| BackgroundAbout 1-2 million newly cases are diagnosed with colorectal cancer annually in the world and it leads to more than 600 thousand of deaths each year.The occurrence of colorectal cancer is a multi-stage and multi-step process,in which the activation of oncogenes and inactivation of tumor suppressor genes are the key factors leading to the occurrence and development of colorectal cancer.Long non-coding RNA is an important epigenetics regulatory factor in the occurrence and metastasis of tumor.LncRNA-MEG3 was the first found to have the function of tumor suppressor and its expression was inhibited in a variety of tumor tissues and cells.MEG3 has been shown to inhibit tumor cell proliferation,invasion and metastasis.Studies have shown that MEG3 could play a role in inhibiting tumor cell proliferation by promoting p53 expression or binding to miRNA molecules.However,studies have shown that lncRNA could be involved in regulation of gene expression at different levels in a variety of ways resulting in tumorigenesis.The role and molecular mechanisms of MEG3 in colorectal cancer need to be further explored.In this study,we will explore the relationship between MEG3 and clinciopathological features in colorectal cancer from the clinical tissue samples,and its role in the prognosis of patients with colorectal cancer.The biological function of MEG3 will be detected by in vivo and in vitro experiments.On this basis,we further explored the regulatory mechanism of MEG3 on colorectal cancer and provide a theoretical basis for further clinical transformation.Experimental method:1.To detect the expression of MEG3 in colorectal cancer and adjacent tissues: the changes of MEG3 expression in colorectal cancer and adjacent tissues were detected by real-time quantitative PCR.The in situ hybridization technique was used to detect the expression of MEG3 in paraffin slices of colorectal cancer and adjacent tissues,respectively.The expression of MEG3 in colorectal cancer tissue microarray was detected by in situ hybridization,and the relationship between MEG3 and clinicopathological features and survival prognosis was analyzed by statist.2.The effects of MEG3 expression on the biological characteristics of colorectal cancer were examined by in vivo and in vitro experiments1.In vitro experiment:The effects of MEG3 on cell proliferation were detected by CCK8 in the experimental group,the control group and the blank control group,respectively.The effects of MEG3 on cell migration and invasion were detected by Transwell assay in the experimental group,the control group and the control group,respectively..The effects of MEG3 on cell apoptosis were detected by flow cytometry in experimental group,control group and blank control group,respectively.In vivo experiment: a nude mouse model of colorectal cancer was established,and used to investigate the effect of MEG3 on tumorigenesis in vivo.A nude mouse model of colorectal cancer to Lung metastasis was established to observe the effect of MEG3 on colorectal cancer metastasis.3.Detection of upstream regulatory genes of MEG3: The bioinformatics analysis was used to detect the likely binding transcription factor in the promoter region of MEG3.The correlation between MEG3 and transcription factor(VDR)was further verified in cell experiments and tissue microarray.We used fluorescein Enzyme reporter gene and chromatin immunoprecipitation technique to further confirm whether VDR could regulate the expression of MEG3.4.The mechanism of MEG3 on the inhibition of colorectal cancer: Potential target protein molecules of MEG3 were identified through RNA-pulldown technology and mass spectrometry analysis.The interactions between target proteins and MEG3 were verified by the Western-blot and RNA immunoprecipitation techniques.EMSA was used to further verify whether MEG3 could directly bind to target protein,and the specific binding region of target protein to MEG3 was detected by Truncation experiment.The CCK8 and Transwell assays verified that MEG3 could affect the proliferation,migration and invasion of colorectal cancer through the downstream target protein.The expression of downstream target protein in MEG3 overexpression colon cancer cell line was detected by real-time quantitative PCR and western blot.Result1.Low expression of MEG3 in colorectal cancer was associated with poor prognosis in patient with colorectal cancer.1.1 MEG3 expression in colorectal cancer tissues: Real-time quantitative PCR and situ hybridization technique were used to detect the expression of MEG3 in colorectal cancer and the adjacent tissues.The results showed that the MEG3 expression in the adjacent tissue was significantly higher than that in colorectal cancer tissues,and the difference was statistically significant(P < 0.05).1.2 The relationship between MEG3 expression,clinical pathological features and prognosis in the patients with overall survival: Using Kaplan-Meier analysis,we found that patients with higher MEG3 level had higher overall survival rate(P = 0.007).MEG3 expression,TNM staging,CEA and CA199 were found to be associated with the prognosis of the colorectal cancer patients through Cox proportional-hazards model(P < 0.05);But clinicopathological features of patients with colorectal cancer of sex,age,tumor depth of invasion,tumor location,tumor differentiation degree were not correlated with the prognosis of colorectal cancer patients.Through the multivariate analysis,we found that MEG3 expression,TNM staging and CEA level were independently associated with the overall survival of colorectal cancer patients1.3The relationship between MEG3 expression,clinicopathological features and prognosis in the patients with tumor-free survival(DFS): Using Kaplan-Meier analysis,we found that patients with higher MEG3 level were also associated with better disease-free survival(P = 0.003).MEG3 expression,TNM staging and CEA level were associated with the DFS of cancer patient(P < 0.05),but not for the other clinical pathological characteristics.Through the multivariate analysis,we found that MEG3 expression,TNM staging and CEA level were independently associated with the disease-free survival of colorectal cancer patients.2.High expression of MEG3 inhibited colorectal cancer tumorigenesis and metastasis2.1 CCK8 experiments showed: Compared with the empty plasmid control group and blank control group,Overexpression of MEG3 in cell line of colorectal cancer significantly decreased the cell proliferation.After siRNA transfection,the MEG3 knockdown cells showed an increased proliferation compared with control group.2.2 Transwell assays showed: the cell migration and invasion ability of MEG3 overexpression group was significantly decreased compared with that of empty plasmid control group and blank control group(P < 0.05).Cell migration and invasion ability of siRNA MEG3 group was significantly higher than that of negative control group and blank control group(P < 0.01).2.3 The effect of MEG3 on apoptosis: MEG3 overexpression can promote the cellular apoptosis.The apoptotic ability of MEG3 interference sequence transfected cell line was significantly decreased.2.4 Subcutaneous tumor forming experiment in nude mice showed:The tumor volume of MEG3 overexpression group was significantly smaller than the empty vector control group.2.5 Lung metastases showed: The number of metastatic tumors was observed,and the number of lung metastases in MEG3 overexpression group was significantly less than that in empty vector control group.3.VDR specifically binded to the upstream promoter region of MEG3 and activated the expression of MEG33.1 Bioinformatics analysis revealed that there was VDR element binding site in the promoter region of MEG3.The luciferase reporter assay showed that VDR was able to activate the luciferase reporter gene plasmid containing MEG3 promoter region.ChIP assay showed that the VDR antibody significantly enriched fragment in the-230~-243 base region before transcription start site,which further confirmed that VDR could bind to the upstream promoter region of MEG33.2 Detection of the correlation level between VDR and MEG3:3.2.1 In SW1116 cells,MEG3 expression was significantly increased in 1α,25-(OH)2D treatment group after 24 hours and 48 hours than anhydrous ethanol control group and blank control group(P < 0.001).The results were similar in the RKO cells(P < 0.05).3.2.2 In SW1116 and RKO cell lines,VDR overexpression significantly increased the MEG3 expression level after 24,48 and 72 h compared wtih the empty vector transfected cells(P < 0.001).The expression of MEG3 was dependent on the transfection time.3.2.3 In colorectal cancer tissues,VDR expression level was highly correlated with the MEG3 expression level as suggested by IHC staining and in situ hybridization(P < 0.001).3.2.4 The patients with higher VDR level were also associated with higher overall survival rate.4.MEG3 specifically binded to downstream target protein CLU to inhibit the proliferation,migration and invasion of colorectal cancer.4.1 RNA sedimentation experiment with biotin labeled MEG3 antisense RNA identified MEG3 specifically aggregated proteins compared with the antisense RNA and the blank controls.A number of MEG3 potential binding protein molecules were found by Gel cutting and mass spectrometry analysis4.2 RNA-pull down and Western blot analysis found that there was no obvious difference between LYAR,the sense of chain,the antisense chain and no magnetic beads control group.However,CLU was significantly enriched in the sense chain group,which suggested that CLU could be combined with MEG3.4.3 MEG3 was significantly enriched in CLU antibody precipitation group by RIP,which suggested that MEG3 could be combined with CLU.4.4 MEG3 could bind to CLU protein and CLU antibody formed RNA-protein-antibody three complexes by EMSA,which confirmed that MEG3 could directly to CLU protein.4.5 MEG3 732-1174 fragment could bind to CLU protein by MEG3 truncation experiment.4.6 The proliferation,migration and invasion ability of SW1116/si-CLU,SW1116/MEG3,SW1116/MEG3/CLU and blank control cells were detected by CCK8 and Transwell assays,which found that the proliferation,migration and invasion ability of SW1116/MEG3/CLU were increased than SW1116/si-CLU and SW1116/MEG3.Its ability was comparable to that of the blank cells.It was confirmed that MEG3 could be involved in the proliferation,migration and invasion of colorectal cancer through CLU.Experimental conclusion:1.MEG3 expression was significantly lower in colorectal cancer tissue than that in the tumor adjacent tissues.MEG3 expression,TNM staging and CEA were associated with the prognosis of the colorectal cancer patients.Low expression of MEG3 was associated with poor prognosis in patients with colorectal cancer.2.MEG3 as a tumor suppressor gene inhibited tumorigenesis and metastasis of colorectal cancer3.VDR could bind to the site of VDR reaction element in the upstream promoter region of MEG3.VDR expression was positively correlated with MEG3 expression in colorectal cancer.The abnormal activation of vitamin D signaling pathway may be an important factor that regulated the MEG3 expression in colorectal cancer tissues and cells.4.MEG3 732-1174 fragment could bind to CLU protein,which formed RNA-protein complex and reduced the stability of CLU in colorectal cancer.It could inhibit the proliferation,migration and metastasis of colorectal cancer. |
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