A Novel Mechanism Of Mir-141-repressed Proliferation And Metastasis In Renal Cell Carcinoma

Renal cell carcinoma (RCC) is the most lethal urological malignancy. Although therapies targeting the vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) pathways have radically improved the natural history of metastatic RCC (mRCC) during the past several years, complete or durable response to therapy is rare, and the majority of patients elect to withdraw drug treatment within about1year. micoRNA (miRNA) is a classic group of non-coding RNA, which is known as one of the most promising and effective therapeutic approaches of human cancers due to its unique characteristics, such as very short length, the contribution to multiple tumorigenic steps and the stability of circulating miRNA. In our previous study, using a microarray platform to study high-throughput miRNA profiles in paired primary tumor and adjacent normal tissues, we identified global downregulation of miRNA expression as a novel feature in ccRCC. Among those differentially expressed miRNAs in ccRCC, miR-141was the most remarkably downregulated miRNA (104-fold) and its roles in RCC have not been fully documented yet. In the present study, we aim to demonstrate the clinical significance and the roles of miR-141in vitro and in vivo, which will facilitate the development of agents against RCC. Our study is composed of the following three parts:Part I Genome-wide miRNA expression profiles and the clinical significance of miR-141in RCCObjective:We aimed to investigate expression profiles of miRNA in ccRCC and assess the clinical significance of miR-141, which may act as a potential and crucial regulator of renal carcinogenesis.Methods:Using a microarray platform which contains851human miRNAs, we studied miRNA profiles in paired primary tumor and adjacent normal tissues (NTs) from5patients. Then, relative miR-141expression levels in distinct kidney tumors and their NTs were analyzed by qRT-PCR.Results:As compared with NTs, we identified74miRNAs dysregulated in ccRCCs (fold change>2.0and p<0.05), among which44were significantly downregulated and the other30were upregulated. Notably, miR-141was the most remarkably downregulated miRNA in ccRCC. In accordance with microarray results, qRT-PCR showed that miR-141was significantly downregulated in92.6%(63/68) ccRCCs (p<0.0001). Receiver operating characteristics (ROC) analysis revealed that miR-141might serve as a useful biomarker for discriminating ccRCC from normal tissues with an area under the ROC curve (AUC) of0.93(95%CI,0.881to0.981). miR-141expression was not associated with tumor stage, grade or size. Unexpectedly and significantly, miR-141was also markedly decreased in other subtypes of kidney tumors, such as chromophobe RCC (chRCC), sarcoma RCC, renal angiomyolipomas (AML). There was no difference between ccRCCs and AMLs.Conclusion:Downregulation of miR-141contributes to the development of tumors originated from kidney independent of specific subtypes. miR-141might serve as a robust biomarker for discriminating between ccRCC and normal tissues. Part Ⅱ Role of intracellular and extracellular miR-141in ccRCCObjective:To explore the biological significance of miR-141in ccRCC in vitro and in vivo, and determine whether miR-141released from the donor tumor cells can be taken up by the recipient tumor cells to function in cell-cell communications during cancer progression.Methods:We stably overexpressed miR-141in two ccRCC cell lines786-0and SN12-PM6with lentiviruses carrying miR-141and its control (miR-NC). The efficacy of infection was tested by qRT-PCR. Cell viability and drug sensitivity were determined by MTT method. Cell cycle was measured by FACS. Cell migration and invasion were analyzed by using transwell assay. To identify antitumorigenic roles of miR-141in RCC in vivo, SN12-PM6cells infected with lentiviral particles carrying miR-141versus miR-NC were injected into the left kidney of nude mice and then tumor growth and metastasis were evaluated. The levels of miR-141in the conditioned media (CM) from miR-141and miR-NC cells were measured at several time points by qRT-PCR. CM from miR-141or miR-NC cells was used to culture miR-NC cells.Results:miR-141expression was elevated up to400-and2400-fold in786-0and SN12-PM6cells, respectively. There were no marked cellular morphologic changes in the miR-141-overexpressed cells, however, the cellular proliferation analyses showed that overexpression of miR-141suppressed ccRCC cell proliferation (p<0.05), caused cell cycle arrest at G0/G1phase and decreased the S phase population. More importantly, overexpression of miR-141markedly impaired ccRCC cell migration and invasiveness compared to miR-NC (p<0.001). Additionally, miR-141overexpression did not directly induce apoptosis in the two tested ccRCC cell lines, with no pronounced alterations in the responsiveness to DDP,5-FU and TRAIL. Over a period of6weeks and8-9weeks postimplantation, there was a more significant decrease in tumor weight and size upon expression of miR-141(p<0.05). More specifically, tumors with miR-141overexpression were commonly encapsulated and confined to kidney parenchyma, whereas tumors of control cells presented more aggressive growth. In addition, no macroscopic metastases were found in tumors with miR-141overexpression. In contrast, miR-NC tumors extensively infiltrated the kidney fascia, especially at8th-9th week after implantation. Compared with miR-141tumors, the miR-NC tumors were prone to local invasion and metastasis. Extracelluar miR-141in CM of miR-141cells was significantly higher than that of miR-NC cells, and the level of intracellular miR-141in the miR-NC cells was increased for about10or7-fold after adding CM of the miR-141cells. Furthermore, co-culture with CM of the miR-141cells decreased migration and invasion of the recipient miR-NC cells. However, the level of extracelluar miR-141was markedly lower than its intracellular counterpart in the parental cells.Conclusion:miR-141functions as a critical tumor-suppressor in RCC by suppressing tumorigenesis and metastasis, and can be excreted into extracellular environment and plays active biological functions in the recipient cells. However, more predominant miR-141activities reside intracellularly. Part Ⅲ EphA2frequently upregulated in ccRCCs is a novel direct target of miR-141Objective:We attempted to identify specific miR-141targets with potential relevance in the regulation of metastasis in RCC, and then extend the studies on the miR-141-target module to further downstream.Methods:We used different miRNA target-predicting algorithms such as TargetScan, Pictar, miRanda, miRDB and miRwalk to identify potential effector (s) of miR-141, and then examined the correlation between miR-141and its target expression in both in vitro and in vivo studies. A208bp fragment from the EphA23’UTR containing the putative miR-141target sites was cloned into a luciferase reporter construct. ccRCC cells were transfected with a synthesized siRNA for EphA2(si-EphA2) versus the negative control (si-NC). A rescue experiment was performed by co-transfecting with EphA2siRNA (versus the negative control) and miR-141inhibitor (versus the negative control) into ccRCC cells. A Medline search was conducted to identify genes, which are functionally and physically important in EphA2signaling. FAK、p-FAK、AKT、p-AKT、MMP-2and MMP-9protein levels were assessed by Western Blot in ccRCC cells overexpressing miR-141(versus negative control) and transfected with EphA2siRNA (versus negative control).Results:We found conserved miR-141sites at the3’UTR of EphA2. Enforced miR-141expression led to a decrease in EphA2mRNA and protein expression in ccRCC cells. Consistently, evaluation of primary tumors in the renal orthotopic xenografts models showed an inversed correlation between miR-141expression and EphA2mRNA and protein levels. miR-141overexpression substantially repressed activity of the reporter that carried the wild-type but not mutant3’UTR of EphA2. Knockdown of EphA2in ccRCC cells attenuated cell growth, induced Go/G1cell-cycle arrest, and suppressed cell migration and invasion, similarly to the phenotypic alterations upon miR-141overexpression. Importantly, the enhancement in ccRCC cell migration and invasion induced by miR-141inhibitor was effectively reversed by EphA2attenuation. Pearson’s correlation analysis showed a good reverse correlation between levels of miR-141and EphA2mRNA in ccRCC tissues (R2=0.3661, p=0.0047). Notably, compared to normal tissues, meaningful overexpression of EphA2protein was observed in17of20and decreased expression of EphA2was showed in1of20ccRCC cases. p-FAK, a critical metastasis regulator, is functionally and physically important in EphA2signaling. p-AKT involved in various cancer cell survive, growth, and migration is associated with EphA2overexpression. Also, MMP-2and MMP-9contribute to FAK/AKT-mediated cell proliferation and metastasis. Accordingly, we reasoned miR-141-EphA2-FAK/Akt-MMP (2/9) pathway regulation in RCC. Lentivirus mediated overexpression of miR-141adequately led to compromised p-FAK, p-AKT, MMP-2in786-0and SN12-PM6cells. Suppression of EphA2by siRNA also resulted in an attenuation of p-FAK, p-AKT, MMP-2. However, a significant downregulation of MMP-9was only observed in SN12-PM6cells but not in786-0cells. Moreover, the rescue experiment showed that upregulation of EphA2expression following miR-141inhibition led to increased phosphorylation of FAK and AKT, while knockdown of EphA2rescued the stimulatory effects of miR-141inhibitor on p-FAK and p-AKT.Conclusion:These findings clearly substantiate that EphA2is a direct and functional target of miR-141. The increased level of EphA2is, at least in part, attributed to loss of miR-141in ccRCC. p-FAK/p-Akt/MMP (2/9) pathway regulation may be the downstream effectors of the miR-141-EphA2module in RCC.