The Molecular Mechanism Of MiR-133a-5p On The Proliferation And Metastasis Of Renal Clear Cell Carcinoma

Clear cell Renal Cell Carcinoma(ccRCC)is the most common type of kidney cancer,accounting for about 80% to 90% of kidney cancers.Advanced clear cell renal carcinoma and metastatic clear cell Renal Cell Carcinoma are very difficult to cure,and the 5-year survival rate is very low.Therefore,it is of great significance to search for the key oncogene/tumor suppressor gene of clear cell Renal Cell Carcinom and elucidate the molecular mechanism of its occurrence and development.Micro RNAs(miRNAs)have attracted more and more attention in tumor research.With the extensive development and deepening of research,the important regulatory role of miRNAs has been gradually clarified.In this study,miR-133A-5p was selected as the study object to conduct a systematic study in clear cell Renal Cell Carcinom.(1)Expression of miR-133A-5p in clinical samples and cells of clear cell Renal Cell CarcinomObjective: To determine the miR-133a-5p expression level in patients with clear cell Renal Cell Carcinoma,to analyze the correlation between miR-133a-5p expression and clinical practice,and to verify the expression of miR-133a-5p at the cellular level.Methods: Real-time quantitative PCR was used to detect the expression of miR-133a-5p in 56 pairs of clear cell Renal Cell Carcinoma tissues and paracancerous tissues obtained from clinical operations.Correlation analysis was used to analyze the correlation between the level of miR-133a-5p expression and clinicopathological data of patients with clear cell Renal Cell Carcinoma.The expression of miR-133a-5p in 5human ccRCC cells(ACHN,Ca Ki-1,769 P,Ca Ki-2,786-O)and one normal renal tubular epithelial cell(HK-2)was detected by real-time fluorescence quantitative PCR.Results: The expression of miR-133A-5p was significantly lower in patients with clear cell Renal Cell Carcinoma and in five ccRCC cell lines(ACHN,Ca Ki-1,769 P,Ca Ki-2,786-O),among which ACHN and 786-O had the lowest expression.The level of miR-133a-5p expression was not correlated with age,gender and patholog ical stage of patients with ccRCC,and was significantly negatively correlated with lymph node metastasis and distal metastasis of patients with ccRCC.The low expression level of miR-133a-5p was significantly positively correlated with the overall survival rate and disease-free survival rate in patients with ccRCC.The lower the expression level of miR-133a-5p,the lower the survival rate in patients with ccRCC and the lower the disease-free survival rate.(2)Effects of miR-133A-5p on proliferation and metastasis of clear cell Renal Cell Carcinoma cellsObjective: To study the effect of miR-133a-5p up-regulation on proliferation and metastasis of clear cell Renal Cell Carcinoma,and to clarify the function of miR-133a-5p in proliferation and metastasis of clear cell Renal Cell Carcinoma.Methods: Lipo2000 was used to transfect miR-133a-5p mimics and NC into clear cell Renal Cell Carcinoma cell lines(ACHN and 786-O),respectively.Realtime quantitative PCR was used to detect the expression of miR-133a-5p,CCK8 assay was used to detect cell proliferation,Transwell assay was used to detect cell invasion and migration,and wound healing assay was used to detect cell migration.Results: Real-time quantitative PCR results showed that the relative expression level of miR-133a-5p in the miR-133a-5p mimics group was significantly increased compared with the NC mimics group;Compared with the NC mimics group,the proliferation,invasion and migration of clear cell Renal Cell Carcinoma cell lines(ACHN and 786-O)in the miR-133a-5p mimics group were significantly reduced.(3)The molecular mechanism of miR-133A-5p targeting MON2 to inhibit the proliferation and metastasis of ccRCCObjective: To study the correlation between MON2 expression and prognosis in patients with clear cell Renal Cell Carcinoma,to clarify the effect of MON2 on proliferation and metastasis of clear cell Renal Cell Carcinoma,and to detect the targeted regulation relationship of miR-133A-5p on MON2.Methods: Real-time quantitative PCR was used to detect the expression of MON2 in 56 pairs of clear cell Renal Cell Carcinoma tissues and paracancerous tissues obtained from clinical operations.SPSS software correlation analysis was used to analyze the correlation between MON2 expression level and prognosis of patients with clear cell Renal Cell Carcinoma.MON2 overexpression and NC plasmid were transfected into clear cell Renal Cell Carcinoma cells(ACHN and786-O)using Lipo2000,respectively.MON2 expression was assessed by real-time quantitative PCR and Western Bloting.Cell proliferation ability was assessed by CCK8 assay,cell invasion and migration ability was assessed by Transwell assay,and cell migration ability was assessed by wound healing assay.The binding sites of miR-133a-5p and MON2 3’UTR were analyzed by bioinformatics,and the targeted regulation of miR-133a-5p on MON2 was detected by Dual Luciferase Reporter Assay.The restorative effects of MON2 overexpressed plasmid and miR-133A-5p mimics on proliferation,invasion and migration of clear cell Renal Cell Carcinoma cells(ACHN and 786-O)were detected by co-transfection.Results: The expression of MON2 was significantly higher in patients with clear cell Renal Cell Carcinoma,and it was negatively correlated with prognosis.Compared with the control group,the proliferation,invasion and migration of clear cell Renal Cell Carcinoma cells(ACHN and 786-O)in MON2 over expression group were significantly increased.Dual-luciferase reporter gene assay results showed that miR-133A-5p was bound to MON2 3’UTR,and the targeted regulation relationship was clear.The co-transfection of MON2 overexpressed plasmid and miR-133a-5p mimics partially restored the inhibitory effects of miR-133a-5p on the proliferation,invasion and migration of clear cell Renal Cell Carcinoma cells(ACHN and 786-O).Conclusions: 1.Clinical studies have confirmed the low expression of miR-133a-5p in patients with clear cell Renal Cell Carcinoma,and a significant positive correlation with the prognosis of patients.2.Upregulating miR-133a-5p can inhibit the proliferation,invasion and migration of clear cell Renal Cell Carcinoma cells,and miR-133a-5p has the function of tumor suppressor gene in the development of renal clear cell carcinoma.3.MON2 was highly expressed in patients with clear cell Renal Cell Carcinoma,and was negatively correlated with miR-133a-5p.4.MON2 overexpression can promote the proliferation,invasion and migration of clear cell Renal Cell Carcinoma cells.5.MON2 is the target gene of miR-133A-5p,and miR-133A-5p negatively regulates MON2 to realize the inhibition of ccRCC proliferation and metastasis.