KLF5通过激活Wnt通路对HpSlyD诱导胃黏膜肠化生作用的影响

KLF5 promotes HpSlyD induced gastric intestinal metaplasia by activating Wnt/β-catenin pathway

背景Krüppel样因子5(krüppel-like factor 5,KLF5)和Wnt/β-Catenin通路都是肿瘤发生的研究热点,目前有研究发现KLF5与肠上皮细胞的分化和增殖有关,推测其可能参与了肠化生的发生.但目前有关KLF5对胃黏膜肠化生组织增殖、凋亡的影响及其分子机制的研究报道相对较少.目的分析KLF5通过激活Wn t通路对幽门螺杆菌(Helicobacter pylori,H.pylori)诱导胃黏膜肠化生(gastric intestinal metaplasia,GIM)作用的影响.方法收集肠化生组织和正常胃黏膜,采用RT-PCR法、Western blot法分别检测KLF5、Wnt3a mRNA和蛋白表达.体外培养胃黏膜上皮细胞GES1,分为空白对照组、HpSlyD组(200 ng/mL的HpSlyD+阴性序列)、干扰KLF5组(200 ng/mL的HpSlyD+KLF5 siRNA)、Wnt激动剂组(200 ng/mL的HpSlyD+KLF5 siRNA+氯化锂)、Wnt激动剂+干扰KLF5组.采用MTT法、流式细胞仪分别检测各组GES1细胞增殖、凋亡情况,采用RT-PCR法检测KLF5、Wnt3a、β-catenin、Villin 1,VIL1)、三叶因子Ⅱ(trefoil factor 2,TFF2)、尾侧同源盒因子2(caudal homeobox factor 2CDX2)mRNA表达.Western blot法检测VIL1、TFF2、CDX2蛋白表达.结果KLF5、Wnt3a mRNA和蛋白表达量在肠化生组织中明显高于在正常胃黏膜中的表达(P<0.01).HpSlyD组、干扰对照组细胞增殖率明显高于空白对照组、凋亡率明显低于空白对照组(P<0.05),干扰KLF5组细胞增殖率明显低于HpSlyD组、凋亡率明显高于HpSlyD组(P<0.05).HpSlyD组、干扰对照组细胞VIL1、TFF2、CDX2 mRNA和蛋白表达明显高于空白对照组(P<0.05),干扰KLF5组细胞VIL1、TFF2、CDX2 mRNA和蛋白表达明显低于HpSlyD组(P<0.05).干扰KLF5组细胞Wnt3a、β-catenin、CDX2 mRNA表达明显低于HpSlyD组(P<0.05).而Wnt激动剂+干扰KLF5组Wnt3a、β-catenin、CDX2 mRNA表达明显高于干扰KLF5组(P<0.05).结论干扰KLF5表达可显著抑制HpSlyD诱导的胃黏膜化生的发生,KLF5可能通过激活Wnt/β-Catenin促进HpSlyD诱导的胃黏膜化生,为胃癌的临床预防提供了新的靶点.

BACKGROUND Krüppel like factor 5(KLF5)and the Wnt/β-catenin pathway are hot topics in the research of tumorigenesis.Some studies have found that KLF5 is related to the differentiation and proliferation of intestinal epithelial cells,suggesting that KLF5 may be involved in the occurrence of intestinal metaplasia.However,there are few reports on the effect of KLF5 on intestinal metaplasia and the underlying molecular mechanism.AIM To explore the effect of KLF5 on gastric intestinal metaplasia(GIM)induced by Helicobacter pylori(H.pylori)and the underlying mechanism.METHODS The mRNA and protein expression of KLF5 and Wnt3a was detected by RT-PCR and Western blot,respectively.Gastric epithelial cell line GES1 was cultured in vitro and divided into a blank control group,HpSlyD group(200 ng/mL HpSlyD+negative sequence),KLF5 interference group(200 ng/mL HpSlyD+KLF5 siRNA),Wnt agonist group(200 ng/mL HpSlyD+KLF5 siRNA+lithium chloride),and Wnt agonist+KLF5 interference group.GES1 cell proliferation and apoptosis were determined by MTT assay and flow cytometry,respectively.RTPCR was used to detect KLF5,Wnt3a,beta-catenin,wool protein 1(VIL1),trefoil factor 2(TFF2),and caudal homeobox factor 2(CDX2)mRNA expression.The expression of VIL1,TFF2,and CDX2 proteins was detected by Western blot.RESULTS The expression of KLF5 and Wnt3a mRNA and protein in intestinal metaplasia was significantly higher than that in the normal gastric mucosa(P<0.01).The cell proliferation rates in the HpSlyD group and interference control group were significantly higher than that of the blank control group,and the apoptosis rates were significantly lower than that of the blank control group(P<0.05).The cell proliferation rate of the KLF5 interference group was significantly lower than that of the HpSlyD group,and the apoptosis rate was significantly higher than that of the HpSlyD group(P<0.05).The mRNA and protein expression of VIL1,TFF2,and CDX2 in the HpSlyD group and interference control group was significantly higher than that of the blank control group(P<0.05),while the mRNA and protein expression of VIL1,TFF2,and CDX2 in the KLF5 interference group were significantly lower than those of the HpSlyD group(P<0.05).The expression of Wnt3a,β-catenin,and CDX2 mRNA in the KLF5 interference group was significantly lower than that of the HpSlyD group(P<0.05).The expression of Wnt3a,β-catenin,and CDX2 mRNA in the Wnt agonist+KLF5 interference group was significantly higher than that of the KLF5 interference group(P<0.05).CONCLUSION Interference of KLF5 expression can significantly inhibit HpSlyD induced gastric metaplasia.KLF5 may promote HpSlyD induced gastric metaplasia by activating the Wnt/β-catenin pathway,which provides a new target for clinical prevention of gastric cancer.