The Expression Of ST6GalⅠ In Clear Cell Renal Cell Carcinoma And Its Effect On The Growth And Apoptosis Of Cancer Cells

Objective:1)Immunohistochemical method was used to detect the expression of humanβGalactosideα-2,6-sialyltransferase I(ST6Gal I)in clear cell renal cell carcinoma(cc RCC)and normal renal tissues,and to explore the correlation between the expression of ST6Gal I and the pathological characteristics of cc RCC.2)To study the effect of ST6Gal I on the polarization of macrophages and explore its role in renal clear cell carcinoma。3)The expression of ST6Gal I and TNFR1 was detected by q RT-PCR and Western-blot and analysis the correlation of ST6Gal I and TNFR1,to Explore the effect and mechanism of ST6Gal I on the apoptosis of renal clear cell carcinoma cells.Methods:1)127 cases of cc RCC patients who came to the hospital from January 2013 to December2019 were collected as the research object.Immunohistochemical methods were used to detect the expression of ST6Gal I in cancer tissues and normal kidney tissues of the same patient.To analyze the differences in the expression level of ST6Gal I in cancer tissues and normal kidney tissues and in different pathological characteristics,the comparison of the positive expression rates of ST6Gal I between the two groups and the comparison of the relationship between the expression of ST6Gal I and clinical pathological characteristics were performed by theχ~2test.2)Transfect si RNA-ST6Gal I to THP-1 cells by liposome method,50 ng/m L PMA and 20 ng/m L IL-4 induce the transfection of THP-1 cells,Real-time fluorescence quantitative PCR and flow cytometry were used to detect the polarization of macrophages.Establish a co-culture system of macrophages and786-O cells,MTT method and flow cytometry to detect cell survival and apoptosis,cell scratch test to detect cell migration changes,ELISA to detect inflammation-related factors in the cell supernatant,Western Blot was used to detect the expression of T cell immunoglobulin mucin-1(TIM-1)and vascular endothelial growth factor(VEGF)in the cells.3)q RT-PCR and Western blot detect the expression of ST6Gal In renal clear cell carcinoma cell lines,then construct a renal clear cell carcinoma cell line with differential expression of ST6Gal I and use flow cytometry to detect the level of apoptosis;q RT-PCR and Western blot Detect the expression of TNFR1 in renal clear cell carcinoma cell lines,and analyze the correlation between the expression of ST6Gal Iand TNFR1,q RT-PCR and Western blot detect the expression of TNFR1 in cell lines that differentially express ST6Gal I;inhibit the expression of ST6Gal In A498 cells At the same time,the expression of TNFR1 was inhibited,and ST6Gal I was over expressed in Caki-1 cells at the same time that TNFR1 was over expressed,and then the apoptosis level of each group of cells was detected.Results:1)The results of immunohistochemistry showed that the expression level of ST6Gal In cancer tissues of patients included in the study was significantly higher than that in normal kidney tissues,and the difference was statistically significant(P<0.05);different tumor stages and tumor sizes(≤7cm and>7cm)and tumor grades(medium-poorly differentiated,well-differentiated)in the positive rate of ST6Gal I expression in cc RCC tissues are statistically significant(P<0.05);2)compared with the control group,the relative expression of ST6Gal I m RNA and protein in THP-1cells in the si RNA-ST6Gal I group were significantly decreased(P<0.05);Compared with the control group,after induction,the expression of Arg-1 m RNA increased,the expression of i NOS m RNA decreased,and the expression of CD206 increased(P<0.05),while in THP-1 cells induced by si RNA-ST6Gal I transfection Arg-1 m RNA expression decreased,i NOS m RNA expression increased,CD206 expression decreased(P<0.05);after 786-O cells were co-cultured with induced THP-1 cells,cell survival rate increased,migration ability increased,and apoptosis Rate drops.The contents of IL-1β,IL-6 and TNF-αin the cell supernatant decreased,the contents of IL-10,TGF-β1 increased,the expression of TIM-1 protein decreased,and the expression of VEGF protein increased(P<0.05);After 786-O cells are co-cultured with THP-1 cells that down-regulate ST6Gal I and induce them,cell survival rate decreases,migration ability decreases,and apoptosis rate increases and decreases.At the same time,the levels of IL-1β,IL-6 and TNF-αin the cell supernatant increased,the levels of IL-10,TGF-β1 decreased,the expression of TIM-1 protein increased,and the expression of VEGF protein decreased(P<0.05).3)ST6Gal I is highly expressed in 5 renal clear cell carcinoma cell lines,and is highest in A498 cells and lowest in Caki-1 cells.After inhibiting ST6Gal In A498 cells,cell apoptosis is significantly increased,and in Caki-1 Cells over expression of ST6Gal I significantly reduced apoptosis;TNFR1 was expressed in 5 renal clear cell carcinoma cell lines,and the expression was negatively correlated with the expression of ST6Gal I;and after ST6Gal I was inhibited,the expression of TNFR1 increased.After expression of ST6Gal I,the expression of TNFR1 decreased;when the expression of ST6Gal I was inhibited in A498 cells and the expression of TNFR1 was inhibited,the level of cell apoptosis was significantly lower than that of the ST6Gal I group alone.When ST6Gal I was over expressed in Caki-1 cells,the level of apoptosis was significantly lower.After over expression of TNFR1,the level of cell apoptosis was significantly higher than that of the ST6Gal I group alone.Conclusions:1)The expression level of ST6Gal I in renal clear cell carcinoma tissues was significantly higher than that in normal kidney tissues.The tumor size was greater than 7cm,and the stage was stage 2.The positive rate of ST6Gal I expression in the tumor graded as moderately and poorly differentiated was high.2)Down-regulating the expression of ST6Gal I can inhibit the polarization of THP-1 cells to M2 macrophages,thereby inhibiting the growth and migration of renal clear cell carcinoma cells,and regulating the expression of inflammatory factors and immune-related proteins TIM-1 and VEGF.3)ST6Gal I can inhibit the apoptosis of renal clear cell carcinoma cells by inhibiting the expression of TNFR1.