甲基化寡核苷酸诱导热休克蛋白A2基因甲基化对胰腺癌增殖及凋亡的影响

Effects of methylation-induced oligonucleotides on heat shock protein A2 methylation on pancreatic cancer proliferation and apoptosis

目的:观察甲基化寡核苷酸诱导热休克蛋白A2(HSPA2)基因甲基化对胰腺癌细胞增殖和凋亡的影响。方法:检测2017年1月至2018年12月桂林医学院附属医院诊治的90例胰腺癌患者(PAN组)和50例胰腺良性疾病患者(CON组)HSPA2基因甲基化,Kaplan-Meier生存分析比较胰腺癌患者中HSPA2基因甲基化和未甲基化生存时间的差异。合成设计不同寡核苷酸(MON、UON、CON-a、CON-b和CON)转染PANC-1胰腺癌细胞,比较转染前、后PANC-1胰腺癌细胞HSPA2基因甲基化、吸光度、细胞增殖和凋亡的差异。结果:PAN组胰腺癌患者HSPA2基因甲基化率显著低于CON组(13.3%比82.05%, χ2=61.537, P<0.05)。PANC-1、SW1990、HPAF-Ⅱ及CFPAC-1胰腺癌细胞中HSPA2为未甲基化状态。PAN组胰腺癌患者中HSPA2基因甲基化者生存期显著高于HSPA2基因未甲基化患者[(16.88±0.67)个月比(11.34±0.51)个月,Logrank=15.195, P<0.01]。MON组可成功诱导PANC-1胰腺癌细胞HSPA2基因甲基化,S期、G 2/M期和增殖指数均显著低于UON组、CON-a组、CON-b组和CON组PANC-1胰腺癌细胞(S期分别为25.8±1.9、32.6±2.7、32.4±2.5、32.7±2.9、32.8±2.3,G 2/M期分别为7.7±1.1、9.5±1.4、9.4±1.5、9.7±1.6、9.5±1.5,增殖指数分别为0.35±0.06、0.43±0.06、0.44±0.05、0.45±0.07、0.46±0.05, F=36.140、45.250、102.210, P<0.05),G 0/G 1期和凋亡率均显著高于UON、CON-a、CON-b和CON组(G 0/G 1期分别为61.3±2.1、55.8±3.2、55.2±3.5、54.9±3.7、55.1±2.9,凋亡率分别为24.7±1.8、21.6±2.1、21.4±2.3、21.5±2.5、21.6±2.4, F=47.280、72.140, P<0.05)。 结论:胰腺癌的发病机制与HSPA2基因去甲基化有关,HSPA2基因去甲基化为胰腺癌患者生存时间影响因素。甲基化寡核苷酸可诱导胰腺癌HSPA2基因甲基化失活而抑制细胞增殖并促进凋亡。

Objective To study the effects of inactivation of heat shock protein A2(HSPA2)gene induced by methylated oligonucleotide on pancreatic cancer proliferation and apoptosis.Methods Methylation specific polymerase chain reaction(PCR)was used to detect the HSPA2 gene methylation in 90 patients with pancreatic cancer and patients with 50 benign pancreatic disease between Jan 2017 to Dec 2018 from Guilin medical college affiliated hospital.Kaplan-Meier survival analysis was used to compare the survival time of patients with methylated and unmethylated HSPA2 gene.The methylated oligonucleotides(MON,UON,CON-a,CON-b and CON)were transfected into PANC-1 pancreatic cancer cell.The HSPA2 gene methylation,absorbance,cell proliferation and apoptosis were studied before and after transfection.Results The HSPA2 gene methylation rate in pancreatic cancer group was significantly higher than in patients with benign pancreatic disease(13.3%vs.82.05%,χ2=61.537,P<0.05).The survival time of patients with methylated HSPA2 gene in pancreatic cancer patients in PAN group was significantly higher than that of patients with unmethylated HSPA2 gene[(16.88±0.67)m vs.(11.34±0.51)m,Logrank=15.195,P<0.01].The MON group can successfully induce the methylation of HSPA2 gene in PANC-1 pancreatic cancer cells.The S phase,G2/M phase and proliferation index in MON group were significantly lower than those in the UON group,CON-a group,CON-b group,and CON group(S stage=25.8±1.9,32.6±2.7,32.4±2.5,32.7±2.9,32.8±2.3,G2/M stage=7.7±1.1,9.5±1.4,9.4±1.5,9.7±1.6,9.5±1.5,proliferation index=0.35±0.06,0.43±0.06,0.44±0.05,0.45±0.07,0.46±0.05,F=36.140,45.250,102.210,P<0.05),while the G0/G1 phase and apoptosis rate were significantly higher(G0/G1 stage=61.3±2.1,55.8±3.2,55.2±3.5,54.9±3.7,55.1±2.9,apoptosis rate=24.7±1.8,21.6±2.1,21.4±2.3,21.5±2.5,21.6±2.4,F=47.280,72.140,P<0.05).Conclusion The pathogenesis of pancreatic cancer is related to the demethylation of HSPA2 gene,which is a factor influencing the survival time.Methylated oligonucleotides can induce inactivation of HSPA2 gene methylation in pancreatic cancer,which can inhibit cell proliferation and promote apoptosis.