The Function And Mechanism Study Of Long Non-coding RNA LncTSG1 In The Suppression Of Hepatocellular Carcinoma Metastasis

AimsHepatocellular carcinoma (HCC) is one of the most malignant tumors in China. HCC is primarily caused by genetic alterations that result in the deregulation of the gene networks which are responsible for the maintenance of cellular homeostasis. Long noncoding RNAs (lncRNAs) are non-protein coding transcripts longer than 200 nucleotides. Notably, lncRNAs affect various aspects of cellular homeostasis, including proliferation, apoptosis, migration, genomic stability and so on. Aberrant expression of lncRNAs has been found associated with many human diseases, including cancer. Therefore, the characterization of HCC-related lncRNAs as well as it regulated signaling pathways will be crucial for understanding HCC carcinogenesis and developing novel cancer therapeutic strategies.MethodsIn this study, microarray test was applied to obtain expression profiles of both protein coding genes and lncRNAs in HCC and paired adjacent non-tumor tissues from 16 patients. As an additional test, we determined the abundance of LncTSG1 in 43 pairs of HCC tissue sample by quantitative real time polymerase chain reaction (qRT-PCR).5’and 3’RACE was performed to identify the sequence of LncTSGl transcript. Knockdown or ectopic expression of the LncTSG1 in HCC cell lines are used to examine its effect on cell proliferation, migration and invasion. RNA pull down and RNA immunoprecipitation (RIP) assays and bioinformatics analysis were used to explore the binding proteins and its downstream signal pathway.ResultsThe global expression profile of both PCGs and lncRNAs in 16 pairs of HCC and adjacent non-cancerous tissues were examined by a custom microarray platform. Bioinformatics analysis showed that many IncRNAs were consistently differentially expressed between cancerous and adjacent benign tissue in HCC. Among these, we observed that LncTSG 1 was down-regulated to more than 90% in HCC compared to the matched normal tissues. As an additional test, we determined the abundance of LncTSG1 in 43 pairs of HCC samples by qRT-PCR.5’-and 3’-RACE analysis was performed to determine the full-length transcript of LncTSG1. The N-terminal sequence of LncTSG1 we verified was nearly identical to one of the annotations in UCSC database, while the C-terminal sequence is 100bp shorter.By analyzing the microarray results, we found that LncTSG1 is closely related to the cell signals of EMT and cell migration. Applying up and down LncTSG1’s expression levels approach, experiments showed that LncTSG1 can significantly inhibit liver cancer cells’migration and EMT, but had no significant effect on liver cancer cells’proliferation.By using RNA fluorescence in situ hybridization (RNA-FISH) and qRT-PCR, we found that approximately 70% of LncTSG1 transcripts were found in the cytoplasm. RIP assay revealed that LncTSG1 could interact with AGO2, indicating that LncTSG1 may function as ceRNA in the cytoplasm. Bioinformatic analysis found a set of miRNAs that putatively bind to LncTSG1. Among these miRNA candidates, we found that has-miR-372 and has-miR-10b involves in cancer metastasis. Knockdown LncTSG1 could inhibit RHGAP6、CELF2、MAPK10、TNS1、SYNPO2 expression, which regulating cancer metastasis. Furthermore, RNA pull-down assay followed by mass spectrometry demonstrated that LncTSG1 binds to hnRNP H.ConclusionLncTSGl is decreased in HCC tissues relative to corresponding adjacent nontumor tissues. The results of this study showed the pathologic and tumorigenic roles of LncTSG1 in suppressing cell migration, invasion and EMT. Thus, our results indicated that LncTSGl acts as a tumor suppressor in HCC and can be considered as a potential therapeutic target for HCC.