原钙黏蛋白10对胃癌顺铂化疗敏感性的影响及作用机制初探

Preliminary study on the influence of protocadherin 10 on cisplatin sensitivity in gastric cancer and the underlying mechanism

目的探究原钙黏蛋白10(PCDH10)对顺铂化疗敏感的影响及机制。方法将pcDNA3.1+PCDH10质粒、pcDNA3.1+空质粒分别转染至对数期人胃癌BGC823细胞株,分别作为转染组和空载组,未做处理的细胞作为空白组,实时荧光定量逆转录聚合酶链反应(RT-PCR)检测PCDH10 mRNA的相对表达量,蛋白质印记法(Western blot)检测PCDH10蛋白的相对表达量,噻唑蓝(MTT)法检测细胞增殖情况,划痕试验检测细胞迁移能力,Transwell小室检测细胞侵袭能力。取转染组、空载组及空白组细胞加入不同浓度的顺铂溶液,比较各组细胞存活率及顺铂对细胞的半数抑制浓度(IC50);取转染组、空白组细胞加入IC50浓度的顺铂溶液,分别作为转染+顺铂组、顺铂组,比较转染组、空白组、转染+顺铂组、顺铂组细胞磷脂酰肌醇-3-羟激酶(PI3K)、蛋白激酶B(AKT)、雷帕霉素靶蛋白(MTOR)、p70核糖体蛋白S6激酶(p70S6)基因及蛋白的相对表达量。结果转染组细胞PCDH10 mRNA及PCDH10蛋白的相对表达量,均高于空载组和空白组细胞(P﹤0.05)。将稳定转染的各组细胞重新接种,转染组细胞培养24、48、72 h时OD值及培养24 h细胞迁移率和穿膜细胞数均低于空载组和空白组细胞(P﹤0.05)。顺铂浓度分别为0.500、1.000、2.000、4.000、8.000μg/ml时,转染组细胞存活率、顺铂对细胞的IC50值均低于空载组和空白组细胞(P﹤0.05)。转染组、顺铂组、转染+顺铂组细胞PI3K、MTOR、p70S6 mRNA和PI3K、MTOR、p70S6蛋白的相对表达量均低于空白组细胞(P﹤0.05),磷酸化蛋白激酶B(pAKT)/AKT低于空白组细胞(P﹤0.05),转染+顺铂组细胞PI3K、MTOR、p70S6 mRNA和PI3K、MTOR、p70S6蛋白的相对表达量均低于空白组细胞(P﹤0.05),pAKT/AKT低于转染组和顺铂组细胞(P﹤0.05)。结论PCDH10过表达可抑制人胃癌BGC823细胞的增殖、迁移、侵袭能力,并可增强细胞顺铂化疗敏感性,其作用机制可能与下调PI3K、MTOR、p70S6 mRNA及PI3K、MTOR、p70S6蛋白表达、抑制AKT磷酸化相关。

Objective To investigate the influence of protocadherin 10(PCDH10)on cisplatin sensitivity in gastric cancer,and to explore the underlying mechanism.Method The pcDNA3.1+PCDH10 plasmid and pcDNA3.1+empty plasmid were transfected into the human gastric cancer BGC823 cells at logarithmic phase as transfection group and empty group,besides,the cells without treatment were included as blank group,respectively.The relative expression of PCDH10 mRNA and protein in each group were detected by real-time fluorescence quantitative reverse transcriptionpolymerase chain reaction(RT-PCR)and protein expression was determined by western blot,cell proliferation was assessed using MTT method,and scratch wound assay was used to evaluate cell migration ability,additionally,the cell invasiveness was analyzed via Transwell chamber assay.Different concentrations of cisplatin were added to transfection group,empty group and blank group,subsequently,the cell survival rate and half inhibitory concentration(IC50)of cisplatin on cells were recorded;the cells in the transfection group and the blank group were treated with cisplatin solution at the concentration of IC50,forming another two groups of transfection+cisplatin group and cisplatin group,respectively.The expressions of phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT),mammalian target of rapamycin(MTOR),p70 ribosomal protein S6 kinase(p70S6)mRNA and protein in transfection group,blank group,transfection+cisplatin group and cisplatin group were compared.Result The relative expression of PCDH10 mRNA and protein in transfection group were significantly higher than that in the blank group and the empty group(P<0.05).Stably transfected cells of each group were re-seeded,the optical density(OD)value of transfected cells cultured for 24,48 and 72 h were lower,and the cell migration rate and the number of transmembrane cells cultured for 24 h in the transfection group were significantly lower than those in the blank group and the empty group(P<0.05).At the concentration of cisplatin of 0.500,1.000,2.000,4.000,and 8.000μg/ml,the cell survival rate and IC50 value of cisplatin in the transfection group were lower compared to empty group and group(P<0.05).The relative expressions of PI3K,MTOR and p70S6 mRNA and PI3K,MTOR,p70S6 protein in the transfection group,cisplatin group and transfection+cisplatin group were evidently lower than those in blank group(P<0.05),and pAKT/AKT was lower than that in blank group(P<0.05).The relative expressions of PI3K,MTOR and p70S6 mRNA and PI3K,MTOR,p70S6 protein in transfection+cisplatin group were lower than those in blank group(P<0.05),and pAKT/AKT were lower than those in transfection group and cisplatin group(P<0.05).Conclusion Overexpression of PCDH10 would significantly inhibit the proliferation,migration and invasion ability of human gastric cancer cell line BGC823,and enhance the sensitivity of cells to cisplatin,which may be related to down-regulation of PI3K,MTOR,p70S6 mRNA and protein expression and inhibition of AKT phosphorylation.