The Role Of Long Noncoding RNA LINC01133 In Colorectal Cancer Metastasis

Colorectal cancer (CRC) is one of the most common cancers that are great threats to human health in the world. Among all the malignant tumors, CRC is one of the three most prevalent cancers in aspects of morbidity and mortality. In recent years, more than 0.6 million patients are died of CRC every year. And more than 90% of cancer-related mortality are caused by tumor metastasis. According to recent studies, Epithelial-mesenchymal transition (EMT) is a key process in tumor metastasis. And through EMT process, the epithelial-derived cancer cells is changed into mesenchymal phenotype and become invasive. However, protein-coding RNAs only accout for less than 20% of the whole human genome, more than 80% proportion of the whole transcriptome are unable to be translated, which also called non-coding RNAs (ncRNAs). Most of the non-coding RNAs are long non-coding RNAs (lncRNAs) that are longer than 200nt. As long non-coding RNAs occupied the most proportion of the transcriptome, they should have irreplaceable roles in biological processes. If they were just rubbish of the transcriptome, they would be a big burden of cells, which would not comply with economic living style of cells acquired from evolution. So we speculate that lncRNAs play an important role in the EMT process of colorectal cancer.We used TGF-p stimulate the CRC cells to establish a TGF-0-induced EMT model. To identify the transcripts associated with the EMT in CRC metastasis, we performed microarray analysis to compare their expression levels between TGF-β-treated and untreated CRC cells. From analysis of the microarray, we identified a differentially expressed lncRNA LINC01133. LINC01133 was significantly downregulated in the TGF-β-induced EMT model. So we chose LINC01133 as target lncRNA to study its role in the EMT and metastasis of colorectal cancer and the main conclusions are as follows:First, LINC01133 was inhibited by TGF-β.In the verification experiment transcriptome microarray analysis, we confirmed that LINC01133 was downregulated by TGF-β. When we blocked the TGF-β receptor or silenced smad a downstream mediator of TGF-β signaling, the expression of LINC01133 was recovered. These results showed that LINC01133 was novel lncRNA inhibited by TGF-p.Second, LINC01133 inhibited the EMT and metastasis of colorectal cancer cells.To study the role of LINC01133 in the EMT and metastasis of colorectal cancer, we silenced LINC01133 expression by siRNAs in the CRC cell lines with high expression of LINC01133 and overexpressed LINC01133 in CRC cell lines with low expression of LINC01133. When LINC01133 was knockdown in CRC cells, the migration and invasion ability of CRC cells was enhanced, the expression of epithelial marker E-cadherin was downregulated and the expression of mesenchymal maker Fibronectin was upregulated. Besides, The lung metastasis model through tail vein injection showed that knockdown of LINC01133 promoted metastasis of CRC cells. Besides, clinical sample experiment showed that LINC01133 was downregulated in CRC tumor tissue. And high expression of LINC01133 in CRC tumor tissues are associated with less metastasis and better prognosis. The expression of LINC01133 in CRC tumor tissues was positive related with E-cadherin and negtive related with Vimentin, All these results indicated that LINC01133 inhibited the EMT and metastasis in colorectal cancer cells.Third, LINC01133 inhibited colorectal cancer EMT and metastasis might through binding SRSF6 and blocking its EMT-promoting role. (1) Long non-congding RNA LINC01133 interacting with protein SRSF6.So we revised ChIRP protocol combined with mass spectrum (MS) to find out proteins binding to LINC01133. The result from MS showed that SRSF6 is binding protein of LINC01133, which was also identified by Western Blot using antibody to SRSF6. And RIP experiment was applied in the verification of interaction between SRSF6 and LINC01133. According to evidence of ChIRP and RIP, we confirmed the interacted between LINC01133 and SRSF6.(2) SRSF6 promoted the EMT and metastasis in colorectal cancer cells.Although it was exciting to identify SRSF6 as a molecule directly interacting with LINC01133 that was involved in the EMT process of CRC cells, we still wondered whether SRSF6 was also involved in the EMT process. Therefore, we knocked down SRSF6 in HT29 cells with high LINC01133 expression, HCT116 cells with low LINC01133 expression, and SW620 cells without LINC01133 expression using lentivirus-mediated shRNA targeting SRSF6 that was very effective in silencing the SRSF6 expression. When SRSF6 was knocked, the migration and invasion ability was significantly reduced. SRSF6 knockdown led to a significant increase in the expression of epithelial marker E-cadherin as well as reductions in the expression of mesenchymal markers such as Vimentin and Fibronectin in CRC cells. All these results indicated that SRSF6 promotes the EMT and metastasis in CRC cells independent ofLINC01133.(3) LINC01133 inhibited the colorectal cancer cell EMT and metastasis depending on interaction with SRSF6.The above results showed that both LINC01133 and SRSF6 were involved in the EMT process of CRC cells, and we also confirmed that LINC01133 bound SRSF6. So we would like to know whether LINC01133 needs SRSF6 to play a role in the EMT. When SRSF6 was knocked down, the influence of LINC01133 on EMT-related markers and the migration ability of CRC cells was attenuated the migration of both CRC cell lines was also dependent on the presence of SRSF6 (Figure 4H). These results implied us LINC01133 inhibited the EMT and metastasis in CRC cells by binding to SRSF6 and blocking its function.According to all the results above, we concluded that long non-coding RNA LINC01133 inhibited the EMT and metastasis of colorectal cancer through interaction with SRSF6.