PLXDC2通过激活PI3K-AKT信号转导通路促进胃癌细胞增殖及克隆形成的机制研究

PLXDC2 promotes the proliferation and clonal formation of gastric cancer cells by activating the PI3K-AKT signal pathway

目的探讨PLXDC2在胃癌组织中的表达特征及其促进胃癌细胞增殖、克隆形成的分子机制。方法通过检索GEPIA和TCGA数据库, 分别筛选在胃癌中高表达(tumor/normal>2)以及与胃癌临床预后显著相关的基因进行重合分析, 通过热图分析及高表达预后差原则确定研究目的基因。采用qRT-PCR法和免疫印迹检测目的基因在30例临床胃癌组织及癌旁正常组织中表达情况。通过转染干扰siRNA敲低目的基因表达后, 利用MTT法细胞增殖实验、平板克隆形成实验和流式细胞周期实验分别验证目的基因对胃癌细胞增殖、克隆形成及细胞周期的影响。通过生物信息学数据库检测分析与目的基因共表达参与的信号通路, 探索目的基因在胃癌中发挥功能的潜在分子机制, 并行进一步验证。结果胃癌组织中PLXDC2 mRNA(5.62±1.35)和蛋白(4.13±0.35)水平显著高于癌旁正常组织中的mRNA(4.22±0.86)和蛋白(1.24±0.23)水平(均P<0.001);随着胃癌恶性程度增加, PLXDC2表达逐渐升高, 且具有高表达预后差的临床特征(P<0.05)。转染培养5d时, siPLXDC2#1组(0.41±0.05)和siPLXDC2#2组(0.35±0.07)BGC-823细胞增殖速率较siCTL组(0.58±0.08)显著减缓(P<0.01)。siPLXDC2#1组(0.49±0.07)和siPLXDC2#2组(0.21±0.04)细胞克隆形成率显著低于siCTL组(1.00±0.01)(F=218.773, P<0.001), 细胞周期显著阻滞在G1期。siPLXDC2#1组和siPLXDC2#2组细胞中p-PI3K和P-AKT水平均较siCTL组显著降低(均P<0.001), 总PI3K和AKT表达不变。在siPLXDC2组细胞中共转染pcDNA3.1-PLXDC2, 显著逆转了siPLXDC2对胃癌细胞增殖、克隆形成及信号通路的抑制作用, 细胞水平基本恢复至正常。结论胃癌中PLXDC2高表达, 其可能通过调控激活PI3K/AKT信号通路, 促进胃癌细胞的增殖和克隆形成, 加快细胞周期进程, 进而参与胃癌的发生发展。

Objective To investigate the profile of PLXDC2 expression in gastric cancer tissues and the molecular mechanism by which PLXDC2 promotes the proliferation and clonal formation of gastric cancer cells.Methods By retrieving the GEPIA and TCGA databases,the genes highly expressed in gastric cancer(tumor/normal>2)and significantly related to the clinical prognosis of gastric cancer were screened for overlap analysis.The target genes were determined by gene expression heatmap and the principle of high-expression-poor-prognosis.QRT-PCR and Western blotting were used to examine the expression of the target gene in 30 pairs of clinical gastric cancer tissue and normal adjacent tissue samples.After knock-down of target gene by transfection with interference siRNA,MTT cell proliferation assay,plate clone formation assay and flow cytometry were used to verify the effect of target genes on proliferation,clonal formation and cell cycle of gastric cancer.By retrieval and analysis of bioinformatics database for signal pathways involved in co-expression of the target gene,the potential molecular mechanism by which the target genes work in gastric cancer was explored and further determined.Results The PLXDC2 mRNA(5.62±1.35)and protein(4.13±0.35)levels in gastric cancer tissues were significantly higher than those in cancer-adjacent normal tissues[mRNA(4.22±0.86),protein(1.24±0.23)(both P<0.001)].With higher malignancy of gastric cancer,the PLXDC2 expression gradually increased,showing a clinical pattern of high-expression-poor-prognosis(P<0.05).On day 5 after culture with transfection,the proliferation rates of BGC-823 cells in the siPLXDC2#1 group(0.41±0.05)and siPLXDC2#2 group(0.35±0.07)were significantly lower than that in the siCTL group(0.58±0.08)(P<0.01).The rates of clonal formation in the siPLXDC2#1 group(0.49±0.07)and siPLXDC2#2 group(0.21±0.04)were significantly lower than that in the siCTL group(1.00±0.01)(F=218.773,P<0.001),and in these two groups,the cell cycle was significantly arrested at G1 phase.In the siPLXDC2#1 and siPLXDC2#2 groups,the levels of p-PI3K and P-AKT were significantly lower than those in the siCTL group(P<0.001),and the total expression of PI3K and AKT remained unchanged.Co-transfection with pcDNA3.1-PLXDC2 to the siPLXDC2 group of cells significantly reversed the inhibitory effects of siPLXDC2 on proliferation,clonal formation and signaling pathways of gastric cancer cells,and the cell level was shown to largely resume normal.Conclusion The highly expressed PLXDC2 in gastric cancer may activate the PI3K/AKT signaling pathway,promote proliferation and clonal formation of gastric cancer cells,accelerate cell cycling,and participate in the development and progression of gastric cancer.