Identification Of Long Non-coding RNAs Differentially Expressed In Gallbladder Caner By Microarray And Preliminary Functional Study

Objective In the past several years, long noncoding RNAs (lncRNAs) havebecome a hot spot of cancer research because their significant regulatory roles in cancerbiology. However, the contributions of lncRNAs to gallbladder cancer (GBC) are stillunknown. To identify the lncRNAs differentially expressed in GBC, we examinedlncRNA expression in9surgically resected GBC tussses and their correspondingnon-tumor tissue by microarray. In addition, preliminary functional study was performedto understand their biological role. This work could help us understand the mechanism ofspecific tumorigenic lncRNA in GBC and provide an experimental basis for searchingnovel targeted medication.Methods We surveyed the lncRNAs differentially expressed in9pairs of GBCand their corresponding non-tumor tissue by lncRNA microarray. Gene-coexpressionnetworks based on differentially expressed lncRNAs and mRNAs were built to identifyinteractions between genes and lncRNAs with critical role in GBC biology. Next, weassess the expression of the Homo sapiens MNX1antisense RNA1，long non-codingRNA (lncRNA-MNX1-AS1) and Long non-coding RNA metastasis associated in lungadenocarcinoma transcript1(MALAT1) in GBC using quantitative real-time polymerasechain reaction (qRT-PCR). To evaluate the biological role of lncRNA-MNX1-AS1andMALAT1, we knockdown them respectively in GBC cell lines by lentiviral siRNA. Then a series of experiments, including CCK-8assay, colony formation assay, transwellmigration and invasion assay, subcutaneous and peritoneal implanted models and westernblot analysis, were performed.Results We identified a total of3769differentially expressed lncRNAs in GBC,including1585upregulated ones and2084downregulated ones. It is observed thatlncRNA-MNX1-AS1might have a critically biological role in GBC by building thecoexpression network. Therefore, we validated its expression level by qRT-PCR andfound that lncRNA-MNX1-AS1was relatively highly expressed in92.3%GBC tissues.Then, lncRNA-MNX1-AS1in GBC cell lines was knockdown by lentiviral siRNA. Aftersilencing lncRNA-MNX1-AS1in GBC cell lines, the proliferation, migration andinvasion was significantly decreased both in vitro and in vivo, and the proportion ofapoptosis was significantly increased. In addition, we also determined the expression,biological functions and mechanism of MALAT1in GBC and found that MALAT1wassignificantly overexpressed in GBC tissues compared to corresponding non-tumor tissues.Knockdown of MALAT1in GBC cell lines using lentivirus-mediated RNA interferencesignificantly inhibited the proliferation and metastasis of the GBC cells both in vitro andin vivo. Furthermore, ERK/MAPK pathway was found to be inactivated in the GBC celllines after MALAT1knockdown.Conclusion A series of differentially expressed lncRNAs in GBC were identified.Among these differentially expressed lncRNAs, lncRNA-MNX1-AS1and MALAT1were found relatively highly expressed in GBC tissues, and the preliminary studyindicated that both of them were necessary to promote proliferation, invasion andmetastasis in GBC cell lines.