| Background:Colorectal cancer is one of the most aggressive malignant tumors in the world,with the morbidity and mortality of this disease in the world increasing year by year.Epidemiological evidence suggests that the global annual newly diagnosed cases ranks third in men and second in women. Among them, the incidence and mortality of colorectal cancer in China also showed an upward trend. The number of new cases in 2015 was 376,1000 and the number of deaths was about 191,1000. At present, the CRC is associated with genetic and environmental (such as diet) and so on a variety of pathogenic factors. It is the result of gene environment interaction. However, the exact etiology and mechanism of colorectal cancer remains to be further clear.MicroRNAs (miRNAs) are endogenously expressed regulatory small noncoding RNAs with a length of 18-25 nucleotides, which is widely distributed in eukaryotes.The research shows that the miRNA gene is located near many loci associated with the disease, and there are expression differences in the process of tumorigenesis. These studies indicate that miRNA is involved in the regulation of tumor progression, with functions of tumor-suppressor genes or proto-oncogene. It has been reported that the expression of miRNA in tumor tissues and adjacent tissues has been changed. The relationship between the changes and the development of tumor is not clear. Our previous microarray data show that miR-206/133b is less expressed in colorectal cancer than in adjacent tissues, and its relationship with colorectal cancer has not been reported at home and abroad. The study of miR-206/133b shows that it plays an important role in the study of macrophage, inflammation and tumor.During the functions of the solid tumor microenvironment, the mononuclear cells go through blood vessels into the tumor tissue,and differentiate into a unique phenotype of macrophages. These macrophages are called tumor-associated macrophages (TAMs),and divided into M1 and M2. In the early days of tumorigenesis, TAMs is given priority to with M1 macrophages, playing the role of anti-tumor cells. During the late stages of tumor development, TAMs is given priority to with M2 type, promoting tumor growth,invasion and metastasis. Our previous studies have shown that miR-206/133b could promote the transformation of M2 type macrophages to M1 and inhibit the proliferation and metastasis of colorectal cancer cells. Therefore, we investigated the inhibitory effect and molecular mechanism of miR-206/133b on colorectal cancer in vitro and in vivo.Objective:1. To detect the expression of miR-206/133b in colorectal carcinoma and its relationship with clinical characteristics;2. To investigate the role of miR-206/133b in the progression of colorectal cancer in vitro and in vivo;3.To investigate and explore the molecular mechanism of miR-206/133b in the progression of colorectal cancer;Methods:1. Taqman qPCR was performed to detect the expression levels of miR-206/133b in clinical specimens; Pearson ?2 was used to analyzed the association between the expression of miR-206/133b and clinical characteristics in patients with colorectal cancer; Kaplan-Meier was applied to draw the survival curves and Log-Rank test was performed for analysis of the statistic difference.2. The colorectal cancer cell model with stable over-expression of miR-206/133b was established. The colony assay, cell proliferation assay, scratch, migration and invasion assays were performed to detect the influence of over-expression of miR-206/133b in colorectal cancer cells. And the apoptosis and cell cycle assays were also performed by flow cytometry.3. Establishing the M1 and M2 subtype model of THP-1 cell differentiation; qPCR was used to detect the markers of M2 subtype after over expression of miR-206/133b. And then we collected the supernatant, centrifuged and filtered it. According to the proportion of 1:1, the supernatant was added to the cells, then the cell cloning, cell proliferation, invasion, migration, and scratch assays were performed following the overexpression of miR-206/133b in M2 macrophages.4. YY1 is a transcription factor that may regulate the expression of miR-206/133b with bioinfomatics prediction; Taqman and SYBR qPCR are used to detect the expression of miR-206/133b after over-expression of YY1; Then luciferase reporter assays were used to detect the luciferase activity after the effect of YY1 on the miR-206/133b promoter.5. CD47 may be a direct target of miR-206/133b with bioinformatics prediction. And then it was verified by dual-luciferase reporter assay, qPCR and Western Blot.Results:1. The correlation between the expression of miR-206/133b and clinical characteristics in patients with colorectal cancerThe expression of miR-206 and miR-133b in colorectal cancer was decreased compared with that in adjacent cancer. Pearson ?2 showed that the low expression levels of miR-206 and miR-133b were significantly associated with metastasis in in patients with colorectal cancer. By using Pearson linear correlation, the results showed that the expression of miR-206 and miR-133b in tumor tissues was highly correlated. The Kaplan-Meier survival curve and survival analysis of Log-Rank showed that miR-206 and miR-133b were negatively correlated with the overall survival of patients with colorectal cancer.2. The role of miR-206 and miR-133b in colorectal cancerThe expression of miR-206 and miR-133b was up-regulated in colorectal cancer cells(HCT116 and SW480), which could inhibit the proliferation, clone formation, scratch and metastasis, but had no effect on cell cycle.3. miR-206 and miR-133b inhibit progression by regulating the expression of CD47 in colorectal cancerThe target genes of miR-206 and miR-133b were predicted by bioinformatics, and the tumor associated protein CD47 was screened in. It was found that CD47 was a common target gene of miR-206 and miR-133b. Dual luciferase reporter assay showed that miR-206 and miR-133b could significantly inhibit the activity of wild-type CD473'-UTR reporter gene, but could not inhibit the activity of mutant CD47 3'-UTR reporter gene. qPCR and Western Blot confirmed that overexpression of miR-206 and miR-133b could down-regulate the expression of mRNA and protein of CD47. miR-206 and miR-133b can inhibit the invasion and metastasis of colorectal cancer and promote the apoptosis of colorectal cancer by targeting the expression of CD47.4. miR-206 and miR-133b inhibit progression through down-regulation of M2 macrophages in colorectal cancerThe M2 macrophage differentiation model was established and verified. The expression of miR-206 and miR-133b could inhibit the polarization of M2 macrophages,respectively, and the supernatant of each group was added to colon cancer cells. The results showed that miR-206 and miR-133b inhibited the ability of the proliferation,clone formation, scratch removal, metastasis and invasion in colorectal cancer cells(HCT116 and SW480) through down regulation of M2 macrophages. And miR-206 and miR-133b promoted the apoptosis of colon cancer cells but no effect on cell cycle in the same principle.5. miR-206 and miR-133b inhibit the progression of colorectal cancer in vivoThe mini-circle plasmid of miR-206, miR-133b and CD47 were prepared and used for the experimental study of colon cancer in mice AOM/DSS model. Each times per week up to 18 weeks. The results showed that the size, number and volume of colon cancer in miR-206 and miR-133b over expression group were less than those in control group,Over expression of CD47 could partly reverse the inhibitory effect of miR-206 and miR-133b on colorectal cancer in mice. The immunohistochemical results of Ki-67 and Caspase-3 showed that overexpression of miR-206 and miR-133b could inhibit the proliferation and promote apoptosis of tumor cells, and overexpression of CD47 could partially reverse the anti-tumor effect. In addition, the immunohistochemical results of F4/80 indicated that overexpression of miR-206 and miR-133b could exert great influence on the number of TAMs.6. The mechanisms of miR-206 and miR-133b inhibition on colorectal cancer miR-206 and miR-133b are regulated by the same promoter region in the genome.Bioinformatics predicts that there is a binding site of transcription factor YY1 in promoter region, which suggests that YY1 may be a negative regulator of miR-206 and miR-13 3b. The over expression of YY1 could downregulate the expression of miR-206 and miR-133b, and decrease the activity of luciferase reporter gene in miR-206 and miR-133b promoter. These results suggest that YY1 can negatively regulate the expression of miR-206 and miR-133b. Therefore, YY1 mediated down-regulation of miR-206 and miR-133b expression may promote the progression of colorectal cancer by regulating the expression of M2 macrophage pathway and CD47 expression.Conclusion:The low expression of miR-206 and miR-133b are negatively correlated with the prognosis of patients with colon cancer. MiR-206 and miR-133b can inhibit the progression of colorectal cancer by regulating M2 differentiation and expression of CD47, which are regulated by the upstream transcription factor YY 1. |
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