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目的探讨高尔基体囊泡转运蛋白(colgi-vesicular transport protein,P115)基因沉默对胃癌细胞株BGC-823中增殖相关因子细胞周期素D1(cyclinD1)、微小染色体维持缺陷蛋白2(mini chromosome maintenance protein 2,Mcm2)和增殖细胞核抗原(proliferating Cell Nuclear Antigen,PCNA)表达的影响及其可能的机制。方法采用脂质体介导法将重组表达质粒P115-shRNA2(P115-shRNA2组)和阴性对照质粒shNC(阴性对照组)转染至高表达P115的胃癌细胞株BGC-823中,并设未转染组,经G418筛选出稳定转染细胞,实时定量PCR(Real-time PCR)法检测细胞中P115、巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)、cyclinD1、Mcm2和PCNA基因mRNA的转录水平;Western blot法检测细胞中P115、MIF、pERK1/2、cyclinD1、Mcm2和PCNA蛋白表达水平;免疫共沉淀法检测BGC-823细胞中P115与MIF间的相互作用;ELISA法检测细胞上清液中MIF的分泌水平。结果与未转染组和阴性对照组相比,P115-shRNA2组细胞中P115、MIF、cyclinD1、Mcm2和PCNA的mRNA和蛋白表达水平均明显降低,ERK1/2的磷酸化水平明显下调,BGC-823细胞培养上清中MIF的含量明显降低;P115蛋白可在抗MIF的免疫沉淀物中检出,P115和MIF在BGC-823细胞中存在特异性的相互作用。结论 P115基因沉默下调胃癌细胞中cyclinD1、Mcm2和PCNA的表达,其分子机制可能与P115通过调控MIF的表达和分泌,进而启动下游的ERK1/2信号通路有关。P115可作为研究胃癌细胞增殖分子机制的新靶点。
Objective To investigate the effects of P115 gene silencing on the expression of cyclin D1 and cyclin D1 in gastric cancer cell line BGC-823, , Mcm2) and proliferating cell nuclear antigen (PCNA) expression and its possible mechanism. Methods The recombinant plasmid p115-shRNA2 (P115-shRNA2) and negative control plasmid shNC (negative control) were transfected into BGC-823 gastric cancer cell line P115 by liposome-mediated method. Group. The stable transfected cells were screened by G418, and the transcription of P115, macrophage migration inhibitory factor (MIF), cyclinD1, Mcm2 and PCNA mRNA were detected by Real-time PCR The expression of P115, MIF, pERK1 / 2, cyclinD1, Mcm2 and PCNA were detected by Western blot. The interaction between P115 and MIF in BGC-823 cells was detected by co-immunoprecipitation. The level of MIF secretion. Results The mRNA and protein expressions of P115, MIF, cyclinD1, Mcm2 and PCNA in P115-shRNA2 group were significantly lower than those in untransfected and negative control groups, while the phosphorylation of ERK1 / 2 was significantly decreased. The expression of BGC- 823 cell culture supernatant MIF content was significantly reduced; P115 protein can be detected in anti-MIF immunoprecipitates P115 and MIF in BGC-823 cells there is a specific interaction. Conclusion P115 silencing down-regulated the expression of cyclinD1, Mcm2 and PCNA in gastric cancer cells. The molecular mechanism may be related to the up-regulation of P115 by regulating the expression and secretion of MIF and then the downstream ERK1 / 2 signaling pathway. P115 can be used as a new target for studying the molecular mechanism of gastric cancer cell proliferation.