Expression Of G1 Phase Protein In Retinoblastoma Cell Line And Its Mechanism Research

Background and purpose:Retinoblastoma(RB)is an intraocular tumor that often occurs in children,which seriously affects the vision even life of patient.In recent years,studies have found that cyclins are closely related to the occurrence and development of many human tumors,especially the G1 phase-associated protein has been reported to be highly expressed in a variety of human tumors.Tumor cells were divided into RB~+cells and RB~-cells according to the expression of RB protein.Studies have shown that knocking down Cyclin D3-CDK6 expression in various tumors can promote cell apoptosis,which is related to RB~+and RB~-.However,there is no research report on the mechanism for action of G1 phase-associated cyclin and CDK in RB cell lines.Therefore,this study is to explore the expression of RB protein and G1 phase protein in RB cell lines,and its impact in cell apoptosis,migration and invasiveness.Methods:Real-time PCR and Western Blot were used to detect the expression of Cyclin D1,Cyclin D2,Cyclin D3,Cyclin E1,Cyclin E2,CDK2,CDK4 and CDK6 in four common RB cell lines(WERI-RB1,SO-RB50,HXO-RB44 and Y79 cell lines).The expression levels of RB proteins in various RB cells were analyzed.After infected WERI-RB1,SO-RB50,HXO-RB44 and Y79 cell lines with the lentiviral plasmids containing CDK2,CDK4 and CDK6 interfering sequences.Transwell assay was used to identify the effect of migration and invasion of WERI-RB1,SO-RB50 and HXO-RB44cell lines after the knockdown of CDK2,CDK4 and CDK6.In addition,Flow cytometry was used to explore the effects of apoptosis in WERI-RB1 after CDK2,CDK4 and CDK6 gene knockdown.Results:In this study,the results showed that WERI-RB1,SO-RB50 and Y79 cell lines were RB~+cell lines,while HXO-RB44 was RB~-cell line.Cyclin D3 is not only highly expressed in RB~+cell lines(WERI-RB1,SO-RB50 and Y79)but also in RB~-cell lines(HXO-RB44)(P<0.05);Cyclin E1 is highly expressed in HXO-RB44 cell lines(P<0.05);whereas CDK6 and Cyclin E2 are only high expressed in WERI-RB1 and SO-RB50 cell lines(P<0.05).After successfully knockdown CDK2,CDK4 and CDK6in WERI-RB1,SO-RB50,HXO-RB44 and Y79 cell lines,respectively,the apoptosis was significantly higher in WERI-RB1 cell line after knocking down the expression of CDK2,CDK4 and CDK6,and SO-RB50 with HXO-RB44 showed a certain degree of reduction in migration and invasion abilityConclusion:The current study found that Cyclin D3 is abnormally highly expressed in WERI-RB1,SO-RB50,HXO-RB44 and Y79 cell lines;whereas CDK6and Cyclin E2 are highly expressed only in WERI-RB1 and SO-RB50 cell lines,and Cyclin E1 is highly expressed only in HXO-RB44.Knockdown the expression of CDK2,CDK4 and CDK6 can significantly increase the apoptosis of WERI-RB1 and reduce the migration and invasion ability of SO-RB50 and HXO-RB44 cell lines.