Objective: To investigate the effects of lncRNA DRAIC on proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells and its mechanism. Methods: Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of DRAIC in lung cancer tissues and corresponding adjacent normal tissues of 40 patients with lung adenocarcinoma who underwent surgery in Tangshan People's Hospital from 2019 to 2020. Lung adenocarcinoma cells A549 and H1299 were cultured in vitro and divided into si-NC group, si-DRAIC group, miR-NC group, let-7i-5p mimics group, si-DRAIC+ inhibitor-NC group, and si-DRAIC+ let-7i-5p inhibitor group. CCK-8 method and clone formation experiment were used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Transwell array was used to detect the cell migration and invasion. Western blot was used to detect the protein expressions of Caspase-3, Caspase-9, Bcl-2 and Bax. The double luciferase reporter gene experiment was used to verify the regulatory relationship between DRAIC and let-7i-5p. Independent sample t test was used for comparison between two groups, one-way ANOVA was used for comparison between multiple groups, and Pearson correlation analysis was used for correlation analysis. Results: Compared with adjacent tissues, the expression level of DRAIC in lung adenocarcinoma tissues increased (P<0.05), but the expression level of let-7i-5p decreased (P<0.05). The expression levels of DRAIC and let-7i-5p in lung adenocarcinoma tissues were negatively correlated (r=-0.737, P<0.05). The absorbance value of A549 and H1299 cells in the si-DRAIC group at 48, 72 and 96 hours were lower than those in the si-NC group (P<0.05), the number of clones formed [(91.00±6.08 vs. 136.67±6.51); (50.67±1.53 vs. 76.67±4.51)], the number of migration [(606.67±31.34 vs. 960.00±33.06); (483.33±45.96 vs. 741.67±29.67)], the number of invasion [(185.00±8.19 vs. 447.33±22.05); (365.00±33.87 vs. 688.00±32.97)] were lower than those in the si-NC group (P<0.05). However, the apoptosis rates of cells [(13.43±2.79)% vs. (4.53±0.42)%; (23.77±1.04)% vs. (6.60±1.42)%] were higher than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC group were higher than those in si-NC group, and the protein expression of Bcl-2 was lower than that in si-NC group (P<0.05). DRAIC is located in the cytoplasm. DRAIC targeted and negatively regulated the expression of let-7i-5p. The absorbance values of A549 and H1299 cells in the let-7i-5p mimics group at 48, 72 and 96 hours were lower than those in the miR-NC group (P<0.05), the number of clones formed [(131.33±14.47 vs. 171.33±6.11); (59.33±4.93 vs. 80.33±7.09)], the number of migration [(137.67±3.06 vs. 579.33±82.03); (425.00±11.14 vs. 669.33±21.13)], the number of invasion [(54.00±4.36 vs. 112.67±11.59); (80.00±4.58 vs. 333.33±16.80)] were lower than those in the miR-NC group (P<0.05). However, the apoptosis rates of cells [(14.57±1.10)% vs. (6.97±1.11)%; (23.97±0.42)% vs. (7.07±1.21)%] were higher than those in the miR-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in let-7i-5p mimics group were higher than those in miR-NC group, and the protein expression of Bcl-2 was lower than that in miR-NC group (P<0.05). The absorbance values of A549 and H1299 cells in the si-DRAIC+ let-7i-5p inhibitor group at 48, 72 and 96 hours were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05), the number of clones formed [(82.00±5.29 vs. 59.00±5.57); (77.67±4.93 vs. 41.33±7.57)], the number of migration [(774.33±35.81 vs. 455.67±19.04); (569.67±18.72 vs. 433.67±16.77)], the number of invasion [(670.33±17.21 vs. 451.00±17.52); (263.67±3.06 vs. 182.33±11.93)] were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05). However, the apoptosis rates of cells [(7.73±0.45)% vs. (19.13±1.50)%; (8.00±0.53)% vs. (28.40±0.53)%] were lower than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC+ let-7i-5p inhibitor group were higher than those in si-DRAIC+ inhibitor-NC group, and the protein expression of Bcl-2 was lower than that in si-DRAIC+ inhibitor-NC group (P<0.05). Conclusion: DRAIC is highly expressed in lung adenocarcinoma, and DRAIC promotes the proliferation, migration and invasion of lung adenocarcinoma cells and inhibits apoptosis by targeting let-7i-5p.
目的： 探讨长链非编码RNA DRAIC对肺腺癌细胞增殖、凋亡和迁移侵袭的影响及其作用机制。 方法： 采用实时荧光定量聚合酶链反应检测2019—2020年于唐山市人民医院行手术治疗的40例肺腺癌患者的肺癌组织及相应癌旁正常组织中DRAIC的表达。体外培养肺腺癌细胞A549和H1299，分为si－NC组、si－DRAIC组、miR－NC组、let－7i－5p mimics组、si－DRAIC+inhibitor－NC组和si－DRAIC+let－7i－5p inhibitor组，细胞计数试剂盒8实验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情况，Transwell实验检测细胞迁移和侵袭能力，Western blot检测细胞中Caspase－3、Caspase－9、Bcl－2和Bax蛋白的表达。核质分离检测DRAIC和let－7i－5p亚细胞定位。双荧光素酶报告基因实验验证DRAIC与let－7i－5p的调控关系。两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，相关分析采用Pearson相关分析。 结果： 肺腺癌组织中DRAIC的表达水平高于癌旁正常组织，let－7i－5p的表达水平低于癌旁正常组织(均P<0.05)。肺腺癌组织中DRAIC和let－7i－5p的表达呈负相关(r＝－0.737，P<0.05)。si－DRAIC组A549和H1299细胞的48、72和96 h吸光度值均低于si－NC组(均P<0.05)，si－DRAIC组A549和H1299细胞克隆数分别为(91.00±6.08)个和(50.67±1.53)个，细胞迁移数分别为(606.67±31.34)个和(483.33±45.96)个，细胞侵袭数分别为(185.00±8.19)个和(365.00±33.87)个，均低于si－NC组[分别为(136.67±6.51)个和(76.67±4.51)个，(960.00±33.06)个和(741.67±29.67)个，(447.33±22.05)个和(688.00±32.97)个，均P<0.05]。si－DRAIC组A549和H1299细胞凋亡率分别为(13.43±2.79)%和(23.77±1.04)%，均高于si－NC组[分别为(4.53±0.42)%和(6.60±1.42)%，均P<0.05]，si－DRAIC组细胞中Caspase－3、Caspase－9和Bax蛋白表达水平高于si－NC组，Bcl－2蛋白表达低于si－NC组(均P<0.05)。DRAIC和let－7i－5p主要位于细胞质，DRAIC靶向负调控let－7i－5p。let－7i－5p mimics组A549和H1299细胞48、72和96 h吸光度值均低于miR－NC组(均P<0.05)，let－7i－5p mimics组A549和H1299细胞克隆数分别为(131.33±14.47)个和(59.33±4.93)个，细胞迁移数分别为(137.67±3.06)个和(425.00±11.14)个，细胞侵袭数分别为(54.00±4.36)个和(80.00±4.58)个，均低于miR－NC组[分别为(171.33±6.11)个和(80.33±7.09)个，(579.33±82.03)个和(669.33±21.13)个，(112.67±11.59)个和(333.33±16.80)个，均P<0.05]。let－7i－5p mimics组A549和H1299细胞凋亡率分别为(14.57±1.10)%和(23.97±0.42)%，均高于miR－NC组[分别为(6.97±1.11)%和(7.07±1.21)%，均P<0.05]，let－7i－5p mimics组细胞中Caspase－3、Caspase－9和Bax蛋白表达高于miR－NC组，Bcl－2蛋白表达低于miR－NC组(均P<0.05)。si－DRAIC+let－7i－5p inhibitor组A549和H1299细胞48、72和96h吸光度值均高于si－DRAIC+inhibitor－NC组(均P<0.05)，si－DRAIC+let－7i－5p inhibitor组A549和H1299细胞克隆数分别为(82.00±5.29)个和(77.67±4.93)个，细胞迁移数分别为(774.33±35.81)个和(569.67±18.72)个，细胞侵袭数分别为(670.33±17.21)个和(263.67±3.06)个，均高于si－DRAIC+inhibitor－NC组[分别为(59.00±5.57)个和(41.33±7.57)个，(455.67±19.04)个和(433.67±16.77)个，(451.00±17.52)个和(182.33±11.93)个，均P<0.05]。si－DRAIC+let－7i－5p inhibitor组细胞凋亡率分别为(7.73±0.45)%和(8.00±0.53)%，均低于si－DRAIC+inhibitor－NC组[分别为(19.13±1.50)%和(28.40±0.53)%，均P<0.05]，si－DRAIC+let－7i－5p inhibitor组细胞中Caspase－3、Caspase－9和Bax蛋白表达低于si－DRAIC+inhibitor－NC组，Bcl－2蛋白表达高于si－DRAIC+inhibitor－NC组(均P<0.05)。 结论： DRAIC在肺腺癌中高表达，DRAIC通过靶向let－7i－5p促进肺腺癌细胞的增殖和迁移侵袭能力，抑制细胞凋亡。.
Keywords: Apoptosis; DRAIC; Lung neoplasms; Migration and invasion; Proliferation; let-7i-5p.