CXCL5 Promotes Progression Of Renal Cell Carcinoma By Inhibiting Autophagy

BackgroundRenal cell carcinoma as one of the most common malignant tumor in urinary system,easy to relapse or metastasis,and is not sensitive to radiation therapy,chemotherapy,and the efficient and duration of targeted therapy is unsatisfactory.In the early of tumor,autophagy can degrade cell synthesis and inhibit tumor proliferation.It was found that autophagy was inhibited in renal cell carcinoma cell lines and tumor tissues,and autophagy level was negatively correlated with tumor stage and grade,suggesting that autophagy may play a role in inducing tumor death.Interestingly,chemokines have recently been shown to be able to influence the growth and survival of breast cancer cells by negatively regulating autophagy.The project team leader preliminary study found that,the PD3CR1(Fractalkine receptor),platelet-derived growth factor(PDGF-B)affect the progression and metastasis of renal cell carcinoma by regulating tumor proliferation,migration and angiogenesis,and the expression level of chemokine receptor has potential value in predicting the prognosis of patients.Therefore,this project was designed to further investigate the role of CXCL5 in the development and progression of renal cell carcinoma and cells,and to reveal whether CXCL5 can affect the progression of renal cell carcinoma by regulating tumor autophagy.Method1.Immunohistochemistry staining of paraffin-fixed sections was performed to detect the expression of CXCL5 from patients with renal cell carcinoma.The patients were divided into low,middle and high expression groups according to the expression of CXCL5.The relationship between the expression of CXCL5 and the prognosis of patients was analyzed by indepth review of the CbioPortal database.2.The expression of CXCL5 in three renal cell lines was confirmed by qRT-PCR.The stable expression of CXCL5 was induced by lentiviral infection.The CXCL5 cell line was knocked out and the CXCL5 was knocked out.The effect of stable expression on cell morphology,proliferation and angiogenesis.3.We used to construct a model of subcutaneous transplanted tumor in nudemice to verify the growth of shRNA-CXCL5 tumor and verify the effect of knockdown of CXCL5 on the growth of renal cell carcinoma.4.The expression of EMT-related protein in tumor cells was detected by Western Blot.The expression of CXCL5 was detected by co-transfection of plasmids of LC3 with GFP and plasmids of shR-CXCL5 or control plasmid.The expression of PI2 K /AKT / mTOR The impact of the pathway.The results1.CXCL5 is expressed in tumor tissues of renal cell carcinoma and has important clinical significance.In this study,According to the expression of CXCL5,the patients were divided into low,middle and high expression groups.The clinicopathological data were collected and the expression of the patients was evaluated.(P <0.05),The Data from the TCGA showed that the overall survival(OS)and disease free survival(DFS)of CXCL5 overexpressing RCC patients were significantly shorter than those of patients with RCC with low expression of CXCL5(P <0.05).This also suggests that our CXCL5 plays an important role in RCC tumor growth.2.The effect of CXCL5 on the biological behavior of renal cell carcinoma cells.2.1 Expression of CXCL5 in Renal Cell Carcinoma CellsWe used qRT-PCR to verify the expression of CXCL5 in three renal cell lines(786-O,A498 and Caki-1 cell lines).The results showed that the expression of CXCL5 in renal cell carcinoma was higher than that in normal More cells.2.2 The effect of CXCL5 expression on in vitro cellsIn order to clarify the role of CXCL5 in tumor cells,we used the lentivirus infection to construct a stable CXCL5 cell line and knock down the stable expression cell line of CXCL5.We evaluated the down-expression of knockout CXCL5 for cell morphology,The effect of angiogenesis.Experiments show that the use of shRNA knockdown CXCL5 A498 cells on the appearance of aging,necrosis,cell proliferation ability decreased significantly,and shRNA-CXCL5 interference of tumor cells to promote the ability of endothelial cells into a significant decline.Apoptotic experiments showed that knockdown of CXCL5 could increase the early apoptosis of tumor cells.2.3 CXCL5 on the growth of renal cell carcinoma cells in vivoWe used the nude mice to observe the growth of shRNA-CXCL5 group.The expression of shRNA-CXCL5 group was significantly inhibited.The tumor tissue of nude mice was immunohistochemically verified that the angiogenesis and shRNA formation of shRNA-CXCL5 group were significantly inhibited The2.4 Effect of CXCL5 on EMT in Renal Cell CarcinomaThe expression of EMCL-related protein in tumor cells was detected by Western Blot.The expression of CXCL5 and E-cadherin was decreased and the expression level of Vimentin was increased.The expression of CXCL5 and E-cadherin was increased and the expression of Vimentin Level down.3.CXCL5 regulates the role and mechanism of autophagy in renal cell carcinomaWe stained the plasmid of LC3 expressed with GFP and stained with shR-CXCL5 plasmid or control plasmid.The results showed that knocking down CXCL5,cell autophagy increased.We used Western blot to detect the effect of CXCL5 on PI3 K / AKT / mTOR signaling pathways.Indicating that knockdown of CXCL5 can inhibit PI3 K and AKT protein phosphorylation levels.ConclusionsThis study proves that the expression level of CXCL5 in RCC cells is positively correlated with tumor staging and tumor T staging,in RCC cell line CXCL5 can up-regulate the expression of CXCL5,and knockout CXCL5 can affect the biological behavior of tumor cells in vivo and in vitro,including inhibition of proliferation and migration,promote apoptosis and inhibit angiogenesis;expression of CXCL5 RCC cell line autophagic effect was significantly inhibited,And knockout CXCL5 can lift the state of autophagy;CXCL5 regulation of autophagy mechanism may be related to PI3 K / AKT pathway.