[Expression and regulatory role of ultraconserved long non-coding RNA uc.77 in lung cancer].

Objective: To investigate the effect and molecular mechanism of ultra-conservative long non-coding RNA uc.77 in lung cancer. Methods: Lung cancer tissues and adjacent normal tissues were obtained from 61 patients with lung cancer who were diagnosed with lung cancer and underwent surgery from 2014 to 2016 in the General Hospital of the Southern Theater Command of the People's Liberation Army. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the uc.77 relative expressions in normal human bronchial epithelial cells 16HBE, lung cancer cell lines, and 61 pair lung cancer tissues. Uc.77 siRNA was transfected into lung cancer cells to interfere with the expression of uc.77, qRT-PCR was used to verify the interference effect, CCK8 method and clone formation experiment were used to detect cell proliferation ability, flow cytometry was used to detect apoptosis and cell cycle changes. H1299 cells transfected with uc.77 siRNA were injected into the subcutaneous right side of BALB/c nude mice to construct a tumor-bearing model for exploring the role of uc.77 on tumor growth. Western blot and qRT-PCR methods were used to detect the protein and mRNA expressions of p21. Results: The relative expression levels of uc.77 in lung cancer cell lines 95D, H1299, A549, H460, H446 and 16HBE-T were significantly higher than that of 16HBE cells (P<0.05). The uc.77 RNA expression levels of lung cancer tissues was significantly higher than that of the adjacent normal tissues (P<0.001). In addition, increased lncRNA uc.77 expression was significantly associated with big tumor size, lymph node metastasis and advanced TNM stage (P<0.05). After transfection with uc.77 siRNA, the expressions of uc.77 in H1299, 95-D and 16HBE-T cells were reduced (P<0.05), and the cell proliferation capacities were reduced at 48 hours and 72 hours (P<0.05). After transfection with uc.77 siRNA-1, the G(0)/G(1) phase cell ratio of H1299 siRNA-1 group [(71.86±3.46)%] was higher than those of H1299-control group [(47.62±5.48)%] and H1299 siRNA-NC group [(61.38±5.62)%, P<0.05], S phase cell ratio of H1299 siRNA-1 group [(14.99±3.61)%] was lower than those of H1299-control group [(34.95±7.05)%] and H1299 siRNA-NC group [(23.75±5.87)%, P<0.05], the apoptosis rate of H1299 siRNA-1 group [(4.90±1.80)%] was higher than those of H1299-control group [(3.30±0.80)%] and H1299 siRNA-NC group [(2.80±1.20)%, P<0.05], the colony formation rate of H1299 siRNA-1 group [(19.20±2.00)%] was lower than those of H1299 control group [(32.60±2.00)%] and H1299 siRNA-NC group [(34.40±1.00)%, P<0.05]. The results of the nude mice tumor formation experiment showed that the tumor volume of the H1299 siRNA-1 group was significantly lower than those of the H1299-control group and the H1299-negative control group (P<0.05), the average tumor weight of H1299 siRNA-1 group was significantly lower than those of H1299-control group and H1299-negative control group (P<0.05), tumor cell growth marker Ki-67 in the H1299 siRNA-1 group showed weak positive, and Ki-67 in the H1299-control group and H1299-negative control group showed positive. The result of qRT-PCR analysis showed that the mRNA expression level of p21 in H1299 siRNA-1 group (2.57±0.45) was higher than those in H1299 control group (1.00±0.00, P=0.001) and H1299 siRNA-NC group (1.52±0.37, P=0.009). The result of western blotting analysis also showed that the expression of p21 protein level in H1299 siRNA-1 group increased. Conclusions: The expression of ultraconserved long non-coding RNA uc.77 is elevated in lung cancer cell lines and lung cancer tissues. Silencing the expression of ultraconservative long noncoding RNA uc.77 can inhibit tumor growth, and blocking uc.77 expression may be a potential therapeutic target for lung cancer.目的：探讨超保守长链非编码RNA uc.77(uc.77)在肺癌中的表达及其分子机制。 方法：肺癌组织及癌旁正常组织来自2014—2016年解放军南部战区总医院确诊肺癌并行手术的61例肺癌患者。采用实时荧光定量聚合酶链反应(qRT-PCR)检测肺癌细胞株中uc.77的相对表达水平，同时在61对肺癌组织及其癌旁正常组织中验证其表达趋势，分析uc.77的表达与肺癌患者临床病理特征的关系。转染uc.77 siRNA敲低uc.77的表达，采用细胞计数试剂盒8法和克隆形成实验检测细胞增殖能力，流式细胞术检测细胞凋亡和细胞周期变化。将转染uc.77 siRNA的肺癌H1299细胞注射至BALB/c裸鼠构建荷瘤模型，观察uc.77对肿瘤生长的影响，对肿瘤组织进行HE染色和免疫组化检测，qRT-PCR和Western blot法检测p21 mRNA和蛋白表达水平。 结果：肺癌细胞系95-D、H1299、A549、H460、H446和16HBE-T中uc.77的表达水平高于正常支气管上皮样细胞16HBE(均P<0.05)；61例肺癌组织中uc.77的表达水平高于旁正常组织(P<0.001)。肿瘤体积、有无淋巴结转移、TNM分期与uc.77的表达有关(均P<0.05)。转染uc.77 siRNA 48和72 h后，siRNA-1组H1299、95-D、16HBE-T细胞A值低于各自对照组和siRNA-NC组(均P<0.05)。转染uc.77 siRNA-1后，H1299 siRNA-1组细胞G(0)/G(1)期细胞比例[(71.86±3.46)%]高于H1299对照组[(47.62±5.48)%]和H1299 siRNA-NC组[(61.38±5.62)%，均P<0.05]；H1299 siRNA-1组细胞S期细胞比例[(14.99±3.61)%]低于H1299对照组[(34.95±7.05)%]和H1299 siRNA-NC组[(23.75±5.87)%，均P<0.05]。H1299 siRNA-1组细胞凋亡率[(4.90±1.80)%]高于H1299对照组[(3.30±0.80)%]和H1299 siRNA-NC组[(2.80±1.20)%，均P<0.05]。H1299 siRNA-1组细胞克隆形成率[(19.20±2.00)%]低于对照组[(32.60±2.00)%]及siRNA-NC组[(34.40±1.00)%，均P<0.05]。H1299 siRNA-1组小鼠肿瘤体积低于H1299对照组和H1299 siRNA-NC组(均P<0.05)，H1299 siRNA-1组小鼠肿瘤重量低于H1299-对照组和H1299 siRNA-NC组(均P<0.05)。Ki-67免疫组化结果显示，H1299对照组和H1299 siRNA-NC组肿瘤细胞增长标志物Ki-67呈阳性，而H1299 siRNA-1组呈弱阳性。qRT-PCR检测结果显示，H1299 siRNA-1组细胞中p21的mRNA表达水平(2.57±0.45)高于对照组(1.00±0.00，P＝0.001)和H1299 siRNA-NC组(1.52±0.37, P＝0.009)。Western blot结果表明，H1299 siRNA-1组细胞p21蛋白表达水平升高。 结论：超保守长链非编码RNA uc.77在肺癌细胞株和肺癌组织中均表达升高，敲低uc.77的表达可抑制肺癌细胞增殖和肿瘤生长，提示uc.77在肺癌中发挥促癌基因的作用，uc.77可能是肺癌的潜在治疗靶标。.