The Investigation Of MiR-4521 Promoting CcRCC Progression And Mechnism By Negatively Regulating FAM129A

Background:Renal cell carcinoma?RCC?is a malignant tumor originating from renal tubular epithelial cells.It is the most common type of kidney cancer,accounting for 3%of all human malignant tumors,accounting for 85%of all kidney cancer.And RCC is one of the most common malignant tumors of the urinary system and the incidence has steadily increased in China over the years.Clear cell RCC?ccRCC?,the most prevalent and aggressive RCC subtype,is characterized with extremely high rates of local invasion,malignancy,and mortality and resistance to chemotherapy and radiotherapy.miR-4521 is abnormally expressed in a variety of malignant tumors and diseases,but there are few studies on its mechanism.Abnormal expression of FAM129A is closely related to a variety of tumors and precancerous lesions.Over expression of FAM129A promoted invasion of thyroid tumor and hyperplasia of thyroid nodules.It is also a potential biomarker for thyroid tumors and differential diagnosis between benign and malignant thyroid nodules.However,there are no study reported about miR-4521 and FAM129A in the progress of ccRCC.Objective:1.To study the expression of miR-4521 and FAM129A in cc RCC patients?tissues and in the ccRCC cell line and the correlation between them.2.To probe into the negative regulatory relationship between miR-4521 and FAM129A in ccRCC.3.Study on the effect of miR-4521 overexpression on the biological behavior of 786-O and ACHN cells and its molecular mechanism.4.To study the effect of down-regulation of FAM129A on biological behavior of 786-O and ACHN cells and its molecular mechanism.Methods:1.qRT-PCR was used to detect the relative expression of miR-429,WB and IHC were used to detect the expression of FAM129A in clinical samples of ccRCC.2.qRT-PCR was used to detect the expression of miR-4521 and FAM129A in normal renal epithelial cells?HK-2?and renal clear cell carcinoma cells?786-O and ACHN?.3.qRT-PCR and WB was used to detect the expression of mi R-4521 and FAM129A after miR-4521 mimic transiently transfeced in 786-O and ACHN cells.4.The recombined vector was transfected into 786-O cells with miR-4521 mimic/NC mimic and the activity value of double luciferase was detected to verify the target relationship between miR-4521 and FAM129A.5.MTT assay determined the effects of mi R-4521overexpression on the proliferation ability of 786-O and ACHN cells;Transwell assay measured the effects of miR-4521 overexpression on the migration and invasion capabilities of 786-O and ACHN cells;Flow cytometry was used to measure the effect of miR-4521 overexpression on apoptosis of 786-O and ACHN cells;WB was used to detect the expression of TIMP-1,MMP2,MMP9,MDM2,P53,Bcl2 and Bax after miR-4521 overexpression.6.MTT assay determined the effects of down-regulation of FAM129A on the proliferation ability of 786-O and ACHN cells;Transwell assay measured the effects of down-regulation of FAM129A on the migration and invasion capabilities of 786-O and ACHN cells;Flow cytometry was used to measure the effect of down-regulation of FAM129A on apoptosis of 786-O and ACHN cells;WB was used to detect the expression of TIMP-1,MMP2,MMP9,MDM2,P53,Bcl2 and Bax after down-regulation of FAM129A.Results:1.mi R-4521 expression were decreased in ccRCC tissues compared to paired normal tissues and its low expression was associated with advanced T stage and higher Fuhrman grade;FAM129A mRNA and protein expression levels were increased in ccRCC tissues compared to paired normal tissues and its high expression was associated with advanced T stage and higher Fuhrman grade;There were the negative correlation between miR-4521 and FAM129A.FAM129A protein was primarily located in cytoplasm.2.miR-4521 expression were decreased and FAM129A were increased in786-O and ACHN cells compared to HK-2.3.After transient transfection of mi R-4521mimic,miR-4521 was up to 1820 times?P=0.01?and 920?P=0.02?times in 786-O and ACHN cells respectively;FAM129A mRNA and protein expression levels were increased in ccRCC tissues compared to NC mimic group.4.Dual luciferase reporter gene assay showed that the detection activity of the two groups was not significantly different with NC mimics or miR-4521 mimic co-transfection in psiCHECK^TM2.0-mutFAM129A-3'UTR group;But in psiCHECK^TM2.0-wtFAM129A-3'UTR group,the activity was decreased by 38.2%in the miR-4521 mimic group compared to the NC mimics group?P=0.0022?.5.miR-4521 overexpression inhibited the proliferation,migration and invasion of 786-O and ACHN cells,and promoted the apoptosis of 786-O and ACHN cells;miR-4521 overexpression promoted the expression of TIMP-1,p53,and Bax to increase and inhibited the expression of MMP2,MMP9,MDM2,and Bcl2.6.Down-regulation of FAM129A inhibited the proliferation,migration and invasion of786-O and ACHN cells,and promoted the apoptosis of 786-O and ACHN cells;Down-regulation of FAM129A promoted the expression of TIMP-1,p53,and Bax to increase and inhibited the expression of MMP2,MMP9,MDM2,and Bcl2.Conclusion:1.The low expression of mi R-4521 and the high expression of FAM129A are closely related to the development of ccRCC,and their expression levels is negatively correlated.FAM129A protein was primarily located in cytoplasm.2.miR-4521 negatively regulates FAM129A expression by binding to FAM129A 3'UTR region.3.Up-regulation of miR-4521 could inhibit the migration and invasion of 786-O and ACH cells by decreasing MMP2 and MMP9 and increasing TIMP-1;It could promote the apoptosis and inhibit the proliferation of 786-O and ACH cells by increasing P53 and Bax and decreasing MDM2 and Bcl2.4.Down-regulation of FAM129A could inhibit the migration and invasion of 786-O and ACHN cells by decreasing MMP2 and MMP9 and increasing TIMP-1;It could also promote the apoptosis and inhibit the proliferation of 786-O and ACH cells by increasing P53 and Bax and decreasing MDM2 and Bcl2.Taken together,mi R-4521 negatively regulated the expression of FAM129A,which affected the expression of TIMP-1,MMP2,MMP9,P53,MDM2,Bcl2 and Bax,and then influences the proliferation,migration,invasion and apoptosis of 786-O and ACHN cells.