LncRNA MALAT-1靶向microRNA-370-3p调节Akt通路对肺癌细胞生物学行为影响的机制研究

Mechanism research of LncRNA MALAT-1 targeting microRNA-370-3p on biological behavior of lung cancer cells by regulating Akt

摘要:

目的 探讨长链非编码RNA肺腺癌转移相关转录因子-1（LncRNA MALAT-1）是否能靶向调节microRNA-370-3p（miR-370-3p）的表达，以及对Akt通路和肺癌细胞生物学行为的影响。方法 选用非小细胞肺癌（NSCLC）A549细胞体外培养，分别抑制LncRNA MALAT-1（转染si-MALAT-1）或过表达miR-370-3p（转染miR-370-3p mimic），抑制LncRNA MALAT-1和干扰miR-370-3p（同时转染si-MALAT-1和anti-miR-370-3p），观察A549细胞的生物学行为和Akt通路蛋白的表达。qRT-PCR检测LncRNA MALAT-1、miR-370-3p mRNA的表达；MMT法检测细胞增殖；流式细胞术检测细胞凋亡；Transwell实验检测细胞迁移与侵袭；Western blotting检测Akt、p-Akt、PI3K、p-PI3K蛋白相对表达量。构建MALAT-1野生型（MALAT-1^WT_1uc）与突变型（MALAT-1^MUT_1uc）荧光素酶报告基因质粒并分别与miR-370-3p、miR-NC转染至A549细胞，观察荧光结合强度并检测miR-370-3p的表达。结果 抑制LncRNA MALAT-1或过表达miR-370-3p能抑制A549细胞迁移、侵袭，促进细胞凋亡，减少A549细胞活性（P <0.05），并下调p-Akt和p-PI3K蛋白的表达（P <0.05）。与单纯抑制LncRNA MALAT-1比较，抑制LncRNA MALAT-1并干扰miR-370-3p能促进A549细胞迁移、侵袭，抑制细胞凋亡，增加A549细胞活性（P <0.05），并上调p-Akt和p-PI3K蛋白的表达（P <0.05）。TargetScan靶基因预测发现LncRNA MALAT-1与miR-370-3p存在结合位点，荧光素酶报告基因实验验证发现，与MALAT-1^WT_1uc+Control组和MALAT-1^WT_1uc+miR NC组比较，MALAT-1^WT_1uc+miR-370-3p组相对荧光强度下降（P <0.05），MALAT-1^MUT_1uc+ miR-370-3p荧光强度无变化（P >0.05），进一步qRT-PCR结果发现，与Control组比较，si-MALAT-1组的miR-370-3p mRNA相对表达量升高（P <0.05），与si-MALAT-1组比较，si-MALAT-1+anti-miR-370-3p组的miR-370-3p mRNA相对表达量降低（P <0.05）。结论 LncRNA MALAT-1可以靶向负调控miR-370-3p。抑制LncRNA MALAT-1可以上调miR-370-3p的表达，抑制A549细胞的增殖、迁移及侵袭，促进A549细胞的凋亡，并下调Akt通路蛋白的磷酸化。

Abstract:

Objective To investigate whether long-non-coding RNA metastasis associated lung adenocarcinoma transcript-1 (LncRNA MALAT-1) can target and regulate the expression of microRNA-370-3p (miR-370-3p) and affect Akt pathway and biological behavior of lung cancer cells.Methods A549 NSCLC cells were cultured in vitro, inhibit LncRNA MALAT-1 (transfection of si- MALAT-1), or overexpression of miR-370-3p (transfection of miR-370-3p mimic), and inhibit LncRNA MALAT-1 and interfere with miR-370-3p (transfection of si- LncRNA MALAT-1 and anti-miR-370-3p), respectively. The biological behavior of A549 cells and the expression of Akt pathway protein were observed. RT-PCR was used to detect the expression of RNA, MMT was used to detect cell proliferation, flow cytometry was used to detect cell apoptosis, Transwell experiment was used to detect cell migration and invasion, and Western blotting was used to detect the expression of Akt, p-Akt, PI3K and p-PI3K proteins. MALAT-1 wild type (MALAT-1^WT_1uc) and mutant (MALAT-1^MUT_1uc) luciferase reporter gene plasmids were constructed and transfected into A549 cells with miR-370-3p and miR-NC respectively. The fluorescence binding intensity was observed and the expression of miR-370-3p was detected.Results Inhibition of LncRNA MALAT-1 or overexpression of miR-370-3p can inhibit the migration and invasion of A549 cells, promote cell apoptosis, reduce the activity of A549 cells (P < 0.05), and down-regulate the expression of p-Akt and p-PI3K proteins (P < 0.05). Compared with single inhibition of LncRNA MALAT-1, simultaneous inhibition of LncRNA MALAT-1 and interference of miR-370-3p can improve the proliferation, migration, and invasion activities of A549 cells, reduce apoptosis, and up-regulate p-Akt and p-PI3K protein expression (P < 0.05). Targetsca target gene prediction found that LncRNA MALAT-1 has a binding site with miR-370-3p, and the luciferase reporter gene experiment verified that the relative fluorescence intensity of MALAT-1^WT_1uc + miR-370-3p group decreased (P < 0.05). Compared with MALAT-1^WT_1uc +control group and MALAT-1^WT_1uc+miR NC group, while the fluorescence intensity of MALAT-1^MUT_1uc +miR-370-3p did not change (P > 0.05). Further qRT-PCR results showed that the relative expression of miR-370-3p mRNA in si-MALAT-1 group was higher than that in control group (P < 0.05), and the relative expression of miR-370-3p mRNA in si-MALAT-1+anti-miR-370-3p group was lower than that in si-MALAT-1 group(P < 0.05).Conclusion LncRNA MALAT-1 can target and negatively regulate the expression of miR-370-3p. Inhibiting LncRNA MALAT-1 can up regulate miR-370-3p, inhibit the proliferation, migration, and invasion of A549 cells, aggravate the apoptosis of A549 cells, and down regulate the phosphorylation of Akt pathway protein.