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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(P<0.05);而后两组比较则无显著差异(P>0.05)。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(P<0.05)。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(P<0.05);后两组则无显著差异(P>0.05)。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)%](P<0.05);后两组则无显著差异(P>0.05)。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。
Objective: To observe the effect of RNA interference on DKK1 gene expression and biological function in JEC cells of endometrial carcinoma. Methods: The DKK1 expression in JEC cells was inhibited by three kinds of RNAi (DKK-homo-366, DKK-homo-811, DKK-homo-886). The interference effect was detected by real- Experimental groups: normal control group (normal fetal bovine serum-cultured JEC cells), NC control group (transfected with non-meaningful sequence of RNA fragments) and siRNA group (transfected DKK-homo-811 interference fragment). Transwell chamber, MTT and flow cytometry were used to detect the changes of cell invasion, migration, proliferation and apoptosis before and after interference. Results: The expression of DKK1 in siRNA group was significantly lower than that in normal control group and NC control group (P <0.05) by real-time PCR (RT-PCR) and Western blotting, but no significant difference was found between the two groups ). The results of Western blot showed that the protein expression of β-catenin in normal control group, NC control group and siRNA group were 0.308 ± 0.035,0.305 ± 0.026 and 0.552 ± 0.018, respectively; the siRNA group was significantly higher than the normal control group and NC control group (P < 0.05). Transwell chamber test results show that interference DKK1 can reduce the invasion and migration of JEC. MTT showed that the absorbance of siRNA group was significantly lower than that of NC control group and normal control group (P <0.05); there was no significant difference between the two groups (P> 0.05). After siRNA interference, the apoptotic rate in siRNA group was significantly higher than that in NC group [(7.34 ± 0.23)%] [(2.24 ± 0.05)%] and normal control group (1.62 ± 0.17)%] (P <0.05) ; There was no significant difference between the latter two groups (P> 0.05). Conclusion: Interference of DKK1 can reduce the invasion and proliferation of JEC and promote apoptosis. DKK1 gene in endometrial cancer JEC cells may play a role in promoting cancer.