| 【Objective】To investigate the biological functions of miRNAs whose expression changes in renal cell carcinoma(RCC)and their target genes on the biological behaviors such as proliferation,invasion and metastasis ability of RCC,as well as the correlation between their target proteins and clinicopathological features,prognosis and survival,providing a new theoretical basis for the molecular targeted therapey of RCC patients and a new marker for the clinical prognosis of RCC.【Methods】Part Ⅰ: 1.The expression of miR-324-3p and miR-4313 in RCC tissues and control renal tissues were detected by q RT-PCR assay.2.The changes of proliferation activity of 786-O and OS-RC-2 cells after transfected miR-324-3p mimic and miR-4313 mimic were detected by MTT assays.3.The changes of migration ability of 786-O cells after transfected miRNA mimics were detected by scratch test.4.The changes of invasion ability of 786-O cells after transfected miRNA mimics were detected by Transwell assay.5.The cell cycle changes of 786-O cells after transfected miR-4313 mimic were detected by PI staining and flow cytometry analysis.6.Using semi-quantative RT-PCR assays and Western-blot assays,we examined the cell cycle related genes and proteins levels in 786-O cells transfected with miR-4313 mimics or mimics control.Part Ⅱ: 1.The candidate target genes of miRNA-4313 were predicted and screened by Target Scan 6.1,DIANA-MICROT,Pic Tar,and miRanda bioinformatics analysis softwares.2.Using q RT-PCR assays,we examined the expression of the candidate gene targets in 786-O cells transfected with increasing concentrations of miR-4313 mimics.3.Using Western-blot assays,we examined the expression of TBX15 target protein in 786-O cells after transfected miR-4313 mimics.4.Using dual luciferase reporter gene system,we investigated whether miR-4313 specifically binds to the 3,-UTR sequence of TBX15 to control the expression of TBX15 gene.Part Ⅲ:1.The RCC tissues,normal kidney tissues and corresponding clinical data were collected,and the kidney cancer tissue chip was prepared.2.The expression of TBX15 protein in human RCC and normal tissues was verified by immunohistochemical staining of tissue microarray,and the clinicopathological correlation and survival analysis were performed.3.The TCGA database was used to analyze the differentia l expression of TBX15 m RNA in RCC and normal kidney tissue,and the survival analysis of TBX15 m RNA expression in RCC was performed.【Results】Part Ⅰ: 1.miR-324-3p and miR-4313 have a significantly low expression levels in RCC compared to normal kidney tissue or adjacent tissues.2.The proliferation ability of renal cell carcinoma cells was weakened after they were transfected with miR-324-3p mimics or miR-4313 mimic.However,miR-4313 had stronger ability to inhibit the cell proliferation.3.The migration and invasion ability of RCC cells were not weakened after they were transfected with miR-324-3p or miR-4313 mimic.4.Transfection of miR-4313 mimics into RCC cell line 786-O caused cell cycle arrest in G0/G1 phase.5.miR-4313 mimics tranfection did reverse cyclin D1 and cyclin D3 genes and their protein expression levels in 786-O cells.miR-4313 mimics tranfection did consistent p21,p27 and p53 genes and their protein expression levels in 786-O cells.Part Ⅱ: 1.By gene target prediction softwares,we found that UCP4,CUX1,MBD6 and TBX15 were candidate target genes of miR-4313.2.After 786-O cells transfected increased concentration of miR-4313 mimics,the expression of candidate gene TBX15 gradually decreased.However,the expression of candidate gene UCP4,CUX1 and MBD6 showed essentially no significant difference.3.miR-4313 mimics transfection did reverse the expression of TBX15 protein.4.miR-4313 specifically binded to the 3,-UTR sequence of TBX15 to control the expression of TBX15 gene.Part Ⅲ:1.The 60 RCC tissues,17 normal kidney tissues from 2009 to 2011 were collected,and the kidney cancer tissue chip was prepared.2.There was a significant difference in the expression of TBX15 in RCC tissues and normal kidney tissues.Compared to normal kidney tissues,the expression level of TBX15 in RCC tissue was significantly higher.There was a signicificant correlation between TBX15 and metastasis,clinical stage in 60 RCC patients.3.Compared to normal kidney tissues using TCGA database analysis,the expression level of TBX15 m RNA in RCC tissue was significantly higher.Patients with high expression of TBX15 m RNA in RCC have a significantly shorter life expectancy than the patients with low expression of TBX15 m RNA,suggesting that the high expression of TBX15 m RNA is closely related to the prognosis evaluation in RCC.【Conclusions】In the development of renal cell carcinoma,the expression of miR-4313 gradually occurs to decline,resulting in disappearance of down-regulation of TBX15.The expression level of TBX15 is increased,Tumor cells ultimately obtain unlimited proliferative and accelerating the malignant progress of renal cell carcinoma.The expression level of TBX15 is closely related to the prognosis evaluation in RCC.In this study,we elucidated the the molecular mechanisms of miR-4313/TBX15 in regulating the development of renal cell carcinoma,and provided a new way for the clinical prognosis and targeted therapy of renal cell carcinoma. |
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