Preliminary Molecular Mechanism Of SR-B1 Affecting The Proliferation Of Renal Clear Cell Carcinoma By HIF-2α In HIF Pathway Through ERK

Objective: The basis of previous work showed that SR-B1(Scavenger receptor class B type 1,SR-B1)was overexpressed in clear cell renal cell carcinoma(cc RCC).Its overexpression is positively correlated with poor patient prognosis.In vitro experiments,overexpression of SR-B1 could significantly promote tumor cell proliferation,and the results of expression profiling and protein profiling chip screening showed that inhibition of SR-B1 could down-regulate the HIF pathway,which may play a role through ERK.Therefore,the purpose of this study is to investigate whether SR-B1 can affect HIF-2α through ERK to promote the proliferation of renal clear cell carcinoma on the basis of previous work.Methods:(1)The gene expression profiling database was used to predict whether SR-B1,ERK/MAPK pathway key molecule ERK and HIF pathway key molecule HIF-2α were correlated in cc RCC.Cell immunofluorescence(IF)and co-immunoprecipitation(Co-IP)were used to verify the predicted results and observe whether the three were combined with each other;(2)The bioinformatics database was used to predict the expression distribution of HIF-2α in cc RCC,and the prediction results were verified by immunohistochemistry(IHC)and Western Blot experiments;(3)SR-B1 was overexpressed in normal renal tubular epithelial cells HK-2,which is a cell line with low SR-B1 expression,and the protein expressions of ERK,p-ERK and HIF-2α were observed;SR-B1 was silenced in cc RCC 786-0 and Caki with high SR-B1 expression,which are cell lines with high SR-B1 expression,and the protein expressions of ERK,p-ERK and HIF-2α were observed;(4)When SR-B1 was overexpressed in HK-2,cells were treated with a gradient concentration of ERK-specific inhibitor(SCH772984),and the protein expressions of SR-B1,ERK,p-ERK,HIF-2α and downstream pathway molecule VEGF were detected before and after drug treatment;While SR-B1 was silenced in 786-0 and Caki,cells were treated with a gradient concentration of ERK agonist(EGF),and the protein expressions of SR-B1,ERK,p-ERK,HIF-2α and the downstream pathway molecule VEGF were detected before and after drug treatment;(5)While overexpressing SR-B1 in HK-2,cells were treated with a gradient concentration of ERK-specific inhibitor(SCH772984),and CCK-8 and cell cloning experiments were used to detect and compare the proliferation ability of cells before and after drug treatment;While SR-B1 was silenced in 786-0 and Caki,cells were treated with gradient concentrations of ERK agonist(EGF),and the proliferation ability of cells before and after drug treatment was detected and compared by CCK-8 and cell cloning experiments.Conclusion:(1)Bioinformatics analysis showed that SR-B1,ERK and HIF-2α protein expressions were positively correlated in renal clear cell carcinoma cells,and the three could be combined with each other(P<0.001 or P<0.01);(2)HIF-2α is highly expressed in renal clear cell carcinoma(P<0.05),and patients with high expression of HIF-2αhave poor survival prognosis(P<0.0001 or P<0.05);(3)Overexpression of SR-B1 can up-regulate the protein expressions of ERK,p-ERK and HIF-2α(P<0.001),and silencing SR-B1 can down-regulate the protein expressions of ERK,p-ERK and HIF-2α(P<0.001);(4)Overexpression of SR-B1 can significantly increase the protein expressions of ERK,p-ERK,HIF-2α and VEGF in HK-2 cells(P<0.001).ERK-specific inhibitor(SCH772984)can reverse the up-regulation effect of SR-B1 on ERK,p-ERK,HIF-2α and VEGF protein(P<0.00);Silencing of SR-B1 significantly reduced the protein expressions of 786-0,ERK,p-ERK,HIF-2α and VEGF in Caki cells(P<0.001).ERK agonists can reverse the down-regulation effect of silencing SR-B1 on ERK,p-ERK,HIF-2α and VEGF proteins(P<0.001);(5)Overexpression of SR-B1 can increase the proliferation ability of HK-2 cells(P<0.01 or P<0.05),and the cell proliferation ability is inhibited by adding ERK inhibitor(P<0.01 or P<0.05);Silencing SR-B1 can attenuate the proliferation of 786-0 cells(P<0.01 or P<0.05),and ERK agonists can reversely inhibit the ability of silencing plasmids to attenuate cell proliferation(P<0.01 or P<0.05).Results: SR-B1 may affect HIF-2α through ERK to promote the proliferation of renal clear cell carcinoma cells.