| Objective:To investigate the mechanism of effect of SIRT6 on proliferation,apoptosis and drug resistance of glioblastoma U87 MG cells by regulating HIF-1α and glycolytic related protein expression under hypoxia condition.Methods:(1)The protein expression of Sirtuin 6(SIRT6)in glioblastoma cells U251,U87 MG,LN229 and human astrocyte NHA was detected by western blot(WB),and the required cell lines were screened.(2)the application of cobalt chloride(Co Cl2)build U87 MG cells in hypoxia model,using the WB experimental detection of oxygen treatment of SIRT6 U87 MG cells,protein involved in glycolysis,glucose transporter protein 1(GLUT1),pyruvate kinase isoform 2(PKM2),lactate Dehydrogenase A,LDHA)and the expression levels of hypoxia induced transcription factor1α(HIF1α).SIRT6 stable overexpression and silencing cell lines were constructed,and named as OE-NC(overexpression control),OE-SIRT6,sh-NC(silencing control)and sh-SIRT6 cells,respectively.Cell counting Kit-8(CCK-8)was used to detect the change of cell proliferation ability under hypoxia condition.(4)WB verified the levels of expression of SIRT6,GLUT1,PKM2,LDHA and HIF1α in cells of OE-NC,OE-SIRT6,sh-NC and sh-SIRT6 under hypoxia.The content of lactic acid and glucose in cells of each group was detected by the kit,and the effect of SIRT6 on the level of cell glycolysis was analyzed.(5)The expression of SIRT6 affected the sensitivity of cells to Temozolomide(TMZ)by plate cloning test and CCK-8.(6)The apoptosis-related proteins Caspase3,Caspase8,Bax and Bcl-2 were detected by flow cytometry.(7)The effect of SIRT6 on U87 MG subcutaneous tumor formation and sensitivity to TMZ was investigated in vivo.Results:(1)WB results showed that the SIRT6 protein of malignant glioma cells U251(t=21.46,P<0.001),U87MG(t=25.74,P<0.001),LN229(t=22.92,P<0.016)was significantly lower than that of astrocytes NHA,while U87 MG was the most significantly lower,with significant difference(P<0.01).Therefore,this cell line was selected for subsequent experiments.(2)The results of hypoxia experiment: under hypoxia conditions,the expression level of SIRT6 protein in Hypoxia group was significantly lower than that in U87 MG cells under normoxic condition(t=9.746,P=0.010),while the glycolytic related protein HIF1α(t=7.963,P=0.016),LDHA(t=9.668,P=0.010),PKM2(t=9.805,P=0.010),GLUT1(t=14.82,P=0.004)were significantly up-regulated under hypoxia.(3)Effect of SIRT6 on cell activity: CCK-8results showed that compared with OE-NC group,the cell activity of OE-SIRT6 group was significantly inhibited at the 3rd day(t=5.047,P=0.007),the 5th day(t=4.869,P=0.008)and the 7th day(t=6.197,P=0.003).Compared with OE-NC group,the cell activity of sh-SIRT6 group was significantly up-regulated on the 5th day(t=3.066,P=0.037)and 7th day(t=5.017,P=0.007).(4)Glycolysis test results:compared with OE-NC group,HIF-1α(t=11.073,P=0.008),LDHA(t=15.84,P=0.004),PKM2(t=5.558,P=0.031),GLUT1(t=6.321,P=0.024)protein expression levels were significantly inhibited.On the contrary,compared with sh-NC group,after U87 MG cells silenced SIRT6,HIF1α(t=6.151,P=0.025),LDHA(t=8.508,P=0.013),PKM2(t=6.378,P=0.024),GLUT1(t=7.161,P=0.019)protein expression was significantly up-regulated.Compared with sh-NC,sh-SIRT6 group increased glucose uptake(t=3.524,P=0.024)and lactate secretion(t=6.071,P=0.004)of U87 MG cells.Overexpression of SIRT6 significantly inhibited the production of lactic acid(compared with OE-NC group,t=5.514,P=0.005).(5)Results of drug sensitivity test:compared with U87 MG cells under normoxic conditions,the cell activity after TMZ treatment was 37 ± 7%(t=9.570,P<0.001);Under hypoxic conditions,the activity of cells treated with TMZ decreased to 62 ± 5% of that of the Normoxia group(t=4.631,P=0.010).In addition,the cell activity of Hypoxia+TMZ group was significantly higher than that of Normoxia+TMZ group(t=5.034,P=0.007).To prove that the decreased sensitivity of U87 MG cells to TMZ caused by hypoxia is related to SIRT6.CCK8 results showed that under the background of hypoxia,OE-SIRT6 could significantly enhance the inhibition of TMZ on U87 MG cells(t=8.457,P=0.001),while sh-SIRT6 enhanced the resistance of cells to TMZ,compared with TMZ group(t=3.377,P=0.028).The results of plate cell clone experiment showed that TMZ could inhibit the formation of OE-SIRT6 cell clone(t=4.771,P=0.008).On the contrary,TMZ can promote the proliferation of sh-SIRT6 cells(t=6.220,P=0.003).(6)Results of apoptosis experiment: Flow cytometry showed that the apoptosis rate of OE-SIRT6+TMZ group was 18.0 ± 2.15%,which was significantly higher than that of TMZ group(t=7.195,P=0.002).The apoptosis rate of sh-SIRT6+TMZ group was1.69 ± 0.96%,which was significantly lower than that of TMZ group(t=10.10,P<0.001).WB results showed that TMZ could induce the increase of apoptotic proteins Bax(t=3.812,P=0.0189)caspase3(t=8.412,P=0.0011)and caspase8(t=16.74,P<0.001)in OE-SIRT6 cells,and reduce the expression of anti-apoptotic protein Bcl-2.TMZ did not cause changes in apoptosis-related proteins in sh-SIRT6 cells(P>0.05).(7)Results of subcutaneous neoplasia in nude mice: compared with the control group,OE-SIRT6 can promote the inhibition of TMZ on U87 MG solid tumor,while sh-SIRT6 has no significant effect on the inhibition of solid tumor.Conclusions:(1)Hypoxia condition can reduce the expression level of SIRT6 protein in U87 MG cells and promote the expression of HIF-1α and glycolysis related protein GLUT1、PKM2、LDHA.(2)Overexpression of SIRT6 can be passed through HIF1α/ Glycolysis pathway inhibits the proliferation of glioblastoma U87 MG.(3)Overexpression of SIRT6 protein can promote the drug sensitivity of U87 MG cells to TMZ,and play an anti-tumor role,silence of SIRT6 did not induce apoptosis.(4)Overexpression of SIRT6 increased the drug sensitivity of the transplanted tumor to TMZ and improved the therapeutic effect of the drug. |
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