Downregulation of miR-193a-3p inhibits cell growth and migration in renal cell carcinoma by targeting PTEN

Abstract

Although miR-193a-3p has been found to be dysregulated in variety of human tumors, little is known about its role in renal cell carcinoma. This study was designed to investigate the function and underlying mechanism of miR-193a-3p in human renal cell carcinoma tissues and cell lines. Here, we demonstrated that the expression of miR-193-3p was increased in renal cell carcinoma tissues and cell lines. In addition, knockdown of miR-193a-3p significantly inhibited cell proliferation and colony formation and induced cells into G1 phase arrest. Meanwhile, the migration potential of 786-O cells was also decreased compared to control group. Furthermore, we identified PTEN as a direct and functional target of miR-193a-3p, at least partly responsible for promoting tumor effect of miR-193a-3p in renal cell carcinoma. Taken together, the findings indicated for the first time that miR-193a-3p functions as a tumor-promoting microRNA by directly targeting PTEN in renal cell carcinoma.

Keywords

miR-193a-3p renal cell carcinoma PTEN proliferation migration

Introduction

Renal cell carcinoma (RCC) is the most common urologic malignancy, accounting for 2%–3%¹ of all adult urologic malignancies and approximately 5%² of epithelial cancers worldwide. Either partial or radical nephrectomy of the affected kidney remains the mainstay of curative treatment for localized disease.³ Although the malignancy generally resists traditional chemotherapy and radiotherapy, the introduction of novel molecular-targeted agents including anti–vascular endothelial growth factor (VEGF) and anti–mechanistic target of rapamycin (mTOR) therapeutic medicines has revolutionized the management of patients with metastatic RCC.^4,5 Because most patients are diagnosed when the tumor is still relatively localized and amenable to surgical removal, the 5-year survival rate for RCC is approximately 73%.⁶ However, the 5-year survival rate of patients who diagnosed at the metastatic stage is <10%.⁷ Therefore, identification of the effector molecules or signal pathways responsible for regulating tumor growth and metastasis is critical for improving the RCC treatment.

MicroRNAs (miRNAs) are an evolutionarily conserved group of small, noncoding RNA molecules (18–24 nucleotides) that are involved in posttranslational regulation of gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner.^8,9 They function via sequence-specific binding of a seed sequence to the 3′ end of the untranslated region (UTR) of a target messenger RNA (mRNA), which causes it to either be degraded or to be translationally inhibited.¹⁰ Increasing evidences suggest that miRNAs play crucial roles in various physiological and pathological processes, including cell differentiation, proliferation, apoptosis, migration, and signal transduction.^11–13 Global miRNA expression studies have identified miRNAs that are consistently dysregulated across various types of human cancers, including gastric cancer, bladder cancer, breast cancer, hepatocellular cancer, RCC, and so on.^14–16 Numerous miRNAs have been reported to perform specific functions in the regulation of tumor progression either as tumor suppressors or as oncogenes.

miR-193a-3p is part of the miR-193 family, together with miR-193a-5p and miR-193b. Nevertheless, the information on miR-193a-3p is limited and its molecular mechanisms and roles in carcinogenesis are still poorly understood. Aberrant expression of miR-193a-3p has been reported in several types of cancer and appears to be significantly associated with the clinical outcome of human cancer patients. For example, miR-193a-3p is predominantly upregulated or overexpressed in both gastric cancer cell lines and human gastric tumors.¹⁷ miR-193a-3p is also differentially upregulated and downregulated between the individual osteosarcoma cells.¹⁸ Moreover, the level of miR-193a-3p is significantly upregulated and positively correlated in both the colorectal cancer patients’ tissue and blood samples.¹⁹ In addition, miR-193a-3p expression was higher in malignant pleural mesothelioma than in adenoma and RCC.²⁰ Additional research demonstrated that miR-193a-3p is a potent promoter of the multichemoresistance in both hepatocellular cancer and bladder cancer via repressing the expression of its three downstream targets.^21,22 However, the expression and role of miR-193a-3p in the RCC development and progression have not yet been reported.

In this study, we determined the expression of miR-193a-3p in human RCC tissue and cell lines and investigated the effects of knockdown of miR-193a-3p on cell proliferation and migration. Moreover, PTEN was identified as a direct target of miR-193a-3p that might mediate its biological effects. The data in this study suggest that miR-193a-3p functions as a tumor-promoting miRNA by regulating PTEN expression, providing a potential target for clinical treatment of RCC.

Materials and methods

Ethics statement and tissue samples

This study was approved by the ethical board of Renmin Hospital of Wuhan University and complied with the Declaration of Helsinki. Informed consents from every patient were obtained. RCC tissues and adjacent normal renal tissues were collected from 30 patients undergoing radical nephrectomy at Renmin Hospital of Wuhan University between January 2012 and December 2015. The clinical samples were immediately stored in liquid nitrogen before RNA extraction.

Cell lines, cell culture, and transfection

The human RCC cell lines A498, Caki-1, and 786-O cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). A normal renal cell line (HK-2) was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 1% glutamine, 1% penicillin/streptomycin (Invitrogen), and maintained in a humidified incubator of 5% CO₂ at 37°C. The miR-193a-3p inhibitor, mimic, and controls which were purchased from GenePharma (China) were transfected into cells by using Lipofectamine 2000 (Invitrogen, Canada) following the manufacturer’s protocol.

Quantitative reverse transcriptase polymerase chain reaction

Total RNA from tissues or cells was extracted using TRIzol reagent (Invitrogen) and purified with an RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The SYBR-Green PCR master mix (Applied Biosystems, Inc., Foster City, CA) was performed on the 7500 Real-time PCR system (Applied Biosystems). All reactions were performed in triplicate. Polymerase chain reaction (PCR) primers included the following: miR-193a-3p—5′-GCATAACTGGCCTACAAAGT-3′ and 5′-GTGCAGGGTCCGAGGT-3′; U6: 5′-CTCGCTTCGGCAGCACA-3′ and 5′-AACGCTTCACGAATTTGCGT-3′; 18S: 5′-CATTCGTATTGCGCCGCT-3′ and 5′-CGACGGTATCTGATCGTC-3′; and PTEN: 5′-CACCTATTCCTCAGCCCTTAT-3′ and 5′-AACCCTCATTCAGACCTTCAC-3′. The relative quantification value for each gene was calculated by the 2^−ΔΔCt method using U6 as an internal control.

The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony formation assay

Cell proliferation was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay performed following the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO, USA). Cells were plated onto 96-well plates at a density of 5000 cells per well and transfected with indicated constructs. Cells were incubated for 72 h, and MTT reagent was added. After incubation for 4 h at 37°C, 150 µL dimethyl sulfoxide (Sigma-Aldrich) was added. The optical density (OD) was measured at 490 nm by an enzyme immunoassay instrument (BioRad, Hercules, CA, USA).

For colony formation assay, cells were transfected with indicated constructs for 48 h, and then cultured in a six-well plate in growth medium. After 7-day incubation, the colonies were fixed with 4% formaldehyde and then stained with 0.5% crystal violet. Stained colonies larger than 1 mm in diameter were counted.

Cell cycle assay

Cells were harvested at 48 h after transfection with indicated constructs and washed three times with cold PBS. Ice-cold 70% ethanol was subsequently added and the cells were fixed at 4°C overnight. After resuspended in fluorescence-activated cell sorting (FACS) solution with RNase and propidium iodide (PI), cell cycle distribution was detected by a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Cell migration assay

Cell migration activity was evaluated with the Transwell system (24-well plates, 8-µm pore size; BD Bioscience). After 48-h transfection with indicated constructs, aliquots of 100,000 786-O cells were resuspended in RPMI-1640 medium without FBS and seeded into the upper chamber coated with collagen IV. The lower chamber was filled with 0.4 mL RPMI-1640 medium containing 10% FBS. Cells were fixed with 10% formalin and stained with 0.1% crystal violet solution after incubation for 24 h. The migration cells were taken pictures and counted in five randomly fields under a light microscope at ×100 magnification. All experiments were performed in triplicate.

Wound-healing assay

The cell migration was determined by wound healing assay. Briefly, the cells were plated in six-well plates (25,000 cells/well) and transfected with indicated constructs 24 h later. After 6 h of transfection, a sterile 200-µL pipette tip was used to make a scratch in the cell monolayer to create a wound area. After removing debris, the cultures were replenished with fresh medium and maintained for 24 h. Cell migration into the would area was examined under phase contrast objectives (×10) on a CK2 inverted microscope (DM 16000; Leica, Germany).

Dual-luciferase reporter assay

The PTEN 3′-UTR containing the putative miR-193a-3p binding site was cloned into the psiCHECK2 vector (Promega, Madison, WI, USA), and the plasmid was confirmed by sequencing. The 786-O cells were seeded in 12-well plates and co-transfected with the reporter construct, control vector, and indicated constructs. At 30-h posttransfection, luciferase activity was measured using the Dual-Luciferase Reporter assay kit (Promega).

Western blotting

Western blotting was performed as preciously described.²³ Briefly, cells were lysed in a modified radioimmunoprecipitation (RIPA) buffer and protein was separated by electrophoresis before transferred to membranes. Membranes were probed with anti-β-actin, anti-PTEN, anti-AKT, and anti-pAKT primary antibodies at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-linked secondary antibodies for 1 h at room temperature. Western blots were developed using the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Piscataway, NJ, USA).

Statistical analysis

The results of multiple experiments are presented as mean ± standard deviation (SD) from three separate experiments. Data were analyzed by Student’s t test or one-way analysis of variance (ANOVA) using SPSS16.0 statistical software. The correlations between miR-193a-3p expression levels and PTEN mRNA levels were analyzed using Spearman’s rank test. The value of p < 0.05 was considered to be statistically significant.

Results

miR-193a-3p is elevated in RCC specimens and RCC cell lines

To examine whether miR-193a-3p is differentially expressed in RCC, we analyzed the expression of miR-193a-3p in 30 paired RCC specimens and pair-matched adjacent normal renal tissues by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The data demonstrated that miR-193a-3p expression was significantly higher in clinical RCC specimens than in normal renal tissues (Figure 1(a)). Moreover, we detected miR-193a-3p expression in a series of human RCC cell lines, and consistent with results in RCC tissues, miR-193a-3p was highly expressed in RCC cell lines (A498, Caki-1, 786-O) than in the control normal renal cell line (HK-2) (Figure 1(b)). In addition, 786-O cells displayed the significant increase in miR-193a-3p expression level. Therefore, 786-O cells were used in the subsequent investigations in this study.

Figure 1.

The expression of miR-193a-3p is upregulated in renal cell carcinoma tissues and cell lines. (a) miR-193a-3p expression was determined by real-time PCR in renal cell carcinoma tissues (RCC) and adjacent noncancerous tissues (Normal). (b) The expression of miR-193a-3p was measured in three human renal cell carcinoma cell lines (A498, Caki-1, 786-O) and one normal renal cell line (HK-2). All data were expressed as mean ± SD.

Downregulation of miR-193a-3p inhibits RCC cell proliferation and disrupts the cell cycle of RCC cells

To better understand the biological function of miR-193a-3p, cell proliferation was investigated. The 786-O cells were transfected with miR-193a-3p inhibitor for 72 h, and cell proliferation was evaluated by MTT assay. The results showed that, in miR-193a-3p downregulated 786-O cells (Figure 2(a)), cell proliferation rate was significantly reduced as compared to control cells (Figure 2(b)), suggesting that inhibition of miR-193a-3p repressed cell proliferation in 786-O cells. As shown in Figure 2(c) and (d), proliferation was also assessed by colony formation assay. The results revealed that colony numbers were significantly decreased after suppression of miR-193a-3p. Taken together, these data clearly indicated that downregulation of miR-193a-3p had anti-tumor effect on RCC cell proliferation.

Figure 2.

Downregulation of miR-193a-3p inhibits RCC cell proliferation. (a) The expression of miR-193a-3p was measured in 786-O cells after transfection with negative control (NC) or miR-193a-3p inhibitor. (b) MTT assay was performed to measure the 786-O cell proliferation after transfection with NC or miR-193a-3p inhibitor. (c) Colony formation analysis was performed to measure the 786-O cell colony formation after transfection with NC or miR-193a-3p inhibitor. (d) Quantification of colony numbers. (e) Cell cycle distribution was analyzed in 786-O cells after transfection with NC or miR-193a-3p inhibitor. All data were expressed as mean ± SD for three independent experiments.

To determine whether downregulation of miR-193a-3p caused cell cycle arrest, cell cycle assay was performed in 786-O cells. Flow cytometer analysis revealed that inhibition of miR-193a-3p increased the number of cells in the G0/G1 phase of the cell cycle, while the S phase population decreased in 786-O cells (Figure 2(e)). These results indicate that inhibition of miR-193a-3p can induce G0/G1 phase arrest among 786-O cells, suggesting that downregulation of miR-193a-3p inhibits cell proliferation by regulating cell cycle.

Downregulation of miR-193a-3p inhibits RCC cell migration

The Transwell migration assay is a useful method to investigate migratory ability. Our results showed that the numbers of migrating cells were significantly less in 786-O cells transfected with miR-193a-3p inhibitor than in those transfected with control (Figure 3(a) and (b)). In addition, the wound healing assay was also performed to evaluate cell migrating capability. As shown in Figure 3(c), migration into the previously wounded area was significantly inhibited by miR-193a-3p downregulation. Therefore, these results demonstrated that the migratory ability of 786-O cells was inhibited by suppression of miR-193a-3p.

Figure 3.

Downregulation of miR-193a-3p inhibits RCC cell migration. (a) Cell migration ability was analyzed by Transwell chamber assay. Representative images (at ×100 magnification) of crystal violet stained 786-O cells after NC or miR-193a-3p inhibitor transfection. (b) Quantification analysis of the number of crystal violet stained 786-O cells. (c) Wound-healing assay was performed to assess cell migration capability. Downregulation of miR-193a-3p alleviated cell migration in 786-O cells. The data represent mean ± SD.

PTEN is the downstream target of miR-193a-3p in RCC cells

The downstream target of miR-193a-3p in regulating RCC cells was further detected. To identify possible target genes of miR-193a-3p, we performed a computational screen using TargetScan software and focused our attention on PTEN as the potential putative target (Figure 4(a)). A luciferase reporter assay revealed a marked increase in luciferase activity in cells co-transfected with miR-193a-3p inhibitor and the reporter vector compared with that in cells co-transfected with the control and the reporter vector, while it failed to affect luciferase activity with the mutated luciferase construct (Figure 4(b)). Interestingly, overexpression of miR-193a-3p reduced luciferase activity in the PTEN wild-type reporter gene but not the mutant type (Figure 4(c)).

Figure 4.

PTEN is a direct target of miR-193a-3p. (a) TargetScan was demonstrated that 3′-UTR of PTEN contained the highly conserved putative miR-193a-3p binding sites. (b) Luciferase reporter analysis showed that downregulation of miR-193a-3p increased luciferase activity in the PTEN wild-type reporter gene but not the mutant type in 786-O cells. (c) Luciferase reporter analysis demonstrated that overexpression of miR-193a-3p decreased luciferase activity in the PTEN wild-type reporter gene but not the mutant type in 786-O cells. (d) Knockdown of miR-193a-3p enhanced PTEN mRNA level, and overexpression of miR-193a-3p inhibited the mRNA level of PTEN in 786-O cells. (e) Western blot analysis of PTEN, pAKT, AKT expression in 786-O cells transfected with NC, miR-193a-3p, scramble, and miR-193a-3p mimic. (f) The relative expression of PTEN in 786-O cells after transfection with NC, miR-193a-3p, scramble, and miR-193a-3p mimic. (g) The relative expression of pAKT/AKT protein levels after transfection with NC, miR-193a-3p, scramble, and miR-193a-3p mimic. (h) PTEN mRNA expression in 30 cases of RCC and adjacent normal tissues were detected by qRT-PCR. (i) Spearman’s correlation analysis was used to determine the correlation between the levels of miR-193a-3p and PTEN in RCC. The data represent mean ± SD.

The regulatory effect of miR-193a-3p on PTEN expression profile in RCC cells was examined by qRT-PCR and Western blot analyses. Our results showed that PTEN expression was significantly increased at mRNA level by miR-193a-3p downregulation (Figure 4(d)); moreover, PTEN protein expression was also enhanced by miR-193a-3p inhibition (Figure 4(e) and (f)). On the contrary, overexpression of miR-193a-3p significantly decreased PTEN expression at both the mRNA and protein levels (Figure 4(d) and (e)). Furthermore, inhibition of miR-193a-3p significantly lowered protein expression of PTEN downstream protein pAKT expression, while overexpression of miR-193a-3p significantly increased pAKT expression (Figure 4(e) and (g)). Thus, these data suggest that PTEN was actively involved in the modulation of miR-193a-3p in RCC cells and they further indicate that PTEN is a direct target of miR-193a-3p in 786-O cells.

To further investigate the correlation between the expression levels of PTEN and miR-193a-3p, expression of PTEN mRNA in 30 RCC and the normal tissues was measured. Results showed that PTEN mRNA was significantly decreased in RCC tissues compared with the normal tissues (Figure 4(h)). Moreover, PTEN was inversely correlated with miR-193a-3p level in RCC tissues (Figure 4(i)).

Inhibition of PTEN reverses the anti-tumor effects induced by miR-193a-3p downregulation in 786-O cells

To evaluate whether PTEN is responsible for the functional effect of miR-193a-3p downregulation in 786-O cells, we co-transfected 786-O cells with miR-193a-3p inhibitor and a PTEN-targeting small interfering RNA (siPTEN), or a control scrambled siRNA. And then, cell proliferation, colony formation, cycle, and migration assays were performed. Compared with cells transfected with negative control, the cells transfected with miR-193a-3p inhibitor exhibited a higher PTEN expression, while cells co-transfected with both miR-193a-3p inhibitor and siPTEN could restore PTEN protein expression (Figure 5(a)). Knockdown of PTEN significantly attenuated the inhibitory effect of miR-193a-3p inhibitor on cell growth in 786-O cells (Figure 5(b) and (c)). Meanwhile, the cell cycle arrest effect of miR-193a-3p inhibitor was also reversed by knockdown of PTEN (Figure 5(d)). Moreover, the restoration of PTEN significantly promoted 786-O cell migration (Figure 5(e) and (f)). Therefore, those functional experiments further demonstrated that PTEN gene is the direct downstream target of miR-193a-3p regulation on RCC proliferation and migration.

Figure 5.

Knockdown of PTEN reverses the anti-tumor effects induced by miR-193a-3p downregulation in 786-O cells. (a) Western blot analysis of PTEN in 786-O cells transfected with indicated constructs. (b) MTT assay was performed to determine the cell proliferation after transfection with indicated constructs. (c) Colony formation analysis was performed to measure the 786-O cell colony formation after transfection with indicated constructs. (d) Cell cycle distribution was analyzed in 786-O cells after transfection with indicated constructs. (e) Wound-healing assay was performed to assess cell migration capability. (f) Cell migration ability was analyzed by Transwell chamber assay. The data represent mean ± SD.

Discussion

Our previous work showed that miR-142-5p can promote cell growth and migration in RCC by targeting BTG3 (B-cell translocation gene3), suggesting the potential use of miRNAs in cancer diagnosis and therapies. In the case of RCC, accumulating evidences have been demonstrated that miRNAs function as either tumor suppressors or oncogenes by regulating various biological procession, and aberrant expression of miRNAs has been reported to be implicated in the tumor initiation and progression. In fact, a number of miRNAs, such as miR-141²⁴ and miR-495,²⁵ have been identified as tumor suppressors, while miR-210²⁶ and miR-155²⁷ were found to act as oncogenes in RCC, suggesting that miRNAs act as new direction in RCC diagnosis and treatment. In this study, we explored cancer relevant biological functions of miR-193a-3p and the potential mechanism in RCC tumorigenesis. Our results revealed that miR-193a-3p was elevated in human RCC tissues and cell lines compared to adjacent non-tumor tissues and normal renal cell. We also found that downregulation of miR-193a-3p significantly inhibited RCC cell proliferation and colony formation and induced the G0/G1 phase arrest in 786-O cells. Consistently, downregulation of miR-193a-3p was found to impair the migration potential of RCC cells. In addition, PTEN was identified as a direct target of miR-193a-3p. Interestingly, restoration of PTEN reversed the effects of miR-193a-3p inhibitor on cell proliferation, cell cycle arrest, and migration in RCC cells. Taken together, our study indicates that miR-193a-3p may play an onco-miRNA role in the progression of RCC. These data provide new insights into RCC research and therapeutic strategies.

miR-193a-3p has been found to be predominantly upregulated or overexpressed in both gastric cancer cell lines and human gastric tumors, which suggested that it mainly plays an oncogenic role in gastric cancer.¹⁷ However, another study found that miR-193a-3p was downregulated in human non–small cell lung cancer (NSCLC) cells, and it acted as a tumor suppressor by inhibiting NSCLC proliferation and migration.²⁸ Meanwhile, miR-193a-3p can induce apoptosis by directly targeting myeloid cell leukemia-1(Mcl-1).²⁹ Based on the above discussion, the results seem to be contradictory in that miR-193a-3p is characterized as an oncogene in certain cancers and a tumor suppressor in others. The reason for this contradiction may be that miR-193a-3p is associated with different signaling pathways in different cancer types to act as either oncogene or tumor suppressive gene. Consistent with previous findings, our present results indicate that miR-193a-3p might function as an oncogene in RCC. The miR-193a-3p expression was significantly higher in clinical RCC specimens and human RCC cell lines than in normal renal tissues and a normal renal cell line, respectively. Moreover, we investigated the effects of miR-193a-3p inhibitor on RCC growth and found that downregulation of miR-193a-3p inhibited RCC cell proliferation, as well as induced G0/G1 phase arrest in 786-O cells. These results agree with previous studies in human gastric cancer, in which miR-193a-3p was aberrantly overexpressed, and knockdown of it inhibited tumor proliferation and migration.¹⁷ Furthermore, we focused on the effects of miR-193a-3p inhibitor on RCC migration and found that inhibition of miR-193a-3p suppressed RCC cell migration. These results demonstrate that miR-193a-3p inhibitor is associated with inhibition of RCC cell growth and migration. This study is in line with some previous study suggesting that miR-193a-3p might function as an oncogene in RCC.

To further determine the involvement of miR-193a-3p in the development of human malignancies, we investigated the molecular mechanism of miR-193a-3p in regulating proliferation and migration in RCC cells. By using TargetScan software, we found that 3′-UTR of PTEN contained the highly conserved putative miR-193a-3p binding sites. Next, we demonstrated that miR-193a-3p overexpression was associated with suppression of luciferase activity, while inhibition of miR-193a-3p led to an increase in luciferase activity. In addition, we observed that overexpression of miR-193a-3p significantly decreased PTEN expression at both the mRNA and protein levels; on the contrary, downregulation of miR-193a-3p significantly increased PTEN expression at both the mRNA and protein levels. Interestingly, we found that miR-193a-3p was positively associated with pAKT, which is a critical signaling pathway in promoting cancer progression. Meanwhile, Pearson correlation analysis revealed a significant inverse correlation between the expression of miR-193a-3p and PTEN in patients with RCC. PTEN has been discovered as a tumor suppressor and plays a critical factor in the development and progression of various cancer including colon cancer, gastric cancer, osteosarcoma, and prostate cancer.^30,31 PTEN, a negative regulator of phosphoinositide 3-kinase (PI3K) signaling pathway, is also demonstrated as a hot target of numbers of miRNAs, including miR-21, miR-214, miR-217, which are involved in the regulation of several cancer types.^32–34 miR-193a-5p, a member of the miR-193 family, was reported to inactivate the AKT/mTOR signaling pathway by directly targeting the class I PI3K regulatory subunit and mTOR, which suppressed NSCLC metastasis.²⁸ However, our data indicate that PTEN is a direct target of miR-193a-3p in RCC cells. This discrepancy suggests that miR-193 family member may regulate different targets via different pathways in different human cancers. Consistent with our above-mentioned results, knockdown of PTEN could abolish the inhibitive effect of miR-193a-3p inhibitor on the proliferation and migration of RCC cells. These results suggest that miR-193a-3p functions as an oncogene in RCC, at least in part, by repressing PTEN expression.

In summary, our study demonstrates that miR-193a-3p is upregulated in RCC specimens and cell lines. Downregulation of miR-193a-3p inhibits RCC cell proliferation, prevents 786-O cell entry into S phase, and attenuates RCC cell migration. The oncogene function of miR-193a-3p is mediated by the downregulation of its downstream target gene PTEN. These findings suggest that miR-193a-3p may be a promising target for the development of new treatment for RCC.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Scientific Foundation of China (No. 81101948) and the Fundamental Research Funds for the Central Universities (No. 2042014kf0624).

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