Role Of ETV6,miR-200 Family And CRKL Feedback Loop On Metastases Of Renal Clear Cell Carcinoma

Background :Renal cell carcinoma(RCC)is a common malignant tumor of high morbidity in the urinary tract,accounting for 2%-3% of adult malignancies.Clear cell renal cell carcinoma(ccRCC)is the most common and most aggressive type of renal cell carcinoma which has no other effective treatment except surgery,but the recurrence rate of postoperative patients is high,accounting for about 20%-40%.Therefore,the study on the tumorigenesis and metastasis of ccRCC has important practical significance.CRK is a type of adaptor protein originally found in chicken tumor 10 virus as an oncogene product and mainly composed of SH2 and SH3 domains.CRKL is a member of the CRK family and is widely expressed in various tissues.Recent studies have shown that: abnormal expression of CRKL is closely related with tumor biological behavior.However,there are few researches on the pathogenesis of CRKL and the possible signaling pathways in renal clear cell carcinoma and that deserve further study.MicroRNAs(miRNAs)are a class of single-stranded,non-coding RNAs that contain 21-25 bases.miRNAs combine with AGO2 to form RNA-induced slicencing complex(RISC),which in turn binds to the 3'-UTR region of the target gene to cause degradation of the target gene mRNA or to inhibit its translation process.The miR-200 family includes miR-200 a,miR-200 b,miR-200 c,miR-429,miR-141.Studies have found that it is involved in the regulation of tumor metastasis as a tumor suppressor in a variety of cancers.However,the miR-200 family is rarely reported in renal cancer and deserves further study.In TargetScan bioinformatics software,it is found that the miR-200 family has a targeted relationship with CRKL 3'-UTR.Therefore,the relationship between mi R-200 family and CRKL in renal clear cell carcinoma is worth further investigation.ETV6,also known as TEL,is a member of the transcription factor ETS family and plays an important role in the development of the disease.Through bioinformatics software analysis,we found that it can regulate the transcription of miR-200 family promoter region.Objective: 1.To explore the relationship between CRKL expression and renal clear cell carcinoma,786-O,ACHN cells;2.to explore the relationship between miR-200 family and clear cell renal cell carcinoma;3.To explore the relationship between CRKL and miR-200 family;4.How does ETV6 regulate miR-200 family and CRKL in clear cell renal cell carcinoma.Methods: 1.The miR-200 family mimic and NC-mimic negative control group were transfected by LipofectamineTM2000,and the transfection efficiency was detected by qRT-PCR.2.To detect786-O and ACHN cell migration and invasion capacity by transwell assay after transfecting miR-200 family,detect CRKL and EMT related molecules expression by WB assay;3.dual luciferase vectors construction of psiCHECK-mutCRKL-site1,psiCHECK-mutCRKL-site2,psiCHECK-wtCRKL-site1 and psiCHECK-wtCRKL-site2,detect the targeting effect of miR-200 family on CRKL.4.The expression of mi R-200 family was detected by qRT-PCR after transfecting.5.To detect786-O and ACHN cell migration and invasion capacity by transwell assay after transfecting si-ETV6.6.To detect CRKL and EMT related molecules expression by WB assay after transfecting siETV6.7.IP,WB assay to detect whether CRKL and ETV6 can direct bonding,to explore the relationship between the CRKL,miR-200 family and ETV6.8.The relative RNA levels of miR-200 family,CRKL and ETV6 in renal clear cell carcinoma samples were detected by qRT-PCR assay.The relative protein levels of CRKL,ETV6 and EMT-related molecules were detected by WB assay.Results: 1.Transfected with miR-200 mimic can inhibit migration and invasion ability in 786-O and ACHN cells,of which miR-429 and miR-200 c inhibit the most obvious effect;2.Transfected with miR-200 mimic can inhibit the protein level of CRKL and inhibit the signal pathway of CRKL / SOS1 / Ras/ Raf / MMP9 by WB assay,of which the inhibitory effect of miR-429 and miR-200 c is the most obvious.3.The successful construction of psiCHECK-mutCRKL-site1,PsiCHECK-mutCRKL-site2,psiCHECK-wtCRKL-site1,psiCHECK-wtCRKL-site2 dual luciferase vectors;4.Dual luciferase reporter assay found that miR-429 and miR-200 c can target CRKL3 '-UTR region.5.Transfected with si-ETV6 can inhibit the relative expression of mi R-200 family and inhibit migration and invasion ability of 786-O and ACHN cells.6.IP assay show that CRKL can directly bind ETV6 and found that transfected with si-CRKL can reduce the expression of CRKL-ETV6 complex in the nucleus and inhibit the expression of miR-200 family.7.WB assay showed that CRKL and ETV6 protein were highly expressed in 40 patients of renal clear cell carcinoma sample,qRT-PCR Detection of 26 patients of renal clear cell carcinoma clinical samples,miR-429 and mi R-200 c are low expression,no correlation with CRKL and ETV6 mRNA levels,but a negative correlation with CRKL and ETV6 protein levels.