The Effect Of HIF-1? Silencing On Oxidative Stress Induced Autophagy And Apoptosis In Renal Carcinoma A498 Cells

Objective:To study the molecule mechanism of autophagy and apoptosis in renal carcinoma cells A498 cells under oxidative stress induced by hydrogen peroxide.The foundation was could be provided for new therapeutical methods of renal carcinoma.Methods:1.A498 cells were treated with different concentration hydrogen peroxide?from 50?M to 400 ?M?for different time?from 2 hous to 8 hours?.The acitivity of CCK-8 was detected and the amount of ROS cells were observed for screening the best concentration and the best treatment time of drug for the cellular oxidative stress model induced by H₂O₂.The level of LC3 ?,p62 or HIF-1? protein of A498 cells treated with 200 ?M hydrogen peroxide was measured by western blot.2.The shRNA sequence of homo HIF-1? gene was designed and synthesized.And the recombinant plasmids were constructed and identified by enzymes.The plasmid and envelope virus were co-transfected into 293T cells.The supernatant of infected 293T cells was collected and transfected into A498 cells.The stable A498 shHIF-1?cell strain was screened by puromycin for two weeks.The expression of gene at mRNA or protein level was detected by QRT-PCR or western blot.3.The expression level of LC3 or p62 protein was measured of above stable transfected cell line under 200 ?M H₂O₂ for 6 hours.The level of HIF-1? or autophagic related proteins?LC311 and p62?of A498 cells was detected respectively,which the cells treated with 3-?5'-hydroxymeethyl-2'-furyl?-1-benzylindazole?YC-1,specific inhibitor of HIF-1?,final concentration was 50 ?M?or N actylcysteine?NAC,anti-oxidative reagent,final concentration was 100 mg/ml?,or combined with H₂O₂.The expression level of cleaved-PARP of A498 shHIF-1? cell strain under 200 ?M H₂O₂ for 6 hours was measured respectively.4.The shRNA sequence of homo LC3 gene was designed and the recombinant plasmids were constructed.The stable A498 shLC3 cell strain was screened by drug and identified.The expression level of cleaved-PARP of cells was detected respectively with 200 ?M hydrogen peroxide.And the level of apoptosis proteins of A498 cells was measured respectively treated with 3-methyl adenine?3-MA,autophagyc inhibitor?or rapamycin?Rapa,autophagic inducer?.5.Data was analysed by the software of SPSS 17.0.Analysis of variance in repeated measurement data or one-way anova test in single time-point results was performed.Results:1.The activity of A498 cell was decreased by 23.9%,58.5%,72.8%,86.7%,96.6%,or 97.7%treated with 50,100,150,200,300,or 400 ?M hydrogen peroxide for 12 h respectively compared with controls.And the percent of ROS cells was 19.8,32.6,35.5,or 83.8 with 200 ?M H₂O₂ for 2,4,or 8hours respectively.The best concentration of hydrogen peroxide was 200 ?M in this test.2.The expression level of LC3? or p62 protein in A498 cells was increased 2.1,3.7,4.5 times or decreased 23.4%,30.1%,52.3%treated with 200 ?M H₂O₂ for 2,4,8 hours respectively compared with that of untreated cells.And the level of HIF-1 a protein was magnified 44.5%,93.3%,140%respectively compared with that of untreated group.3.The stable A498 shHIF-1? cell strain was constructed and screened.Theexpression level of LC3? of control or test was elevated by 1.06 or 1.03 times respectively under hydrogen peroxide for 6 hours compared with that of untreated cells,and the magnification of test group was low by 19.4%copared with that of control group?p value is 0.035?.However,The expression level of p62 of control or test was decreased by 25.0%or 13.8%respectively under the same treatment,and the magnification of test group was low by 72.8%copared with that of control group?p value is 0.027?.4.The expression level of p62 was down-regulated 38.7%,and the level of LC3 ?or HIF-1? was not changed of A498 cells with hydrogen peroxide single compared that of cells with hydrogen peroxide combined NAC?p<0.0001?.However,the level of p62 was up-regulated 18.7%,and the level of LC3 ? or HIF-1? was down-regulated 17.4%or 5.8%of A498 cells with hydrogen peroxide single compared that of cells with hydrogen peroxide combined with YC-1 pretreated?p<0.05?.5.The expression level of cleaved-PARP ofA498 shHIF-1? cellstreated with hydrogen peroxide for 6 or 12 hours respectivelywas decreased by 36.2%or 21.0%,compared with that of control.6.The expression level of cleaved-PARP was increased 7.3 or 14.2 times,however,the expression of caspase3,caspase8,or caspase 12 was not shown significant change in cells which human LC3 gene was knockdown by shRNA,treated with H₂O₂ for six,or twelve hours respectively compared with that of control.The expression of cleaved-PARP of A498 cells was down-regulated 62.1%for pretreating with 3-MA and H₂O₂,but that was up-regulated 92.6%for with rapamycin and H₂O₂ compared with that of control group.Conclusion:1.The autophagy of A498 cells was induced and the expression level of HIF-1? was up-regulated in a time-dependent manner treated with 200 ?M hydrogen peroxide.2.The expression level of LC3? of A498 cells was decreased,and the level of p62 was increased after HIF-1? gene was knockdown by shRNA.Autophagy in A498 cells was down-regulated by HIF-1? protein that was inhibited specificly with YC-1,however,autophagy was partial inhibited by ROS which was reduced with NAC.These results demonstrated that HIF-1? was correlated with autophagy tightly.3.Apoptosis could be caused by oxidative stress which induced with hydrogen peroxide in A498 cells,but apoptosis was alleviated by HIF-1? gene was knock-down by shRNA.And the apoptosis of cells was also abated by LC3 gene was interfered by shRNA.This suggested that oxidative stress induced autophagy mediated by HIF-1? could promot the apoptosis of A498 cells.