Function And Mechanism Study Of Long Noncoding RNA CLMAT1 In Colorectal Liver Metastasis

Liver metastasis is a primary factor on colorectal cancer prognosis, and the relative research is a focal point for metastatic colorectal cancer. In recent years, although noncoding RNA have received much attention in cancer processes and brought a series of achievements, most studies still focused on small RNAs (e.g. miRNA) and neglected the longer noncoding RNA (named IncRNA). LncRNA has shown kinds of biomedical functions especially in tumor metastasis though the research is only beginning. However, little is known about the function of IncRNA in the colorectal liver metastasis (CLM) process.Based on the above backgrounds, this study (divided into four parts) intended to investigate the role of IncRNA in CLM and the underlying mechanism:LncRNA arrays and qRT-PCR were used respectively for screening and testing IncRNAs in samples of colorectal cancer with various liver metastases. After CLM associated IncRNAs were validated, a series of analysis and experiments including clinical values assessment, colorectal cancer cell function experiments in vitro and in vivo, and initial mechanism exploration were performed to understand the effect of the target IncRNA on colorectal cancer metastasis and the potential mechanism.Part I:Screen and validation of candidate IncRNAs related to CLM and clinical value assessmentObjective:To seek CLM related IncRNAs and further assess their clinical values.Methods:LncRNA arrays were employed to screen differentially expressed IncRNAs in CRC tissues with synchronous, metachronous and non-liver metastases. Based on the data from biomedical informatics science, qRT-PCR was used to identify target IncRNAs in expanded CRC samples with various liver metastases. The relationships between target IncRNA and clinical characters or patients prognoses were further analyzed.Results:After achieved CLM differential expression profile of IncRNAs (n= 1332),40 IncRNAs possibly related to CLM were chosen to further detest in expanded clinical samples, and 3 novel target IncRNAs were verified, named IncRNA-CLMAT1-3. High expression of CLMAT1 was strongly correlated with CRC patients with liver metastases or positive lymph node metastases (P<0.05). Moreover, patients with high CLMAT1 expression had less median survival time than those with low CLMAT1 expression (33.7 vs.30.1 months, P=0.04). Conclusion:There were different expressions of lncRNAs in CRC with various liver metastases. LncRNA-CLMAT1 with high expression may be related to CLM, and brings poor survival for patients with CRCPart II:The effect of lncRNA-CLMAT1 on growth, migration and invasion of colorectal cancer cell in vitroObjective:To study the effects of IncRNA-CLMAT1 on the proliferation, migration, and invasion of CRC cells in vitro.Methods:Different expression levels of lncRNA-CLMAT1 in various CRC cell were detected by qRT-PCR. The CLMAT1 shRNA and overexpression lentivirus vectors were constructed and respectively transfected into CRC cells with high and low expression of CLMAT1. After down- or up-regulation of CLMAT1 expression, cellular proliferation inhibitory activity was determined by CCK8 assay, the cell cycle or apoptosis activity was tested by flow cytometry. Additionally, transwell assay and wound healing were used to assess the effect of CLMAT1 on cell migration and invasion.Results:Both the CLMAT1 shRNAand overexpression lentivirus vectors were successfully transfected into LOVO and HCT116 cells respectively. After CLMAT1 expression significantly down-regulated by shRNA, the abilities of migration and invasion decreased significantly, and apoptosis rate also significantly increased (P<0.05) though proliferation ability or cell cycle rate no changed obviously when compared with control group. Above results were further verified when CLMAT1 expression was up-regulated.Conclusion:IncRNA-CLMAT1 significantly promotes CRC cell metastasis in vitro, though the proliferation ability of CRC cells could not be significantly affected by CLMAT1.Part Ⅲ:The effect of lncRNA-CLMAT1 on growth and liver metastasis of colorectal cancer in vivoObjective:To study the effects of IncRNA-CLMAT1 on the proliferation and invasion of CRC cells in vivo by used tumor-bearing and CLM nude mice model.Methods:The CLMAT1 overexpression (experimental group) or blank lentivirus vectors (control group) were transfected into HCT116 cell and screened by puromycin selection. The nude mice were subcutaneously inoculated with above cell (2×106/per mice) to establish the tumor-bearing animal model. Meanwhile, the CLM animal model was established through injecting 2×106 HCT116 cells under the spleen involucrum. Fluorescence imaging system was used to monitor the process of CLM in vivo. Tumor volume/weight and growth curve were recorded in tumor-bearing mice model. Metastatic number/size in liver and liver weight were recorded in CLM mice model.Results:There was no significant difference between experimental group and control group regarding to tumor volume/weight and growth curve in tumor-bearing mice model. Nude mice built with CLM model in experimental group had more and larger metastatic nodes, compared to those in control group (P<0.05)Conclusion:Based on tumor-bearing and CLM nude mice model, IncRNA-CLMAT1 is further proved to have no obvious effect on the CRC cell proliferation but to promote CLM occurrence in vivo.Part Ⅳ:The preliminary mechanism study of IncRNA-CLMAT1 involved in the promotion of colorectal cancer metastasisObjective:To initially investigate the mechanism of IncRNA-CLMAT1 involvement in the CRC metastasis.Methods:To explore the effect of IncRNA-CLMAT1 on the EMT biomarkers expression, EMT related PCR array was used, and other methods (e.g. qRT-PCR, Western blot, cell immunofluorescence and Immunohistochemistry) were also used. Western blot and transwell assays were used to further explore the effect of down-regulated SNAIL expression on promoting EMT process or cell metastasis brought by IncRNA-CLMAT1.Results:LncRNA-CLMAT1 induced CRC cell to experience EMT analogic morphological changes. CLMAT1 could down-regulate the expression of epithelial biomarker (e.g. E-cad), while up-regulate the expression of mesenchymal biomarkers ( e.g. N-cad, VIM, et al) and EMT-inducing transcription factors (e.g. SNAIL). The abilities of CLMAT1 to promote EMT process and cell metastases were weaken when SNAIL expression inhibited by siRNA. Conclusion:EMT is the possible mechanism how IncRNA-CLMAT1 promotes CRC cell metastasis. Furthermore, how CLMAT1 drives the EMT process is possibly through up-regulating expression of SNAIL.