LncRNA DRAIC regulates the proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells by targeting let-7i-5p / 中华肿瘤杂志

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.

Highly expressed miRNA let-7i-5p in renal cell carcinoma tissues regulates the malignant biological behaviors of 769-P cells through hyaluronan-binding protein 4 / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨miRNA let-7i-5p在肾细胞癌（renal cell carcinoma，RCC）组织中的表达水平及其对RCC细胞769-P的增殖、迁移、侵袭和透明质酸结合蛋白4（hyaluronan-binding protein 4，HABP4）表达的影响。方法：利用TCGA RCC数据库及GEO数据库对let-7i-5p在RCC组织中的表达进行meta分析。体外常规培养人RCC细胞769-P并对其进行转染，根据转染物不同分为过表达组（转染let-7i-5p模拟物）、抑制组（转染let-7i-5p抑制物）和对照组（转染NC序列）。采用CCK-8、细胞划痕实验、Transwell实验及WB法检测let-7i-5p对769-P细胞增殖活力、划痕愈合率、穿膜侵袭细胞数及HABP4表达水平的影响。双荧光素酶报告基因实验验证let-7i-5p与HABP4 mRNA的靶向关系。结果：分析TCGA RCC数据库及5个GEO数据集（GSE23085、GSE47582、GSE95385、GSE16441和GSE71302）中数据结果显示，let-7i-5p在RCC组织中的表达水平显著高于正常肾组织（均P<0.05）。体外实验结果显示，与对照组相比，过表达组在24、48及72 h时细胞增殖活力显著升高，而抑制组显著降低（均P<0.01）；过表达组的划痕愈合率[（37.276±2.058）% vs（15.663±2.949）%，P<0.01]和穿膜侵袭细胞数[（377.000±34.044） vs （255.667±25.384）个，P<0.01]均显著升高，而抑制组的划痕愈合率[（8.791±2.568）% vs（15.663±2.949）%，P<0.05]和穿膜侵袭细胞数[（170.333±14.978）vs（255.667±25.384）个，P<0.01]均显著降低。在野生型HABP4-3’ URT质粒组中，过表达let-7i-5p可显著抑制细胞的荧光素酶活性（P<0.01）；而在突变型HABP4-3’ URT质粒组中，过表达let-7i-5p对细胞的荧光素酶活性无影响（NS，P>0.05）。WB检测结果显示，与对照组相比，过表达组的HABP4和E-cadherin的水平均降低（均P<0.01）、CDK2的水平升高（P<0.01），而抑制组则相反（均P<0.01）。结论：Let-7i-5p在RCC组织中呈高表达，其可能通过靶向HABP4基因来促进769-P细胞的增殖、迁移和侵袭。