干扰LncRNA表达减轻非小细胞肺癌细胞对紫杉醇耐药的机制研究

Study on mechanism of interfering with LncRNA expressing to reduce paclitaxel resistance in non-small cell lung cancer cells

目的研究干扰长链非编码RNA烟酰胺核苷酸转氢酶反义RNA1(LncRNA NNT-AS1)表达减轻非小细胞肺癌(NSCLC)细胞对紫杉醇(TAX)耐药的机制。方法构建NSCLC TAX耐药细胞系(A549/TAX),检测正常、亲本、耐药细胞中LncRNA NNT-AS1的表达情况,并验证miR-582-5p与LncRNA NNT-AS1、HMGB2的靶向关系;体外培养A549/TAX细胞,观察单独干扰LncRNA NNT-AS1或同时干扰LncRNA NNT-AS1、miR-582-5p对细胞中LncRNA NNT-AS1、miR-582-5p、HMGB2 mRNA及其蛋白表达以及细胞活力、克隆形成、凋亡的影响;通过裸鼠成瘤实验观察干扰LncRNA NNT-AS1对肿瘤生长及肿瘤组织中miR-582-5p、HMGB2 mRNA及其蛋白表达的影响。结果与正常细胞比较,LncRNA NNT-AS1在亲本、耐药细胞中均呈高表达(P<0.05),且有递增趋势。经验证,miR-582-5p与LncRNA NNT-AS1、HMGB2均存在靶向关系。干扰LncRNA NNT-AS1表达后,A549/TAX细胞中LncRNA NNT-AS1、HMGB2 mRNA及其蛋白的表达水平以及细胞活力、克隆形成数均显著降低,而miR-582-5p的表达水平、细胞凋亡率均显著升高(P<0.05);同时干扰miR-582-5p表达可使上述改变得以逆转(P<0.05)。干扰肿瘤细胞中LncRNA NNT-AS1的表达后,荷瘤裸鼠的肿瘤体积和肿瘤质量均显著降低,miR-582-5p的表达水平显著升高,HMGB2 mRNA及其蛋白的表达水平均显著降低(P<0.05)。结论干扰LncRNA NNT-AS1的表达可靶向上调miR-582-5p的表达并下调HMGB2的表达,进而减轻NSCLC TAX化疗耐药。

OBJECTIVE To study the mechanism of interfering with long non-coding RNA nicotinamide nucleotide transhydrogenase-antisense RNA1(LncRNA NNT-AS1)expressing to reduce paclitaxel(TAX)resistance in non-small cell lung cancer(NSCLC)cells.METHODS NSCLC TAX-resistant cell line(A549/TAX)was constructed,and the expressions of LncRNA NNT-AS1 in normal,parental,and drug-resistant cells were observed.The targeting relationship of microRNA-582-5p(miR-582-5p)with LncRNA NNT-AS1 and high mobility group box2(HMGB2)was verified.A549/TAX cells were cultured in vitro to observe the effects of interfering with LncRNA NNT-AS1 alone or interfering with LncRNA NNT-AS1 and miR-582-5p on the expressions of LncRNA NNT-AS1 and miR-582-5p,the mRNA and protein expressions of HMGB2,cell viability,clone formation and apoptosis.The effects of interfering with LncRNA NNT-AS1 on tumor growth and the expression of miR-582-5p and the mRNA and protein expressions of HMGB2 in tumor tissue were observed in nude mice.RESULTS Compared with normal cells,LncRNA NNT-AS1 was highly expressed in parental and drug-resistant cells(P<0.05),showing an increasing trend.It was validated that miR-582-5p had a targeting relationship with LncRNA NNT-AS1 and HMGB2.After interfering with the expression of LncRNA NNT-AS1,the expression of LncRNA NNT-AS1 and the mRNA and protein expressions of HMGB2,cell viability and the number of cloned cells in A549/TAX cell,decreased significantly,while the expression of miR-582-5p and the apoptotic rate increased significantly(P<0.05);simultaneously interfering with the expression of miR-582-5p could reverse above changes(P<0.05).Interfering with the expression of LncRNA NNT-AS1 in tumor cell could significantly reduce tumor volume and tumor weight of nude mice bearing tumors;at the same time,the expression of miR-582-5p was up-regulated significantly and the mRNA and protein expressions of HMGB2 were down-regulated significantly(P<0.05).CONCLUSIONS Interfering with the expression of LncRNA NNT-AS1 may alleviate TAX chemotherapy resistance in NSCLC through targeted up-regulation of miR-582-5p and down-regulation of HMGB2.