DKK1对子宫内膜癌JEC细胞生物学功能的影响

目的:观察RNA干扰对子宫内膜癌JEC细胞DKK1基因表达及生物学功能的影响。方法:用3种RNAi(DKK-homo-366、DKK-homo-811、DKK-homo-886)抑制JEC细胞中DKK1的表达,采用实时荧光定量PCR和Western blot检测干扰效果。实验分组:正常对照组(正常胎牛血清培养的JEC细胞)、NC对照组(转染无意义序列的RNA片段)和siRNA组(转染DKK-homo-811干扰片段)。Transwell小室、MTT、流式细胞仪分别检测干扰前后细胞侵袭、迁移、增殖和细胞凋亡的变化。结果:real-time PCR(RT-PCR)和Western blot结果显示,siRNA组的DKK1表达显著低于正常对照组和NC对照组(P0.05);而后两组比较则无显著差异(P0.05)。Western blot结果显示,正常对照组、NC对照组和siRNA组中β-catenin蛋白表达分别为0.308±0.035、0.305±0.026和0.552±0.018;siRNA组显著高于正常对照组和NC对照组(P0.05)。Transwell小室检测结果显示,干扰DKK1可降低JEC的侵袭、迁移能力。MTT显示,siRNA组吸光度值显著低于NC对照组和正常对照组(P0.05);后两组则无显著差异(P0.05)。siRNA干扰后,siRNA组的细胞凋亡率(7.34±0.23)%]显著高于NC对照组(2.24±0.05)%]和正常对照组(1.62±0.17)%](P0.05);后两组则无显著差异(P0.05)。结论:干扰DKK1可降低JEC的侵袭和增殖能力,促进细胞凋亡。DKK1基因在子宫内膜癌JEC细胞中可能发挥促癌作用。

Effection of RNA interference on DKK1 expression in JEC and its mechanism

Objective:To observe effect of RNA interference of DKK1 gene on its expression in endometrial carcinoma cell line JEC,and to explore its functional mechanism.Methods:Three siRNAs targeting DKK1(DKK-homo-366,DKK-homo-811,DKK-homo-886) and a negative siRNA were designed and transfected into JEC cells using liposome mediated methods as described before.The transfected efficiency were proved by real-time fluorescence quantitative RT-PCR and Western blot assay.The cells were divided into three group:blank control group(JEC cells cultured with normal fetal calf serum),NC control group(JEC cells transfected with nonsence sequence fragment) and the siRNA group(JEC cells transfected with DKK-homo-811 fragment).The difference of invasiveness,migration,proliferation and apoptosis of JEC cells before and after DKK1 gene silencing were detected with transwell chamber assay,MTT assay and flow cytometry respectively.Results:Both mRNA and protein expression of DKK1 in all three siRNA groups were significantly decreased than those in NC control group(P < 0.05),meanwhile there had no significant difference between blank control group and NC control group(P > 0.05).The expression of β-catenin in blank control group,NC control group and siRNA group were 0.308 ± 0.035,0.305 ± 0.026,0.552 ± 0.018 respectively.And β-catenin expression of siRNA group were significantly increased than that of the former two group(P < 0.05).Transwell chamber assay showed that the ability of invasiveness and migration of JEC cells were lower after silencing of DKK1.MTT assay showed that the absorbance value of siRNA group were significantly increased than that of blank and NC control group(P < 0.05),meanwhile there had no significantly difference of that between the two control groups(P > 0.05).Flow cytometry showed that apoptosis rate of siRNA group((7.34 ± 0.23) %) were significantly increased than that of blank(1.62 ± 0.17) % and NC(2.24 ± 0.05) % control group(P <0.05),meanwhile there had no significantly difference of that between the two control groups(P >0.05).Conclusion:Silencing of DKK1 gene could inhibit the proliferation,invasion and migration of JEC cells and promote the apoptosis of JEC cells.DKK1 gene may have positive efforts on proliferation and invasion of endometrial carcinoma cell line JEC.