Liu B L, Cui Y S, Tian Y P, Zhu Y Z, Hong Z Q, Zhang X M, Sun G G
School of Public Health, North China University of Science and Technology, Tangshan 063000, China.
School of Clinical Medicine, North China University of Science and Technology, Tangshan 063000, China.
Zhonghua Zhong Liu Za Zhi. 2023 Jun 23;45(6):471-481. doi: 10.3760/cma.j.cn112152-20220331-00225.
To investigate the effects of lncRNA DRAIC on proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells and its mechanism. 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 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. Compared with adjacent tissues, the expression level of DRAIC in lung adenocarcinoma tissues increased (<0.05), but the expression level of let-7i-5p decreased (<0.05). The expression levels of DRAIC and let-7i-5p in lung adenocarcinoma tissues were negatively correlated (=-0.737, <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 (<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 (<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 (<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 (<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 (<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 (<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 (<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 (<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 (<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 (<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 (<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 (<0.05). 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.
探讨长链非编码RNA DRAIC对肺腺癌细胞增殖、凋亡、迁移和侵袭的影响及其机制。采用逆转录-定量实时聚合酶链反应（RT-qPCR）检测2019年至2020年在唐山市人民医院接受手术的40例肺腺癌患者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模拟物组、si-DRAIC+抑制剂-NC组和si-DRAIC+ let-7i-5p抑制剂组。采用CCK-8法和克隆形成实验检测细胞增殖。采用流式细胞术检测细胞凋亡。采用Transwell小室检测细胞迁移和侵袭。采用蛋白质免疫印迹法检测Caspase-3、Caspase-9、Bcl-2和Bax的蛋白表达。采用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，相关性分析采用Pearson相关分析。与癌旁组织相比，肺腺癌组织中DRAIC表达水平升高（P<0.05），而let-7i-5p表达水平降低（P<0.05）。肺腺癌组织中DRAIC与let-7i-5p的表达水平呈负相关（r=-0.737，P<0.05）。si-DRAIC组A549和H1299细胞在48、72和96小时的吸光度值低于si-NC组（P<0.05），克隆形成数[（91.00±6.08对136.67±6.51）；（50.67±1.53对76.67±4.51）]、迁移数[（606.67±31.34对960.00±33.06）；（483.33±45.96对741.67±29.67）]、侵袭数[（185.00±8.19对447.33±22.05）；（365.00±33.87对688.00±32.97）]均低于si-NC组（P<0.05）。然而，细胞凋亡率[（13.43±2.79）%对（4.53±0.42）%；（23.77±1.04）%对（6.60±1.42）%]高于si-NC组（P<0.05）。si-DRAIC组Caspase-3、Caspase-9和Bax的蛋白表达高于si-NC组，Bcl-2的蛋白表达低于si-NC组（P<0.05）。DRAIC位于细胞质中。DRAIC靶向并负调控let-7i-5p的表达。let-7i-5p模拟物组A549和H1299细胞在48、72和96小时的吸光度值低于miR-NC组（P<0.05），克隆形成数[（131.33±14.47对171.33±6.11）；（59.33±4.93对80.33±7.09）]、迁移数[（137.67±3.06对579.33±82.03）；（425.00±11.14对669.33±21.13）]、侵袭数[（54.00±4.36对112.67±11.59）；（80.00±4.58对333.33±16.80）]均低于miR-NC组（P<0.05）。然而，细胞凋亡率[（14.57±1.10）%对（6.97±1.11）%；（23.97±0.42）%对（7.07±1.21）%]高于miR-NC组（P<0.05）。let-7i-5p模拟物组Caspase-3、Caspase-9和Bax的蛋白表达高于miR-NC组，Bcl-2的蛋白表达低于miR-NC组（P<0.05）。si-DRAIC+ let-7i-5p抑制剂组A549和H1299细胞在48、72和96小时的吸光度值高于si-DRAIC+抑制剂-NC组（P<0.05），克隆形成数[（82.00±5.29对59.00±5.57）；（77.67±4.93对41.33±7.57）]、迁移数[（774.33±35.81对455.67±19.04）；（569.67±18.72对433.67±16.77）]、侵袭数[（670.33±17.21对451.00±17.52）；（263.67±3.06对182.33±11.93）]均高于si-DRAIC+抑制剂-NC组（P<0.05）。然而，细胞凋亡率[（7.73±0.45）%对（19.13±1.50）%；（8.00±0.53）%对（28.40±0.53）%]低于si-NC组（P<0.05）。si-DRAIC+ let-7i-5p抑制剂组Caspase-3、Caspase-9和Bax的蛋白表达高于si-DRAIC+抑制剂-NC组，Bcl-2的蛋白表达低于si-DRAIC+抑制剂-NC组（P<0.05）。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、迁移和侵袭并抑制凋亡。
