| Background and purposeRenal cell carcinoma is one of the most common malignancies of urinary system in the global adult population.Because the patients with clinically localized renal cell carcinoma often have no clinical obvious symptoms and signs,it may easily lead to misdiagnosis.The data showed that about one-third of patients have had distant metastases when they first diagnosed with renal cell carcinoma.And once in the stage of advanced metastatic tumors,the prognosis of patients with metastatic renal cell carcinoma is very poor.Clear cell renal cell carcinoma(ccRCC)is the most common histological type of renal cell carcinoma which is also the most invasive and mortal one.Therefore,the study of the metastatic mechanism of ccRCC and the key molecules involved in the disease progression was very meaningful and clinical valuable for the tumor molecular classification and the therapeutic measures to prevent tumor metastasis.MiRNAs are a class of molecules that have been extensively studied which could play an important role in multiple biological processes of the tumorigenesis,progression and drug resistance in various tumors.Many previous studies have reported that miR-193b-3p is an key suppressor in various tumors,and the expression of mi R-193b-3p is down-regulated in many tumor tissues compared with pericarcinous tissue.In this study,we screened differentially expressed miRNAs between primary metastatic and non-metastatic ccRCC with miRNAs expression profiling technique,and found that the expression of miR-193b-3p was down-regulated in primary metastatic ccRCC.Furthermore,we evaluate the expressive status of miR-193b-3p in a large clinical samples to confirm the result of miR-193b-3p in miRNAs expression profiles.Then combined with the clinical pathological parameters and follow-up data in patients with ccRCC,we confirmed that the expression of miR-193b-3p could be used as a prognostic factor for the patients with ccRCC.We found that miR-193b-3p could inhibit the proliferative and metastatic ability in ccRCC in vitro and in vivo,and confirmed the potential target gene of miR-193b-3p.Materials and Methods1.According to metastatic status of preoperative diagnosis of renal cell carcinoma,we divided into two groups,each group of 3 cases,including primary metastatic and primary non-metastatic tumors,and only selected the cases with postoperative pathology confirmed as ccRCC.Using miRNAs expression gene chip technology,we screened out the miRNAs with significant differences between the above two groups.2.Using the method of qRT-PCR assay,we evaluate the expressive status of miR-193b-3p in a large clinical samples including the primary metastatic and primary non-metastatic renal clear cell carcinoma tissues and their matching adjacent tissues to confirm the result of miR-193b-3p in miRNAs expression profiles.Combining with the clinical pathological parameters and follow-up data in patients with ccRCC,we wanted to find out the relationship between the expression of miR-193b-3p and the common clinicopathological parameters or he prognosis of patients with ccRCC.3.In vitro,miR-193b-3p inhibitor and miR-193b-3p mimics were respectively transfected with 786-O and ACHN cell lines.Stably overexpression of miR-193b-3p was performed in vivo,which was constructed using lentivirus.The effect of mi R-193b-3p on the proliferation of renal cancer cells was investigated by CCK-8 assay,western blot,Brdu assay and colony formation assay.Constructing tumor growth model by subcutaneously injection,we observed the tumor growth,and the result was further confirmed with immunohistochemical staining.In addition,we investigated the effect of miR-193b-3p on the migration and invasion of renal cancer cells using wound healing assay and transwell migration and invasion assay in vitro.Constructing metastatic renal cell carcinoma model in nude mice by tail vein injection,fluorescence monitoring and HE staining to further observe the effect of miR-193b-3p on the metastasis of renal cell carcinoma in vivo.4.Combing with the result of differentially expressed mRNAs in the transcriptome microarray and miRNA targeting algorithms,we determined the potential targeted gene of miR-193b-3p by dual-luciferase reporter system,qRT-PCR,western blot and immunofluorescence assay.Results1.A total of 27 differentially expressed miRNAs were screened by miRNAs expression profiles between the primary metastatic and primary non-metastatic renal clear cell carcinoma tissues,of which 12 were down-regulated and 15 were up-regulated.The mi R-193b-3p was further studied by combining with the Fold change,the FDR value and literature research.2.According to the results of qRT-PCR assay,we found that miR-193b-3p showed a significant down-regulation in renal clear cell carcinoma(P<0.05)and was further reduced in primary metastatic ccRCC(P<0.05).In addition,the down-regulated expression of miR-193b-3p was found to be significantly associated with advanced clinical stage,higher pT stage and positive metastatic status.Kaplan-Meier survival analysis showed that the low expression of miR-193b-3p in tumor tissues of ccRCC was association with poor prognosis of patients of ccRCC.Multivariate analysis which was done by COX proportional hazard model showed that the expression level of miR-193b-3p could be an independent prognostic risk factor for disease progression and survival in patients with ccRCC.3.CCK-8 study showed that the proliferation of renal cancer cells was enhanced by being transfected with miR-193b-3p inhibitor,and was significantly inhibited by transfection with miR-193b-3p mimics.Western blot showed that transfection with miR-193b-3p inhibitor could enhance the expression of two specific proliferative antigen,Ki-67 and PCNA,and transfection with mi-193b-3p mimics could significantly inhibit the expression of Ki-67 and PCNA in renal cancer cells.Brdu assay showed that knockdown of miR-193b-3p could increase the ratio of Brdu-positive cells,whereas overexpression of mi R-193b-3p reduced the percentage of Brdu-positive cells.Colony formation assay also showed that knockdown of miR-193b-3p increased colony formation in renal cancer cells,whereas overexpression of miR-193b-3p reduced colony formation of renal cancer cells.In vivo,we found that stable expression of miR-193b-3p significantly inhibited the growth of subcutaneous tumor growth in nude mice.By immunohistochemical staining of subcutaneous tumor,we found that the expression of Ki-67,PCNA and CD31 was significantly inhibited.4.Wound healing assay showed a significant increase in the healing ability of renal cancer cells transfected with miR-193b-3p inhibitor,whereas the healing ability was significantly inhibited transfected with miR-193b-3p mimics.Transwell migration and invasion assay showed that knockdown of miR-193b-3p enhanced the migration and invasion ability of renal cancer cells,while overexpression of miR-193b-3p significantly inhibited the migration and invasion ability of renal cancer cells.It was found that stable expression of miR-193b-3p could inhibit the formation of lung micrometastases of renal cell carcinoma in metastatic model by tail vein injection in vivo.5.The dual-luciferase reporter assay confirmed that miR-193b-3p was able to directly target the 3'UTR region of MCL1,MMP19 and E2F6.In addition,qRT-PCR,western blot and immunofluorescence assay confirmed that miR-193b-3p could inhibit the mRNA and protein expression level of MCL1,MMP19 and E2F6.Therefore,miR-193b-3p may play an important role in inhibiting the proliferation and metastasis of renal cell carcinoma by regulating the expression of MCL1,MMP19 and E2F6.Conclusions1.Compared with adjacent normal renal tissues,miR-193b-3p was down-regulated in tumor tissues and further down-regulated in primary metastatic ccRCC.The clinical data analysis found that miR-193b-3p could be used as a prognostic biomarker for the patients with ccRCC.2.MiR-193b-3p can significantly inhibit the proliferation,migration and invasion of renal cancer cells,and may play a key role in the pathogenesis of ccRCC.3.In ccRCC,miR-193b-3p has been proved to be capable of targeting the 3'UTR region of MCL1,MMP19 and E2F6,and could negatively regulate the expression of these target genes. |
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