lncRNA HOTTIP通过miR⁃637/KLK4轴促进肺癌SPC⁃A⁃1细胞的恶性 生物学行为

目的：探讨 lncRNA HOTTIP 对肺癌细胞增殖、凋亡及 EMT 的影响及其作用机制。方法：利用 qPCR 检测 lncRNAHOTTIP、miR-637和KLK4在肺癌SPC-A-1、正常肺上皮BEAS-2B细胞中的表达量；siRNA干扰SPC-A-1细胞中lncRNA HOTTIP的表达后，分别通过CCK-8、Transwell、流式细胞术和WB法检测SPC-A-1细胞增殖、侵袭、凋亡和EMT能力的变化。miRanda软件和双荧光素酶报告基因实验分析 lncRNA HOTTIP 和 miR-637 之间的靶向关系，RNA pull-down 实验检测 lncRNA HOTTIP 和miR-637的吸附作用，检测lncRNA HOTTIP通过miR-637对SPC-A-1细胞增殖、侵袭、凋亡和EMT的调控。利用TargetScan软件分析 miR-637 与 KLK4 的相关性，双荧光素酶报告基因实验检测 miR-637 与 KLK4 mRNA 之间的相互作用；检测 miR-637 通过KLK4 mRNA对SPC-A-1细胞增殖、侵袭、凋亡和EMT的调控。下调lncRNA HOTTIP和miR-637表达后，利用qPCR和WB检测KLK4 mRNA 和蛋白表达水平的变化。结果：与 BEAS-2B 细胞比，在 SPC-A-1 细胞中 lncRNA HOTTIP 呈高表达（P<0.01），miR-637呈低表达（P<0.01），KLK4呈高表达（P<0.01）。下调lncRNA HOTTIP后，SPC-A-1细胞增殖、侵袭与EMT能力显著减弱，细胞凋亡率显著上升（P<0.01）；lncRNA HOTTIP与miR-637具有靶向关系；下调miR-637表达后，SPC-A-1细胞增殖、侵袭与EMT能力显著上升，细胞凋亡率显著降低（P<0.01）。miR-637与 KLK4 3''UTR特异性结合。miR-637通过 KLK4显著促进了 SPC-A-1细胞增殖、侵袭与 EMT，细胞凋亡率显著上升（P<0.01）。下调 lncRNA HOTTIP 使 KLK4 表达显著降低，而下调 miR-637 可促进KLK4表达（P<0.05）。结论：上调lncRNA HOTTIP可通过miR-637/KLK4轴促进肺癌SPC-A-1细胞的增殖、侵袭与EMT而抑制癌细胞凋亡。

Up-regulation of lncRNA HOTTIP promotes the malignant biological behaviors of lung cancer SPC-A-1 cells through miR-637/KLK4 axis

Objective: To investigate the effect and mechanism of lncRNA HOTTIP on proliferation, apoptosis and EMT of lung cancer cells. Methods: The expressions of lncRNA HOTTIP, miR-637 and KLK4 in SPC-A-1, BEAS-2B cells were detected by qPCR.After siRNA interference with the expression of lncRNA HOTTIP, the proliferation, invasion, apoptosis and EMT of SPC-A-1 cells were detected by CCK-8, Transwell, flow cytometry, and WB, respectively. The targeting relationship between lncRNA HOTTIP and miR-637 was analyzed by miRanda software and dual-luciferase reporter gene assay. RNA pull-down assay was used to detect the adsorption of lncRNA HOTTIP and miR-637, and to detect the effects of lncRNA HOTTIP regulating miR-637 on proliferation,invasion, apoptosis, and EMT of SPC-A-1 cells. The correlation between miR-637 and KLK4 was analyzed by TargetScan software,and the interaction between miR-637 and KLK4 was detected by dual-luciferase reporter gene assay. After siRNA interference with the expression of KLK4, the proliferation, invasion, apoptosis, and EMT of SPC-A-1 cells were detected. After down regulation of lncRNA HOTTIP and miR-637 expression, the levels of KLK4 mRNA and protein expression were detected by qPCR and WB. Results:Compared with BEAS-2B cells, the expression of lncRNA HOTTIP in SPC-A-1 cells was significantly up-regulated (P<0.01), the expression of miR-637 was down-regulated (P<0.01), the KLK4 expression was up-regulated (P<0.01). Down-regulation of lncRNA HOTTIP could significantly reduce the proliferation, invasion, and EMT capacity of SPC-A-1 cells, and increase the apoptosis rate (P<0.01). lncRNA HOTTIP had a targeting relationship with miR-637. Down-regulation of miR-637 expression could significantly promote the proliferation, invasion and EMT capacity of SPC-A-1 cells, and inhibit the apoptosis rate (P<0.01). miR-637 specifically bound to KLK4 3''UTR. Down-regulation of KLK4 could significantly inhibit the proliferation, invasion, and EMT capacity of SPC-A-1 cells, and increase the apoptosis rate (P<0.01). Down-regulation of lncRNA HOTTIP could significantly decrease KLK4 expression,while down-regulation of miR-637 could promote KLK4 expression (P<0.05). Conclusion: Up-regulation of lncRNA HOTTIP promotes proliferation, invasion, and EMT of lung cancer SPC-A-1 cells through miR-637/KLK4 axis, and inhibits the apoptosis of cancer cells.