Moleculer Mechanism Research Of Overexpression Of TFE3 Gene In Xp11.2 Translocation Renal Cell Carcinoma

Backround and Objective:Xp11.2 translocation renal cell carcinoma was delineated as a new disease in WHO renal tumor classification scheme,while often present onset age is light and prognosis is poor.The main characteristics are TFE3 gene fractured and translocated,which lead to TFE3 overexpression.But TFE3 gene overexpressed on renal carcinoma cell lines remained unclear and the signal pathway need to be researched.We attemped to explore and study the mechanism,providing theoretical basis for the diagnosis and treatment of this carcinoma.Objective:1.To screen one of the renal cell carcinoma cell lines suitable for constructing lentiviruses that overexpress TFE3 gene.Constructed specific lentivirus and detected drop degree.2.To investigate the effects of overexpression of TFE3 on proliferation,colony formation and cell cycle by constructing overexpressing vectors and infecting renal cancer cell.3.To investigate the expressions of mTOR and pS6 in Xp11.2 translocation renal cell carcinoma and non-Xp11.2 translocation renal cell carcinoma,and to detect their expressions on cell line overexpressing TFE3 gene.4.To detect differential genes associated with Xp11.2-translocated renal cell carcinoma by means of Affymetrix human microarray so as to screen more accurate therapeutic targets.Methods:1.The expressions of TFE3 in 8 renal cell carcinoma cell lines were detected using Real-time PCR and Western blot,and one cell line was selected for subsequent experiments.Construct specific lentivirus transfection overexpression vector and identify its infection efficiency.2.Lentivirus transfection was used to construct the TFE3-overexpressing group(OE),the negative control group(NC)and the empty control group(CON),and the expression of TFE3 was verified by Western blot.3.Cell proliferation and colony formation were detected using MTT assay and platelet cloning assay,and cell cycle was detected by flow cytometry.4.The expressions of mTOR and pS6 in Xp11.2 translocation renal cell carcinoma and non-Xpll.2 translocation renal cell carcinoma were detected by immunohistochemistry in paraffin specimens,and their differences in different experiment groups were detected using Western blot.5.Genomic microarray was used to detect the differentially expressed associated genes in Xp11.2 translocation renal cell carcinoma patients,and the changes in gene expression profile between cancer tissues and adjacent tissues were analyzed by Affymetrix human microarray.And a comprehensive analysis and comparison were conducted to detect the gene expression differences between cancer tissues and paracancerous tissues.Results:1.Real-time PCR results showed that the mRNA of the TFE3 gene was moderate in ACHN,and Western blot results showed that no TFE3 target bands were produced in ACHN.Therefore,the ACHN cell line was selected as the target cell line for lentiviral transfection.At the same time,the human TFE3 gene-specific recombinant lentiviral vector was constructed successfully and the corresponding lentivirus was obtained.2.In the experimental group,the virus drops degree of lentiviral vector was 2×108 TU/ml,MOI=10.95kDa band of antibody fusing with TFE3 was detected in the OE group,which was consistent with the results of plasmid expression detection.This indicated that the TFE3-overexpressing vector was successfully transfected into ACHN cells.The proliferation level of cells in the OE group was significantly higher than that in the NC group(P<0.05),indicating that overexpression of TFE3 could promote the proliferation of ACHN cells.The number of OE cell clones was significantly increased compared to the NC group(P<0.05),proving that overexpression of TFE3 promoted the clone formation of ACHN cells.The cell cycle was detected using flow cytometry.Compared with the NC group increase in S phase was noted in the OE group(P<0.05),indicating that overexpression of TFE3 could regulate the cycle of ACHN cells.3.The positive rate of mTOR and pS6 in Xp11.2 translocation renal cell carcinoma was significantly higher than that in the non-Xp11.2 translocation renal cell carcinoma(P<0.05),and the expressions of the two proteins in TFE3-overexpressing cells were significantly increased(P<0.05).The results suggested that the Xp11.2 translocation renal cell carcinoma excessive activation of PI3K/AKT/mTOR pathway in histology and cytology level.4.Genomic microarray was used to detect the differentially expressed associated genes in Xp11.2 translocation renal cell carcinoma patients.A total of 1479 differentially expressed genes were screened,including 790 up-regulated genes and 689 down-regulated genes in cancer tissues.Specifically,RNF180 gene related to ubiquitination was down-regulated at least 5-fold,which might become a new research target and suggested the development of Xp11.2 translocation renal cell carcinoma may relate to UPP pathway.Conclusions:1.Overexpression of TFE3 can promote proliferation of ACHN cells and regulate their cell cycle.2.Xp11.2 translocation renal cell carcinoma exists in PI3K/AKT/mTOR pathway over activation.3.Xp11.2 translocation renal cell carcinoma is associated with UPP pathway.