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目的 :研究沉默磷脂酶Cε1(phospholipase C epsilon 1,PLCε1)基因对食管癌Eca109细胞凋亡和侵袭的影响,并探讨其可能的作用机制。方法 :采用阳离子脂质体法将构建有特异性针对PLCε1基因的短发夹RNA(short hairpin RNA,sh RNA)表达载体(p Genesil-1-PLCε1-sh RNA1、p Genesil-1-PLCε1-sh RNA2和p Genesil-1-PLCε1-sh RNA3)转入Eca109细胞,并筛选出干扰效果最好的表达载体。分别采用FCM和Transwell小室法于PLCε1-sh RNA转染48 h后检测PLCε1基因沉默后对Eca109细胞凋亡和侵袭能力的影响。半定量反转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)法检测PLCε1基因沉默后Eca109细胞中自杀相关因子(factor-associated suicide,Fas)及其配体Fas L(Fas ligand)、CD44、基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)和血管内皮生长因子(vascular endothelial growth factor,VEGF)m RNA表达的改变。结果 :PLCε1-sh RNA2对PLCε1基因的干扰效果最好。PLCε1-sh RNA2转入Eca109细胞48 h后,PLCε1-sh RNA2组Eca109细胞的凋亡率为(30.27±5.13)%,明显高于阴性对照组的(22.06±4.47)%(P<0.05);阴性对照组和PLCε1-sh RNA2组穿过Transwell小室聚碳酸酯膜的Eca109细胞数分别为(82.00±2.00)个和(62.67±3.06)个,PLCε1-sh RNA2组穿过Transwell小室膜的细胞数明显低于阴性对照组(P<0.05)。PLCε1-sh RNA2组Eca109细胞中Fas m RNA的表达水平较阴性对照组明显上调(P<0.05),而Fas L、MMP-9和VEGF m RNA的表达水平均较阴性对照组明显下调(P均<0.05),CD44 m RNA的表达水平未发生改变。结论:沉默PLCε1基因的表达可能通过上调Fas的表达和下调Fas L的表达而促进Eca109细胞的凋亡;并通过下调MMP-9和VEGF的表达抑制Eca109细胞的侵袭能力。
Objective: To investigate the effect of phospholipase C epsilon 1 (PLCε1) gene on the apoptosis and invasion of esophageal cancer Eca109 cells and to explore its possible mechanism. Methods: The short hairpin RNA (shRNA) expression vector (p Genesil-1-PLCε1-sh RNA1, p Genesil-1-PLCε1-sh) was constructed by cationic liposome method RNA2 and p Genesil-1-PLCε1-shRNA3) were transfected into Eca109 cells, and the expression vector with the best interference effect was selected. The effects of PLCε1 gene silencing on the apoptosis and invasion of Eca109 cells were detected by FCM and Transwell chamber method after 48 hours of PLCε1-sh RNA transfection. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of Fas ligand-associated Fas ligand (FasL) in Eca109 cells after PLCε1 gene silencing ), CD44, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) m RNA expression. Results: PLCε1-sh RNA2 had the best interference effect on PLCε1 gene. The apoptosis rate of Eca109 cells in PLCε1-sh RNA2 group was (30.27 ± 5.13)% after PLCε1-sh RNA2 transfection into Eca109 cells for 48 h, which was significantly higher than that in the negative control group (22.06 ± 4.47)% (P <0.05) The numbers of Eca109 cells in the negative control group and the PLCε1-sh RNA2 group passing through the Transwell chamber polycarbonate membrane were (82.00 ± 2.00) and (62.67 ± 3.06), respectively. The number of cells passing through the Transwell chamber membrane in the PLCε1-sh RNA2 group Significantly lower than the negative control group (P <0.05). The expression level of Fas m RNA in PLCε1-sh RNA2 group was significantly higher than that in the negative control group (P <0.05), while the expressions of Fas L, MMP-9 and VEGF m RNA were significantly lower than those in the negative control group <0.05), the expression level of CD44 m RNA did not change. Conclusion: The silencing of PLCε1 gene may promote the apoptosis of Eca109 cells by up-regulating the expression of Fas and down-regulating the expression of Fas L, and inhibiting the invasion ability of Eca109 cells by down-regulating the expression of MMP-9 and VEGF.