机构地区:[1]河北医科大学第四医院肿瘤免疫科,河北石家庄050035 [2]河北医科大学国际合作研究干细胞实验室,河北石家庄050035
出 处:《中国肿瘤生物治疗杂志》2020年第4期359-364,共6页Chinese Journal of Cancer Biotherapy
基 金:国家自然科学基金资助项目(No.81871894);河北省自然科学基金资助项目(No.H2018206318)。
摘 要:目的:探讨长链非编码RNA(long non-coding RNA,lncRNA)DNA损伤激活的非编码RNA(non-coding RNA-activated by DNA damage,NORAD)对食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)细胞株EC9706增殖和迁移能力的影响及其机制。方法:采用RT-PCR法检测不同ESCC细胞(EC9706、TE1、YES-2、KYSE150)中NORAD m RNA表达水平,通过RNA干扰技术将NORAD的小干扰RNA(siRNA)转染到EC9706细胞(si-NORAD组)以建立NORAD低表达细胞,另设置空白对照组(Ctrl组,不转染任何序列)及阴性对照组(NC组,转染siRNA阴性对照序列),qPCR验证其转染效果。用MTT、平板克隆形成和划痕愈合实验检测敲低NORAD前后EC9706细胞增殖和迁移能力的变化,Western blotting检测敲低NORAD前后EC9706细胞中上皮钙黏蛋白(E-cadherin)、神经钙黏蛋白(N-cadherin)和锌指转录因子Snail的表达变化。结果:在4种ESCC细胞中NORAD mRNA均呈高表达状态,同时与TE1、YES-2、KYSE150细胞相比,EC9706细胞中NORAD mRNA呈显著高表达(P<0.01)。与Ctrl组和NC组比较,转染NORAD-siRNA后,si-NORAD组EC9706细胞中NORAD表达水平显著降低(均P<0.01),EC9706细胞的增殖和迁移能力显著降低(均P<0.05);敲低NORAD表达后,EC9706细胞中E-cadherin表达升高而N-cadherin和Snail表达降低(均P<0.05)。结论:NORAD在EC9706细胞中呈高表达状态,敲低NORAD表达可通过上调E-cadherin、下调N-cadherin和Snail表达而抑制EC9706细胞的增殖和迁移能力。Objective: To investigate the effects and mechanisms of long non-coding RNA(lncRNA) non-coding RNA-activated by DNA damage(NORAD) on the proliferation and migration of esophageal squamous cell carcinoma(ESCC) EC9706 cells. Methods:RT-PCR was used to detect the mRNA expression level of NORAD in different ESCC cells(EC9706, TE1, YES-2, KYSE150). Small interfering RNA(siRNA) targeting NORAD gene was transfected into EC9706 cells(as si-NORAD group) with RNA interference technique to knockdown NORAD expression;in addition, blank control group(as Ctrl group, without any transfection) as well as negative control group(as NC group, transfected with siRNA negative control sequence) were also established. qPCR was used to verify the transfection efficiency. MTT, Colony formation assay and Wound-healing test were used to detect the abilities of proliferation and migration of EC9706 cells before and after NORAD knockdown. Western blotting was used to detect the expressions of E-cadherin,N-cadherin and Snail in EC9706 cells before and after NORAD knockdown. Results: NORAD mRNA was highly expressed in 4 ESCC cell lines. Comparing with TE1, YES-2 and KYSE150 cells, the expression of NORAD mRNA was significantly higher in EC9706 cells(P<0.01). After transfection of NORAD-siRNA into EC9706 cells, the expression of NORAD was down-regulated significantly as comparing with Ctrl group and NC group(all P<0.01), in the meanwhile, the proliferation and migration abilities of EC9706 cells were also significantly suppressed(P<0.05). After NORAD knockdown, the expression of E-cadherin was up-regulated while the expressions of N-cadherin and Snail were down-regulated in EC9706 cells(all P<0.05). Conclusion: NORAD is highly expressed in EC9706 cells;knockdown of NORAD expression can inhibit the proliferation and migration ability of EC9706 probably through up-regulating E-cadherin and down-regulating N-cadherin and Snail.
关 键 词:DNA损伤激活的非编码RNA 食管鳞状细胞癌 EC9706细胞 增殖 迁移
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