miR-323a-3p通过靶向结合TM4SF1而调控NSCLC A549细胞的增殖、 迁移和侵袭

目的：探究miR-323a-3p、四次穿膜蛋白超家族成员1（TM4SF1）在NSCLC组织和细胞中的表达及两者间的靶向调控关系，观察两者表达对A549细胞增殖、迁移、侵袭和裸鼠移植瘤生长的影响。方法：收集2014年1月至12月间青海省人民医院手术切除的20例NSCLC组织及其相应的癌旁组织，qPCR和WB法检测癌组织中miR-323a-3p、TM4SF1 mRNA 和TM4SF1蛋白的表达。向A549细胞转染miR-323a-3p mimic，采用MTT法、Transwell法、WB法检测miR-323a-3p过表达对细胞的增殖、迁移和侵袭以及TM4SF1、细胞周期蛋白 D1（cyclin D1）、p21、MMP-2、MMP-9蛋白表达的影响。采用生物信息学预测工具 StarBase 和双荧光素酶报告基因实验分析 miR-323a-3p 与 TM4SF1 靶向关系。将 si-TM4SF1 转染至 A549 细胞，以及分别将 miR323a-3p mimic 与 pcDNA 或 pcDNA-TM4SF1 共转染 A549 细胞，评估细胞增殖、迁移和侵袭能力的变化；同时建立各组细胞的BALB/c裸鼠移植瘤模型，在14、21和28 d时测量并计算移植瘤体积。结果：与癌旁组织相比，NSCLC组织中miR-323a-3p表达水平明显下调，TM4SF1 mRNA和蛋白表达水平显著上调（均P<0.01）。miR-323a-3p过表达或抑制TM4SF1表达都会降低A549细胞的增殖、迁移、侵袭能力及cyclin D1、MMP-2、MMP-9蛋白表达而促进p21蛋白表达，并且抑制A549细胞裸鼠移植瘤的生长（均P<0.01）。生物信息学StarBase工具预测和双荧光素酶基因报告实验结果显示miR-323a-3p能够靶向结合TM4SF1基因并调控 TM4SF1的表达。上调TM4SF1表达后，miR-323a-3p过表达对A549细胞恶性生物学行为及cyclin D1、MMP-2、MMP-9蛋白表达、移植瘤生长的抑制作用均被部分逆转（均P<0.01），对p21蛋白表达的促进作用也被逆转（P<0.01）。结论：miR-323a-3p 通过靶向下调肺癌A549细胞中TM4SF1的表达抑制细胞的增殖、迁移、侵袭和裸鼠移植瘤生长。

miR-323a-3p regulats the proliferation, migration and invasion of NSCLC A549 cells through targeting TM4SF1

Objective: To explore the expression of miR-323a-3p and transmembrane 4 super family member 1 (TM4SF1) in lung cancer tissues and cells, and the target regulatory relationship between them, and to observe their effects on the proliferation, migration and invasion of lung cancer A549 cells, as well as their effects on the growth of A549 cell transplanted tumors in nude mice. Methods:Twenty pairs of lung cancer tissues and their corresponding paracancerous tissues were collected from January 2014 to December 2014 in Qinghai Provincial People''s Hospital. The expressions of miR-323a-3p, mRNA and TM4SF1 expression of TM4SF1 in lung cancer tissues were detected by qPCR and WB methods. A549 cells were transfected with miR-323a-3p mimic, and the effects of miR-323a-3p overexpression on cell proliferation, migration and invasion as well as protein expression of TM4SF1, cyclin D1, p21, MMP-2 and MMP-9 were detected by MTT method, Transwell method and WB method, respectively. The targeting relationship between miR-323a3p and TM4SF1 was analyzed using the bioinformatics prediction tool StarBase and dual-luciferase reporter gene experiments. A549 cells were transfected with si-TM4SF1, miR-323a-3p+pcDNA or miR-323a-3p+pcDNA-TM4SF1, respectively, and the changes in proliferation, migration and invasion of A549 cells were evaluated. In the meanwhile, xenograft model was established in BALB/c nude mice by transplanting A549 cells with various treatment, and the xenograft volume was measured and calculated on day 14, 21 and 28.Results: Compared with paracancerous tissues, the expression levels of miR-323a-3p in lung cancer tissues were significantly downregulated, while the mRNA and protein levels of TM4SF1 were significantly up-regulated (all P<0.01). Overexpression of miR-323a-3por inhibition of TM4SF1 expression reduced the proliferation, migration, and invasion of A549 cells, as well as the expression of cyclin D1, MMP-2, and MMP-9 proteins, but promoted the expression of p21 protein (all P<0.01). StarBase prediction and dual-luciferase gene reporter experiment showed that miR-323a-3p could complimentarily bind with TM4SF1 and regulate its expression. After upregulation of TM4SF1 expression, the inhibitory effects of miR-323a-3p overexpression on malignant biological behaviors of A549 cells and protein expression of cyclin D1, MMP-2 and MMP-9, as well as tumor growth in nude mice were reversed (all P<0.01);moreover, the promotive effect of miR-323a-3p overexpression on the protein expression of p21 was also reversed (P<0.01).Conclusion: miR-323a-3p inhibits proliferation, migration and invasion of A549 cells and suppresses xenograft growth in nude mice by downregulating TM4SF1 expression.