下调miR-32对幽门螺杆菌诱导的肠上皮细胞凋亡及TAK1-p38通路的影响

Effects of down-regulating miR-32 on the apoptosis of intestinal epithelial cells induced by Helicobacter pylori and the TAK1-p38 pathway in vitro

目的探究下调微小RNA-32(miR-32)对幽门螺杆菌(H.pylori)诱导的肠上皮细胞凋亡及转化生长因子β激活激酶1-p38丝裂原激活蛋白激酶(TAK1-p38)通路的影响。方法体外培养正常人肠上皮细胞系HIEC-6细胞,分为空白对照组(BC组,无H.pylory感染无转染正常培养HIEC-6细胞)、H.pylory组(H.pylory感染)、H.pylory+NC组(inhibitor-NC转染+H.pylory感染)、H.pylory+inhibitor组(miR-32 inhibitor转染+H.pylory感染),CCK-8法检测各组HIEC-6细胞活性,Annexin V-FITC/PI检测各组HIEC-6细胞凋亡,酶联免疫吸附试验(ELISA)检测上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)表达量,实时荧光定量PCR(RT-qPCR)检测各组细胞miR-32、TAK1、p38 mRNA表达水平,免疫印迹(WB)检测各组细胞TAK1、p38蛋白表达水平。结果与BC组比较,H.pylory组HIEC-6细胞miR-32表达水平、凋亡率、上清液TNF-α、IL-6表达量、细胞TAK1、p38 mRNA及蛋白表达水平显著增加,细胞活性显著降低,差异有统计学意义(P<0.05);与H.pylory+NC组比较,H.pylory+inhibitor组miR-32表达水平、凋亡率、上清液TNF-α、IL-6表达量、细胞TAK1、p38 mRNA及蛋白表达水平显著降低,差异有统计学意义(P<0.05),细胞活性显著增加,差异有统计学意义(P<0.05)。结论下调miR-32可抑制H.pylory感染诱导的HIEC-6细胞凋亡,可能与抑制TAK1-p38通路,减轻细胞炎性反应有关。

Objective To investigate the effects of down-regulating microRNA-32(miR-32)on the apoptosis of intestinal epithelial cells induced by Helicobacter pylori(H.pylory)and the pathway of transforming growth factorβactivated kinase 1-p38 mitogen activated protein kinase(TAK1-p38).Methods The normal human intestinal epithelial cell line HIEC-6 cells were cultured in vitro and divided into three groups:blank control group(BC group,no H.pylory infection,no transfection of normal HIEC-6 cells),H.pylory group(H.pylory infection),H.pylory+NC group(inhibitor-NC transfection+H.pylory infection),H.pylory+inhibitor group(miR-32 inhibitor transfection+H.pylory infection).The activity of HIEC-6 cells was detected by CCK-8,and the apoptosis of HIEC-6 cells was detected by Annexin V-FITC/PI,and the expressions of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in supernatant were detected by enzyme-linked immunosorbent assay(ELISA),the expression levels of miR-32,TAK1 and p38 mRNA were detected by RT-qPCR,moreover,Western Blot(WB)was used to detect the protein expressions of TAK1 and p38.Results Compared with those in BC group,the expression levels of miR-32,apoptosis rate,and the expressions of TNF-αand IL-6 in supernatant,expression levels of TAK1,p38 mRNA and protein in HIEC-6 cells in H.pylory group were significantly increased,however,the cell activity was significantly decreased(P<0.05).Compared with those in H.pylory+NC group,the expression levels of miR-32,apoptosis rate,expressions of TNF-αand IL-6 in supernatant,expression levels of TAK1,p38 mRNA and protein in HIEC-6 cells in H.pylory+inhibitor group were significantly decreased(P<0.05),but,the cell activity was significantly increased(P<0.05).Conclusion To down-regulate the expression of miR-32 can inhibit the apoptosis of HIEC-6 cells induced by H.pylory infection,which may be related to the inhibition of TAK1-p38 pathway and the alleviation of cell inflammatory response.