层粘连蛋白β-1在胃癌侵袭转移中的作用及其机制

Effect and mechanism of laminin β-1 in the invasion and metastasis of gastric cancer

目的探讨层粘连蛋白β-1(LAMB1)在胃癌癌结节表达及在肿瘤侵袭迁移过程中作用及其机制。方法收集河北医科大学第四医院35例胃癌患者的胃癌根治术后的病理标本, 包括胃癌癌结节、原发灶及癌旁组织。以免疫组织化学(IHC)技术检测LAMB1蛋白在胃癌癌结节、原发灶及癌旁组织中的表达情况。荧光实时定量聚合酶链反应(RT-qPCR)和蛋白印迹(Western blot)技术检测LAMB1 mRNA和蛋白在人胃癌细胞株AGS、MKN74及正常胃上皮细胞株GES-1中表达。合成抑制LAMB1的小干扰RNA(siRNA)并转染AGS细胞;噻唑蓝(MTT)法检测细胞的活性;划痕实验(Wound assay)和Transwell小室实验检测细胞的迁移侵袭能力;Western blot检测上皮-间充质转化(EMT)相关蛋白基质金属蛋白酶-2(MMP-2)、E-钙粘蛋白(E-cadherin)、N-cadherin、波形蛋白(Vimentin)表达, 组间比较采用t检验。结果 LAMB1蛋白在胃癌癌结节、原发灶及癌旁组织中的强表达率分别为82.86%(29/35)、60.00%(21/35)、14.29%(5/35)(χ^(2)=34.211, P<0.01)。AGS、MKN74、GES-1细胞LAMB1 mRNA表达水平为1.996±0.225、1.519±0.162、0.670±0.230, 差异有统计学意义(F=63.602, P<0.01);蛋白的表达水平为1.044±0.138、0.693±0.106、0.348±0.089, 差异有统计学意义(F=58.028, P<0.01)。LAMB1-siRNA转染后AGS细胞LAMB1 mRNA表达(0.345±0.092)明显低于对照组(1.066±0.149), 差异有统计学意义(t=10.085, P<0.01)。MTT结果显示LAMB1-siRNA转染组AGS经过24 h后细胞活性(0.491±0.097)低于对照组(0.751±0.090), 差异有统计学意义(t=4.813, P<0.01)。LAMB1-siRNA转染组AGS细胞迁移率[(37.017±6.043)%]明显低于对照组[(66.533±11.567)%], 差异有统计学意义(t=5.540, P<0.01);Transwell小室实验结果显示LAMB1-siRNA转染组侵袭细胞数[(57.667±10.152)个]明显低于对照组[(109.667±17.592)个], 差异有统计学意义(t=6.271, P<0.01)。LAMB1-siRNA转染组细胞MMP-2、N-cadherin、Vimentin蛋白表达(0.615±0.126、0.409±0.065、0.734±0.103)均低于对照组(1.117±0.115、1.032±0.128、1.115±0.101), 差异有统计学意义(t=5.097、7.517、4.575, P<0.05), 而E-cadherin表达水平(1.323±0.189)则高于对照组(0.817±0.139), 差异有统计学意义(t=-3.736, P<0.05)。结论 LAMB1在胃癌转移过程中水平增高, 可能是通过调控肿瘤细胞的EMT过程而参与了胃癌的进展转移。

Objective To investigate the expression of lamininβ-1(LAMB1)in gastric cancer nodules and its role and mechanism in tumor invasion and migration.Methods The pathological specimens of 35 patients with gastric cancer after radical gastrectomy in the Fourth Hospital of Hebei Medical University were collected,including gastric cancer nodules,primary lesions and adjacent tissues.The expression of LAMB1 protein in gastric cancer nodules,primary lesions and adjacent tissues was detected by immunohistochemistry(IHC).Quantitative real-time polymerase chain reaction(RT-qPCR)and Western blotting techniques were used to detect the expression of LAMB1 mRNA and protein in human gastric cancer cell lines AGS,MKN74 and normal gastric epithelial cell line GES-1,respectively.LAMB1-small interfering RNA(siRNA)was synthesized and transfected into AGS cells.The cell activity was detected by thiazolyl blue(MTT)assay.The migration and invasion ability of cells was detected by Wound assay and Transwell chamber assay.Western blotting was used to detect epithelial-mesenchymal transition(EMT)-related proteins matrix metalloproteinase-2(MMP-2),E-cadherin,N-cadherin,and Vimentin.Data of results were analyzed by t-test with SPSS 25.0 statistical software.Results The high expression rate of LAMB1 protein in gastric cancer nodules,primary lesions and adjacent tissues was 82.86%(29/35),60.00%(21/35)and 14.29%(5/35)(χ^(2)=34.211,P<0.01)respectively.The expression levels of LAMB1 mRNA in AGS,MKN74,and GES-1 cells were 1.996±0.225,1.519±0.162,and 0.670±0.230 respectively,and the difference was statistically significant(F=63.602,P<0.01).The protein expression levels of LAMB1 in AGS,MKN74,and GES-1 cells were 1.044±0.138,0.693±0.106 and 0.348±0.089 respectively,and the difference was statistically significant(F=58.028,P<0.01).The expression of LAMB1 mRNA in AGS cells after LAMB1-siRNA transfection(0.345±0.092)was significantly lower than that in the control group(1.066±0.149),and the difference was statistically significant(t=10.085,P<0.01).MTT results showed that the cellular activity of AGS in the LAMB1-siRNA transfected group was significantly lower than that in the control group(0.751±0.090)after 24 h(t=4.813,P<0.01).The results of Wound assay showed that the migration rate of AGS cells in the LAMB1-siRNA transfection group[(37.017±6.043)%]was significantly lower than that in the control group[(66.533±11.567)%],and the difference was statistically significant(t=5.540,P<0.01).The Transwell assay revealed that the number of invasive cells in the LAMB1-siRNA transfection group[(57.667±10.152)cells]was significantly less than that in the control group[(109.667±17.592)cells],and the difference was statistically significant(t=6.271,P<0.01).The protein expression of MMP-2,N-cadherin and Vimentin in the LAMB1-siRNA transfection group(0.615±0.126,0.409±0.065,0.734±0.103)was significantly lower than that in the control group(1.117±0.115,1.032±0.128,1.115±0.101;t=5.097,7.517,4.575,P<0.05),and the expression level of E-cadherin(1.323±0.189)was significantly higher than that in the control group(0.817±0.139)(t=-3.736,P<0.05).Conclusion The expression of LAMB1 increased during the process of gastric cancer metastasis,which might be involved in the progression and metastasis of gastric cancer by regulating the EMT of cancer cells.