PAK1在幽门螺杆菌联合MNNG诱导人食管上皮细胞分泌IL-8和GRO-α中的作用

The role of Pak1 in Helicobacter pylori and MNNG-induced secretion of IL-8 and GRO-αin human esophageal epithelial cells

目的构建PAK1低、高表达人食管上皮细胞(HEEC)稳定细胞株,探究PAK1在幽门螺杆菌(HP)联合N-甲基-N’-硝基-N-亚硝基胍(MNNG)诱导HEEC分泌趋化因子IL-8和GRO-α中的作用,为食管癌发病机制提供理论依据。方法利用RNAi技术转染HEEC构建PAK1低表达(PAK1-KD)稳定细胞株,利用过表达慢病毒转染HEEC构建PAK1高表达(PAK1-OE)稳定细胞株;两种细胞分别分为HP+MNNG染毒组、HP染毒组和MNNG染毒组,并设立空白对照组。按感染复数(MOI)=100∶1,即细菌∶细胞=100∶1将HP加入细胞中进行共培养,采用CCK8法计算MNNG半数抑制浓度(IC50)。收集染毒后3 h、6 h和9 h的细胞上清液,采用ELISA法测定IL-8和GRO-α含量。结果相较于空载体对照组(PAK1-NC),PAK1-KD组PAK1基因敲减效率达到84.1%(t=14.627,P=0.004),PAK1-OE组PAK1基因的表达丰度是PAK1-NC组的1,101.646倍(t=16.104,P=0.004)。表明成功构建了PAK1低、高表达HEEC稳定细胞株。在PAK1-KD和PAK1-OE细胞中,各染毒组细胞IL-8和GRO-α分泌含量均随染毒时间呈趋势性增加(均P<0.01)。在PAK1-KD和PAK1-OE细胞中,相同染毒时间,各组细胞IL-8和GRO-α含量均出现联合染毒组>HP染毒组>MNNG染毒组>对照组,差异有统计学意义(均P<0.05)。在相同染毒时间内,与PAK1低表达(PAK1-KD)相比,PAK1高表达(PAK1-OE)显著促进联合染毒组、HP染毒组和MNNG染毒组细胞分泌IL-8和GRO-α,差异有统计学意义(均P<0.05)。结论PAK1作为幽门螺杆菌感染上皮信号通路关键因子,其高表达可促进HP联合MNNG染毒诱导人食管上皮细胞分泌趋化因子IL-8和GRO-α,在食管癌发生中起重要作用。

Objective To construct stable cell lines with low and high expression of PAK1 in human esophageal epithelial cells(HEEC),and to explore the effects of PAK1 on IL-8,GRO-α of HEEC induced by Helicobacter pylori(HP)combined with N-methyl-N'-nitro-n-nitroguanidine(MNNG),providing theoretical basis for the pathogenesis of esophageal cancer.Methods PAK1 low expression stable cell lines(PAK1-KD)were constructed by transfecting HEEC with RNAi technology,and PAK1 high expression stable cell lines(PAK1-OE)were constructed by transfecting HEEC with overexpressed lentivirus.The two cells were divided into the HP+MNNG group,the HP group,the MNNG group and the blank control group.The multiple of infection(MOI)was 100:1,that was,bacteria:cell=100:1,and HP was added into the cells for co-culture.CCK8 method was used to calculate the half inhibitory concentration of MNNG(IC50).The contents of IL-8 and GRO-α were determined by ELISA,and the supernatants were collected at 3 h,6 h and 9 h after exposure.Results Compared with the empty vector control group(PAK1-NC),PAK1 gene knockout efficiency in the PAK1-KD group reached 84.1%(t=14.627,P=0.004),and the expression abundance of PAK1 gene in the PAK1-OE group was 1101.646 times compared with in the PAK1-NC group(t=16.104,P=0.004).The results showed that PAK1 low and high expression stable cell lines of HEEC were successfully constructed.In PAK1-KD and PAK1-OE cells,the secretion levels of IL-8 and GRO-α in each exposed group tended to increase with the time of exposure(all P<0.01).In PAK1-KD and PAK1-OE cells,the contents of IL-8 and GRO-αwere found to be the highest in the HP+MNNG group,second in the HP group,third in the MNNG group and last in the control group at the same exposure time(all P<0.05).During the same exposure time,compared with PAK1 low expression(PAK1-KD),PAK1 high expression(PAK1-OE)significantly promoted the secretion of IL-8 and GRO-α in the HP+MNNG group,the HP group and the MNNG group,and the differences were statistically significant(all P<0.05).Conclusion PAK1 is a key factor in the epithelial signaling pathway of HP,and its high expression could promote the secretion of chemokines IL-8 and GRO-α in HEEC induced by HP comdined with MNNG,and play an important role in the development of esophageal cancer.