LncRNA Malat1调控miR-124影响肺腺癌细胞侵袭转移的机制研究

Dysfunctions of various non-coding RNAs are common in the process of invasion and metastasis of lung adenocarcinoma. MALAT1 is a long non-coding RNA which has been proved to have powerful functions in a wide variety of tumors. By preliminary experiments, we found that: MALAT1 has no effects on the lung adenocarcinoma cell proliferation or apoptosis, but can significantly inhibit lung adenocarcinoma cancer cell migration and invasion ability, suppress the expression of miR-124 and block the EMT process. According to the results of online biology prediction software analysis and qRT-PCR analysis, we designed a dual luciferase reporter gene experiment, which confirmed the MALAT1 can be directly combined with miR-124 in lung adenocarcinoma cell. Finally, we conducted the rescue experiment and proved that functions of low expression of MALAT1 in lung adenocarcinoma cell can be partly reversed by inhibition of miR-124. In conclusion, we propose a hypothesis: MALAT1 can serve as a competing endogenous RNA (ceRNA) by a competitive combination of miR-124, and in turn affect the lung adenocarcinoma cell invasion and metastasis and EMT process.This study is expected to theoretically enrich the functions and mechanisms of MALAT1 in the invasion and metastasis of lung adenocarcinoma, and provide new ideas for clinical diagnosis and treatment of lung adenocarcinoma.

肺腺癌侵袭转移过程中普遍存在各种非编码RNA功能异常，其中MALAT1是一种在多种肿瘤中被证实具有强大功能的长链非编码RNA。通过前期实验我们发现：MALAT1不影响肺腺癌细胞增殖能力，而能够显著抑制肺腺癌细胞迁移和侵袭能力，同时抑制miR-124的表达并抑制EMT进程。结合在线生物学预测软件分析结果和qRT-PCR分析结果，我们设计了双荧光素酶报告基因实验，证实了在肺腺癌细胞中MALAT1的确可与miR-124直接结合。最后，我们设计了拯救实验发现：低表达MALAT1对肺腺癌细胞的作用可被敲除miR-124部分逆转。综上，我们提出假说：MALAT1可作为竞争性内源RNA通过竞争性结合miR-124，进而影响肺腺癌细胞的侵袭转移和EMT进程。此研究可望从理论上丰富MALAT1在肺腺癌侵袭转移中的功能及分子机制，并为临床肺腺癌诊治提供新思路。

近些年的大量研究表明长链非编码RNA（LncRNA）和微小RNA（miRNA）在肿瘤的发生发展过程中起重要作用。LncRNA肺腺癌相关转录本1（MALAT1）和miR-429已分别被证明与肺腺癌（LUAD）的进展有关，但其是否由互相作用仍不清楚。此研究中，我们进行qRT-PCR实验检测了LUAD组织或细胞中miR-429，MALAT1，ZEB1，E-钙粘着蛋白，N-钙粘着蛋白和波形蛋白等基因的mRNA表达水平。用蛋白质印迹分析检测ZEB1，E-钙粘着蛋白，N-钙粘着蛋白和波形蛋白的蛋白水平。使用Cell Counting Kit-8（CCK-8）和Transwell侵袭试验来评估细胞增殖和侵袭的能力。采用双荧光素酶报告基因分析法研究MALAT1，miR-429和ZEB1的靶标作用关系。首先在LUAD组织和细胞系中检测到miR-429的表达减少和MALAT1的表达增加。进一步的研究发现，miR-429通过直接结合ZEB1的3'UTR抑制LUAD细胞的增殖，侵袭和EMT过程。动物实验结果表明：靶向MALAT1的治疗可减少肺癌转移瘤的形成，其作用亦可部分被miR-429所消除。总之，我们揭示了MALAT1 / miR-429 / ZEB1通路对于LUAD发展的促进作用，为LUAD的精准治疗提供了一个潜在的靶通路。