miR-582-5p靶向抑制Rab27a对肺癌细胞生物学行为的影响

目的探讨microRNA-582-5p(miR-582-5p)在非小细胞肺癌(NSCLC)中表达及其对A549细胞增殖、侵袭的影响,以及对Rab27a基因的靶向调控作用。方法采用实时荧光定量PCR(QPCR)检测33例行手术治疗的NSCLC患者的癌组织、对应癌旁组织以及A549细胞株、人正常支气管肺上皮细胞株BEAS-2B中miR-582-5p的表达水平。采用miR-582-5p inhibitor(anti-miR-582-5p)和miR-582-5p mimics(mim-miR-582-5p)转染A549细胞,另分别转染miRNA inhibitor无关系列(antiNC)和miRNA mimics无关系列(mim-NC)作阴性对照。采用双荧光素酶报告实验验证Rab27a与miR-582-5p的关系。采用MTT法和Transwell小室法检测各组细胞增殖、侵袭能力;采用QPCR和Western blotting检测各组A549细胞中Rab27a mRNA和蛋白水平。结果 NSCLC组织和癌旁组织中miR-582-5p表达水平分别为0. 26±0. 09和0. 83±0. 10; A549细胞株和BEAS-2B细胞株中miR-582-5p表达水平分别为0. 63±0. 08和1. 17±0. 09,差异均有统计学意义(P<0. 05)。anti-miR-582-5p组A549细胞24、48、72、96 h后的增殖率分别为(115. 68±4. 34)%、(130. 48±5. 48)%、(138. 95±5. 55)%、(147. 03±5. 69)%,明显高于anti-NC组; mim-miR-582-5p组A549细胞24、48、72、96 h后的增殖率分别为(91. 31±4. 18)%、(86. 74±3. 23)%、(79. 45±3. 20)%、(75. 22±4. 09)%,明显低于mim-NC组,差异均有统计学意义(P<0. 05)。Transwell小室结果显示,anti-miR-582-5p组A549细胞侵袭数为189±19,明显高于anti-NC组; mim-miR-582-5p组A549细胞侵袭数为55±8,明显低于mim-NC组,差异均有统计学意义(P<0. 05)。miR-582-5p可抑制野生型Rab27a 3’-UTR报告基因载体的荧光素酶活性,而对突变型Rab27a 3’-UTR的荧光素酶活性无影响。anti-miR-582-5p组A549细胞Rab27a mRNA和蛋白表达水平分别为2. 01±0. 29和0. 85±0. 12,高于anti-NC组; mimi-miR-582-5p组A549细胞Rab27a mRNA和蛋白表达水平分别为0. 35±0. 08和0. 21±0. 05,明显低于mim-NC组,差异均有统计学意义(P<0. 05)。结论上调miR-582-5p表达可抑制Rab27a表达,从而抑制肺癌A549细胞增殖、侵袭活性,miR-582-5p有望成为肺癌新的治疗靶点。

Targeted inhibition of Rab27a by microRNA-582-5p on biological behavior of lung cancer cells

Objective To investigate the expression of microRNA-582-5p （miR-582-5p） in non-small cell lung cancer （NSCLC） and its effect on proliferation and invasion of A549 cells， as well as the targeting regulation of Rab27a gene. Methods The expression of miR-582-5p in 33 NSCLC tissues and adjacent tissues， A549 cell line and human normal bronchopulmonary epithelial cell line BEAS-2B was detected by real-time fluorescence quantitative polymerase chain reaction （QPCR）. MiR-582-5p inhibitor （anti-miR-582-5p） and miR-582-5p mimics （mim-miR-582-5p） transfected into A549 cells， and anti-NC and mim-NC were used as negative control. The relationship between Rab27a and miR-582-5p was validated by double luciferase reporter assay. MTT assay and Transwell chamber assay were used to detect the proliferation and invasion ability of A549 cells， QPCR and Western blotting were used to detect the expression of Rab27a gene and protein. Results The expression of miR-582-5p in NSCLC tissues and adjacent tissues were 0.26±0.09 and 0.83±0.10， respectively. The expression of miR-582-5p in A549 cell lines and BEAS-2B cell lines were 0.63±0.08 and 1.17±0.09， respectively， with statistical significance （P＜0.05）. The proliferation rates of A549 cells in anti-miR-582-5p group after 24， 48， 72 and 96 hours were （115.68±4.34）％， （130.48±5.48）％， （138.95±5.55）％， （147.03±5.69）％， respectively， which were significantly higher than those in anti-NC group （P＜0.05）. The proliferation rates of A549 cells in mim-miR-582-5p group after 24， 48， 72 and 96 hours were （91.31±4.18）％， （86.74±3.23）％， （79.45±3.20）％， （75.22±4.09）％ respectively， which were significantly lower than those in mim-NC group （P＜0.05）. Transwell chamber results showed that the invasion number of A549 cells in anti-miR-582-5p group was 189±19， which was significantly higher than that in anti-NC group； the invasion number of A549 cells in mim-miR-582-5p group was 55±8， which was significantly lower than that in mim-NC group （P＜0.05）. MiR-582-5p inhibited the luciferase activity of wild type Rab27a 3’-UTR reporter gene vector， but had no effect on mutant Rab27a 3’-UTR luciferase activity. The expression of Rab27a mRNA and protein in anti-miR-582-5p group were （2.01±0.29） and （0.85±0.12）， which were higher than anti-NC group （P＜0.05）. The expression of Rab27a mRNA and protein in mim-miR-582-5p group were （0.35±0.08） and （0.21±0.05）， which were lower than mim-NC group （P＜0.05）. Conclusion Up-regulation of the expression of miR-582-5p can inhibit the expression of Rab27a， thus inhibiting the proliferation and invasive activity of lung cancer A549 cells. MicroRNA-582-5p is expected to become a new therapeutic target for lung cancer.