190a-3p inhibitor, the expression ofYOD1 in U87 and U251 cell lines increased. ∗∗p < 0:01.


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Figure 5: Continued.


3.5. Silencing YOD1 Reverses the Effect of miR-190a-3pInhibitor. For confirming the relationship between YOD1and miR-190a-3p, we first designed shRNA to silenceYOD1 expression, as shown in Figure 5(a). Next, we usedCCK-8 and colony formation assays to confirm that sh-YOD1 could reverse the inhibited proliferation of gliomacells caused by miR-190a-3p inhibitor (Figures 5(b) and5(c)). Besides, the silenced YOD1 alleviated the apoptosisinduced by miR-190a-3p inhibitor as well as promotedmigration and invasion of glioma cells to a certain extent(Figures 5(d)–5(f)). These results verified the negative rela-tionship between miR-190a-3p and YOD1.

4. Discussion

Glioma is a malignant intracranial tumor. Nowadays, on aglobal scale, the treatment of this disease is still based on tra-ditional treatment, such as surgery-assisted radiotherapy orchemotherapy. However, the prognosis for patients under-going surgery is poor, mainly because this tumor is invasiveand it is difficult to completely remove the lesion after sur-gery [20]. At the same time, the occurrence and develop-ment of glioma are a comprehensive process involvingmultiple factors, genes, and molecules; the specific patho-genesis of the disease is still unclear, so glioma is still amalignant tumor with poor prognosis and easy recurrence[21]. In recent years, more and more scholars focused theirresearch on molecular-targeted therapy and proposed manynew treatment schemes for various tumors.

Recent studies on glioma have found that miRNAs playan essential part in the progress or apoptosis of tumor cellsvia controlling the expression of some key genes, such asp53 [22, 23]. Research has found that the upregulated miR-NAs in glioma cells include the miR-183 [24], miR-21 [25],and miR-221 [26], while the downregulated miRNAsinclude miR-122 [27], miR-145 [28], and miR-26a [29].Ding et al. found that in SHG-44 cell line, when the levelof intracellular miR-122 was increased, the level of cas-pase9/3 was significantly increased, as well as the apoptosisrate. Further analyzing the target genes of miR-122 found

that it may regulate apoptosis in glioma cells by inhibitingthe expression of endogenous Bcl-w protein, which impliedthat the increased miR-122 inhibits the expression of endog-enous Bcl-w and thus regulating the apoptosis process of gli-oma cells, playing an essential part in the appearance andprogress of glioma [27]. Chen et al. validated that miR-105,miR-16, and miR-195 are low-expressed in U251, U87,SHG44, and MO59K cell lines. These three miRNAs couldinhibit the metastasis of glioma cells and induced cell apo-ptosis. Besides, they combine with and inhibit the expressionof SALL4, implying that these three miRNAs take part in theprogress of glioma by regulating SALL4 [30].

In this paper, we explored the function of miR-190a-3pand its target YOD1 in glioma. The expression of miR-190a-3p was found markedly upregulated in glioma, whereasthe level of YOD1 was markedly decreased. Suppression ofmiR-190a-3p restrained the progress and accelerated apo-ptosis of glioma cells. In addition, the expression of miR-190a-3p is negatively related to the expression of its targetgene YOD1, and miR-190a-3p negatively controls theexpression of YOD1. The rescue assay further validated therelationship between miR-190a-3p and YOD1, as well astheir role in the progression of glioma.

Our research demonstrated that miR-190a-3p could pro-mote the proliferation and migration of glioma through itstarget gene YOD1. The progression of glioma could be sup-pressed by inhibiting miR-190a-3p or overexpressing its tar-get YOD1. This inhibition effect might be achieved bypromoting cell apoptosis of glioma. In the future, we willcontinue to investigate the mechanism of apoptosis initia-tion after inhibition of miR-190a-3p. Besides, the in vivoexperiments also need to validate. In general, our findingsprovide a novel approach for molecular therapy of gliomathat can attempt to inhibit the proliferation and metastasisof glioma by inhibiting the expression of miR-190a-3p.

Data Availability

All data generated or analyzed during this study are includedin this published article.

U87 U251 U87 U2510

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NC inhibitor+sh-NC



NC inhibitor+sh-NCmiR-190a-3p inhibitor+sh-NCmiR-190a+3p-inhibitor+sh-YOD1

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(g)

Figure 5: Silencing YOD1 can reverse the effect of miR-190a-3p inhibitor. (a) YOD1 was silenced after being transfected with sh-YOD1.(b, c) The suppression of cell proliferation caused by miR-190a-3p inhibitor could be relieved by silencing of YOD1. (d, e) Aftercotransfection with miR-190a-3p inhibitor and sh-YOD1, the apoptosis of cells was relieved, compared with the cotransfected miR-190a-3p inhibitor and sh-NC group. (f, g) Silencing YOD1 could improve the reduced metastasis capacity of glioma cells caused bymiR-190a-3p inhibitor. ∗p < 0:05 and ∗∗p < 0:01 vs. miR-190a-3p inhibitor+sh-NC group, ##p<0.01 vs. miR-190a-3p inhibitor+sh-YOD1group. Scale bar = 100μm for (d), (f), and (g).


Ethical Approval

The experimental protocol was established, according to theethical guidelines of the Helsinki Declaration, and wasapproved by the Animal Ethics Committee of The AffiliatedHuai’an Hospital of Xuzhou Medical University.

Consent

Written informed consent was obtained from all patients.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

L. Z. and Y. W. conceived and designed the study. L. L. con-ducted most of the experiments. Y. C. and B. L. analyzed thedata. C. C. performed the literature search and data extrac-tion. Z. Z. drafted the manuscript. Q. Z. and L. Y. finalizedthe manuscript. All authors read and approved the finalmanuscript. Lili Zhou, Lingzhi Li, and Yan Chen contrib-uted equally to this work.

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mir-190a-3p promotes proliferation and migration in glioma

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