miR‐143 inhibits renal cell carcinoma cells metastatic potential by suppressing ABL2

Abstract

Although micro RNA (miRNA) expression profiles are widely investigated in renal cell carcinoma (RCC), their potential roles for affecting RCC initiation and progression remain largely unknown. Here, we examined the aberrant expression profiles of miRNAs inhuman metastatic RCC tissues based on Gene Expression Omnibus (GSE37989). We further validated them iRNAs expression data in the largest clinical dataset: The Cancer Genome Atlas (TCGA). And cell adhesion and migration abilities and epithelial me senchymal transition (EMT) related proteins were assessed in both normal and tumor RCC cell lines. We suggest that hsa‐miR‐143 is a potential tumor suppressor in RCC as its down regulation positively correlated with adverse prognosis. Biologically, cell adhesion, migration, and EMT were dramatically inhibited by miR‐143. Mechanistically, we found that miR‐143 targets ABL proto‐oncogene 2 (ABL2), which was also found to be an indicator for poor survival in TCGA database. Our results have important implications in understanding functions of miRNAs in metastatic RCC and will provide a basis for further clinical application.

1. INTRODUCTION

In 2015, a total of 61 560 new renal cancer cases were estimated in the United States and 14 080 deaths were related to these malignancies.1 A rapid rise of the incidence of renal cell carcinoma (RCC) was observed in some epidemiologic analyses. Clear cell renal cell carcinoma (ccRCC) accounting for about 80% of RCC cases, remains the most common subtype of RCC. Approximate 30% of RCC patients have metastatic disease at the time of diagnosis, who have a poor prognosis with a 5‐year survival rate of less than 20%.2, 3

Although there has been widespread research into genetic biomarkers for RCC, epigenetic biomarkers including microRNAs (miRNAs) have also received considerable attention as their biological and clinical utility in diagnosis and treatment. MiRNAs are small noncoding RNA strands that post‐transcriptionally regulate gene expression4, 5 and appear to be modulators of urologic cancers. Specific miRNA profiles have been observed previously in ccRCC.6 Downregulation of miRNAs expression has been identified as a feature in ccRCC in previous studies. Similarly, dysregulated miRNAs in RCC, such as miR‐210, miR‐141 and 200c, have been found to give rise to renal carcinogenesis.7

Previous studies have shown that miR‐143 was significantly downregulated in several types of human cancers. For example, Su et al found that miR‐143 was downregulated in 86.67% of the melanoma tissue samples compared to their matched normal control tissues and miR‐143 could inhibit cell proliferation, repress cell cycle progression and promote apoptosis in melanoma cells by targeting Syn‐1.8 Moreover, overexpression of miR‐143 inhibited cell proliferation, migration, tumor growth in vivo and angiogenesis and increased chemosensitivity to oxaliplatin treatment in an IGF‐IR‐dependent manner in colorectal cancer.9 However, there is rare study apparently has been conducted to investigate the role of miR‐143 in ccRCC.

In this study, our data demonstrated that miR‐143 was underexpressed in ccRCC tissues and cell lines and the result was confirmed in The Cancer Genome Atlas (TCGA) database. Overexpression of miR‐143 in 786‐O cells could inhibit cell attachment, migration, and epithelial mesenchymal transition (EMT) transformation. We identified ABL2 was a direct target of miR‐143 in ccRCC, providing a potential diagnostic and therapeutic target for the treatment of ccRCC.

2. MATERIALS AND METHODS

2.1. Cell culture and transfection

786‐O which is a human RCC cell line with metastatic potential and HK‐2 which is an immortalized proximal tubule epithelial cell line were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). HK‐2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 786‐O cells were cultured in RPMI 1640 supplemented with 10% FBS. 786‐O cells were transiently transfected with miR‐143 and negative control (NC) mimics (GenePharma) using Lipofectamine 2000 (Invitrogen). Cells were seeded in 6‐well plates and transfected with 5 nM of miR‐143 mimics or NC mimics.

2.2. TCGA, ONCOMINE databases Gene Expression Omnibus GSE37989 dataset retrieve

MiRNAs expression profiles were retrieved from Gene Expression Omnibus (GSE37989).10 MiR‐143 and ABL2 expression and clinical data were retrieved from TCGA database (http://cancergenome.nih.gov) and ONCOMINE Cancer Profiling Database (www.oncomine.org). These datasets were utilized to investigate the clinical relevance of miR‐143 and ABL2 expression with patient traits.

2.3. Clinical sample collection

Human RCC tissues and the normal adjacent tissues were obtained from 18 patients that underwent radical nephrectomy for localized clear cell RCC at Nantong First Hospital from 2011 to 2012. The use of the samples was reviewed and approved by the Institutional Ethics Committee of Zhongda hospital. Histological diagnosis was established according to the guidelines of the World Health Organization.

2.4. Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR)

Total RNA was extracted from cells and tissues by using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The expression of miR‐143 and U6 (the internal control) were measured using the Hairpin‐it miRNAs qPCR Quantitation Kit (GenePharma). The relative expression of miR‐143 in cells and tissues were calculated by the 2‐dt method.

2.5. Western blotting

Cells were lysed by RIPA buffer (Beyotime) supplemented with protease inhibitors cocktail (Roche). The protein samples were electrophoresed in 10% sodium dodecyl sulfate polyacrylamide gels. Electrophoresed proteins were transferred to a polyvinylidene fluoride membrane (Millipore) and blocked with 5% skim milk at room temperature. After the proteins were incubated with primary antibodies at 4°C overnight, the blots were washed, incubated with HRP‐labeled secondary antibody at room temperature for 1 hour and visualized through enhanced chemiluminescence. Protein levels were determined by normalizing against GAPDH. The related antibodies were rabbit anti‐GAPDH (1:1000, Santa Cruz), anti‐vimentin (1:1000, Proteintech), anti‐N‐cadherin (1:200, Proteintech), anti‐E‐cadherin (1:500, Proteintech), anti‐Fibronectin (1:500, Proteintech), anti‐ABL2(1:500, Proteintech), HRP‐labeled goat anti‐rabbit secondary antibody (1:3000, Zhongshan Goldenbridge Biotechnology), and HRP‐labeled goat anti‐mouse secondary antibody (1:3000, Zhongshan Goldenbridge Biotechnology).

2.6. Cell adhesion assay

Ninety‐six‐well cell culture plates were coated with culture medium containing 10% matrigel in 4°C overnight. Cells transfected with either miR‐143 mimics or NC mimics after 24 hours were plated at a density of 10⁴ cells/well. The cells were cultured for 4 hours in RPMI‐1640 with 5% FBS, washed twice with PBS, and then fixed with 4% formaldehyde. Cell numbers in four fields per well were counted using an inverted microscope.

2.7. Cell migration assay

786‐O cells were transfected with miR‐143 mimics or NC mimics, cultivated for 48 hours, and transferred on the top of chambers (24‐well insert, 8‐μm pore size, BD) in 100 μL serum‐free RPMI 1640 and the medium containing 20% FBS was added to the lower chamber. After 12 hours, nonmigrated cells were removed from the upper chamber and the migrated cells were fixed by 4% paraformaldehyde and consequently stained with 0.1% crystal violet, and photographed in four independent fields for each well.

2.8. Luciferase assay

The 3′UTR of human ABL2 gene that was predicted to interact with miR‐143 was synthesized and inserted into pMIR‐REPORT (Ambion). Cells were transfected with the pMIR‐REPORT vectors containing the 3′UTR and miR‐143 mimics for 48 hours. Luciferase values were determined by using the Dual‐luciferase Reporter Assay System (Promega).

2.9. Statistics

All statistical analyses were performed using SPSS version 16.0. The significance of differences between groups was estimated by Student's t test. Overall survival rates were calculated by the Kaplan‐Meier method with the log‐rank test applied for comparison. Two‐sided P‐values were calculated and a level of <.05 was considered statistical significance.

3. RESULTS

3.1. miR‐143 expression was underexpressed in ccRCC

After retrieving the raw data from GSE37989 concerning miRNAs expression in metastatic and primary RCC, we found that miR‐143 was underexpressed in metastatic RCC (Figure 1A). And qPCR was performed to detect the miR‐143 expression in RCC cell line (786‐O) and normal human renal tubule epithelial cell line HK‐2. We also found that miR‐143 expression was downregulated in 786‐O cells compared to HK‐2 cells (Figure 1B). To explore whether miR‐143 was also downregulated in RCC tissues, we first detected miR‐143 expression in 18 paired ccRCC tissues and NAT tissues and as expected, miR‐143 expression in ccRCC tissues was significantly underexpressed (Figure 1C). In order to confirm the results, we queried miR‐143 expression in TCGA RCC database, and in accordance with the results above, in 67 paired ccRCC tissues, miR‐143 expression was also found downregulated (Figure 1D). Although not significant, miR‐143 trended to have a prognostic role in ccRCC overall survival in TCGA (Figure 1E, P = .065).

Figure 1.

A, GSE37989 showed that miR‐143 was downregulated in metastatic RCC. B, realtime PCR demonstrated that miR‐143 was also underexpressed in 786‐O cell lines compared with HK‐2 cell lines. C, In our own clinical samples, compared with normal tissues, miR‐143 expression in tumor tissues was low. D, in TCGA dataset, miR‐143 was found underexpressed in 67 paired RCC tissues. E, Also in TCGA dataset, low level of miR‐143 trended to be a poor overall survival indicator in RCC patients. *P < .05. PCR, polymerase chain reaction; RCC, renal cell carcinoma; TCGA, The Cancer Genome Atlas

3.2. miR‐143 inhibited RCC cells adhesion and migration

Cells adhesion and migration are of most importance in tumor metastasis process. We further investigated whether miR‐143 influenced ccRCC cells adhesion and migration. The adhesion assay revealed that miR‐143 mimics remarkably inhibited 786‐O cells adhesion (Figure 2A). And consistent with this result, silencing of miR‐143 in HK‐2 cells resulted in a significant increase in cells adhesion (Figure 2B). Moreover, transwell migration assays revealed that the migration potential of 786‐O cells transfected with miR‐143 mimics was dramatically decreased (Figure 2C), whereas silencing of miR‐143 in HK‐2 cells significantly enhanced cells migration (Figure 2D). Taken together, miR‐143 may contribute to inhibition of RCC metastasis.

Figure 2.

A and C, Overexpression of miR‐143 in 786‐O cells inhibited cells adhesion and migration abilities. B and D, Knockdown of miR‐143 in HK‐2 cells promoted cells adhesion and migration. *P < .05. E‐cad, VIM, and fibronectin proteins were downregulated while N‐cad was upregulated in 786‐O cells after transfected with miR‐143 mimics, showing that miR‐143 suppressed EMT process in 786‐O cells. EMT, epithelial mesenchymal transition

3.3. EMT inhibition by miR‐143 in RCC cells

EMT can change cell migration ability. To identify whether miR‐143 can affect EMT, we evaluated some common EMT markers through Western blot analysis. Compared with cells transfected with control miRNA, a decreased expression of N‐cadherin, vimentin and fibronectin proteins was observed in cells transfected with miR‐143 mimics in 786‐O cells, while E‐cadherin expression increased in cells transfected with miR‐143 mimics (Figure 2E). These results suggested that miR‐143 may impede cells migration by inhibiting EMT.

3.4. ABL2 was a target of miR‐143 in RCC

As one of the tyrosine kinases, ABL2 is found elevated in ccRCC tissues and is supposed to be an oncogene in RCC formation and progression. When using miRNA.org online software, we revealed that ABL2 was a potential target for miR‐143 (Figure 3A). To confirm that, we first found that ABL2 expression level was markedly downregulated in miR‐143 mimics transfectants in comparison with NC transfectants in 786‐O cells while knockdown miR‐143 in HK‐2 cells promoted ABL2 expression (Figure 3B). Moreover, in luciferase assay, when cotransfected with miR‐143 mimics, the plasmid containing ABL2‐3′UTR had a decreased luciferase activity compared with that cotransfected with NC mimics, while cotransfected with anti‐miR‐143 in HK‐2 cells increased luciferase activity, indicating that miR‐143 could bind to ABL2‐3′UTR directly (Figure 3C).

Figure 3.

A, ABL2 was predicted to be a miR‐143 target in miRNA.org online software. B, Overexpression miR‐143 decreased ABL2 expression in 786‐O cells, while knockdown miR‐143 increased ABL2 expression in HK‐2 cells. C, Cotransfection of miR‐143 and pMIR‐reporter‐ABL2 plasmid inhibited luciferase ability in 786‐O cells. However, cotransfecting anti‐miR‐143 and pMIR‐reporter‐ABL2 plasmid in HK‐2 cells led to increased luciferase activity. *P < .05

3.5. ABL2 in RCC

Consistent with the lower level of miR‐143 in RCC (which may induce higher expression of ABL2), we investigated ONCOMINE RCC datasets. We found that all datasets demonstrated either a ABL2 upregulation or a copy number increase in RCC samples (Figure 4A, P = .020). Additionally, higher expression of ABL2 represents lower survival rate, indicating that ABL2 has a prognostic role in RCC overall survival in TCGA dataset (Figure 4B, P = .001).

Figure 4.

A, In ONCOMINE RCC database, pooled analysis indicated that ABL2 expression in RCC was upregulated. B, In TCGA dataset, patients with higher level of ABL2 expression had a poor overall survival rate. RCC, renal cell carcinoma; TCGA, The Cancer Genome Atlas

4. DISCUSSION

Accumulating evidence of abnormally expressed miRNAs in human cancers has confirmed the existence of miRNA‐mediated functions and has shown that differentially expressed miRNAs contribute to cancer initiation, development and metastasis. What is more, it may be an effective therapeutic strategy for tumor treatment. Recently, many papers have reported that a panel of miRNAs were altered in RCC tissues, suggesting that the aberrant expression of miRNAs are common events in renal tumorigenesis. By retrieving GSE37989 dataset from GEO databases, miR‐143 was found downregulated in ccRCC metastatic tissues. In the present study, we investigated the biological role of miR‐143 in human RCC. Our data demonstrate that miR‐143 is remarkably underexpressed in RCC cells and surgically excised ccRCC tumors. And the results were confirmed by miR‐143 expression data in TCGA RCC database. Particularly, low level of miR‐143 is significantly associated with improved survival in ccRCC patients. In the present study, we first described that experimental restoration of miR‐143 expression in RCC cells led to suppression of ABL2, which consequently reduced RCC cells adhesion, migration and EMT process, while silencing miR‐143 in normal epithelial HK‐2 cells promoted cells adhesion and migration.

MiR‐143 is frequently and consistently downregulated in various types of cancer and targeted multiple oncogenes and pathways. MiR‐143 was downregulated and acted as a tumor suppressor gene in bladder cancer,11 prostate cancer,11, 12 gastric cancer,13 and colorectal cancer14 by targeting KRAS, hexokinase 2, IGF1R, bcl2, and so on. However, to date, only one study concerned about role of miR‐143 in RCC. In Yoshino's study, they found that both miR‐143 and miR‐145 could inhibit RCC cells proliferation by targeting HK2.15 Different from their results, we firstly demonstrated that miR‐143 could reduce RCC cells adhesion, migration and EMT process by targeting ABL2 which was highly expressed in most RCC tissues. Tyrosine kinase signaling networks are required for the regulation of multiple cellular processes including growth, survival, invasion and angiogenesis during tumor initiation and progression.16 Activation of ABL kinases in solid tumors is driven by enhanced ABL1 or ABL2 expression or other oncogenic tyrosine kinases, chemokine receptors, oxidative stress or inactivation of negative regulatory proteins.16 Recently, activation of ABL2 has been detected in several tumor types including RCC. During the past two decades, tyrosine kinase inhibitors have been the hot spots in drug development. However, few studies are concerning about this topic. In our present study, it is interesting that ABL2 can be targeted by a tumor suppressor miRNA indicating that as a ABL tyrosine kinase inhibitor, miR‐143 could be a great treatment strategy in RCC. And besides ABL2 tyrosine kinase networks, miR‐143 can also target multiple oncogenic pathways as MAPK which makes it more efficient.

In conclusion, our study indicates that overexpression of miR‐143 would be a more effective therapeutic option for RCC patients.

CONFLICT OF INTEREST

All authors declare no conflicts of interests.

Xu B, Wang C, Wang Y‐L, et al. miR‐143 inhibits renal cell carcinoma cells metastatic potential by suppressing ABL2. Kaohsiung J Med Sci. 2020;36:592–598. 10.1002/kjm2.12207