| Part ⅠExpressions of miR-223 and p120 in colon cancer and the relationship between themObjectiveThe purpose is to investigate the expressions of miR-223 and p120 catenin (p120) in colon cancer, analyze their relationship with clinicopathological features and the correlation between their expressions, to predict the possibility of p120 as a miR-223 target gene.MethodsThe expression and distribution of p120 in 80 cases of colon cancer tissue and 42 cases of adjacent normal colon tissue was evaluated by immunohistochemistry; the expression of miR-223 in 18 cases of colon cancer tissue and the corresponding normal colon tissue was detected by Real Time PCR, The relationship between their expression and clinicalpathological features was analyzed, the correlation between their expressions was also analyzed. Bioinformatics analysis was used to predict the possibility of p120 as a miR-223 target gene. And then the possibility was verified with dual-luciferase reporter assay and transfecting miR-223 mimics into the LoVo human colon cancer cells. The mRNA and protein levels of p120 were detected by Real Time PCR and Western blot, respectively.Results(1) The immunohistochemistry showed:the abmormal expression rates of p120 in colon carcinoma and normal colon tissues were 71.25% and 0%(P<0.05), respectively, there was a statistically significant difference between the two groups.(2) The abnormal expression rates of p120 in poorly differentiated, moderately differentiated and well differentiated carcinoma tissues were 91.67%,43.48% and 33.33%(P<0.05), respectively; the abnormal expression rates of p120 in tissues with lymph node metastasis and without lymph node metastasis were 85.29% and 60.87% (P<0.05), respectively; the abnormal expression rates of p120 in patients with Dukes stage C-D and stage A-B were 85.71% and 58.70%(P<0.05), respectively; the differences between groups were all statistically significant. But the differences of abnormal p120 expression rates between different age, gender, tumor size, tumor location, depth of invasion, etc. all had no statistical significance (P> 0.05).(3) Real Time PCR showed:the expression of miR-223 in colon carcinoma tissues was higher than normal colon tissues, the difference between the two groups was statistically significant (P<0.05). And the expression of miR-223 in poorly differentiated carcinoma was significantly higher than that in moderately differentiated and well differentiated carcinoma (P<0.05). Expression in the T4 tissues was significantly higher than that in T1-T3 tissues (P<0.05), as well as in the tissues with and without lymph node metastasis (P<0.05), and in the tissues with Dukes stage C-D compared with that in stage A-B (P<0.05).(4) In colon cancer tissue, p120 expression was negatively correlated with miR-223 expression (r=-0.575, P=0.013).(5) The bioinformatics database TargetScan and MicroRNA.org predicted that, the 5’ seed region of miR-223 may complementarily bind with the 3’UTR region of p120 mRNA. Dual luciferase reporter assay showed that:when co-transfected with miR-223 mimics, the luciferase activity was decreased in p120-3’UTR WT plasmid group (P<0.05), while the luciferase activity has no significant difference in p120-3’UTR Mut plasmid group, compared with the negative control (P> 0.05).(6) When transfected with miR-223 mimics in LoVo human colon cancer cells, Real Time PCR showed the expression of p120 mRNA was significantly decreased (P <0.001), Western blot showed the expression of p120 protein also significantly decreased (P<0.05), compared with the negative control group.ConclusionsThe increased expression of miR-223 and abnormal expression of p120 are associated with the malignancy of colon cancer, p120 expression was negatively correlated with miR-223 expression. miR-223 may promote invasion and metastasis of colon cancer cells by targeting the tumor negative regulator p120 and downregulating its expression in the development of colon cancer.Part ⅡEffects of miR-223 on biological behavior of LoVo colon cancer cells and its mechanismsObjectiveThe purpose is to observe the effects of miR-223 on biological behavior of LoVo cells, and explore its regulation of downstream signaling pathways in order to identify the molecular mechanism of miR-223 as an oncogene.MethodsmiR-223 mimics or inhibitor were transfected into LoVo cells, MTT assay was used to observe its effect on cell proliferation; wound healing assay and Transwell cell migration assay to observe its effect on cell migration; matrigel invasion assay to observe its effect on cell invasion. The expression of E-cad and VIM was detected by Western blot; the RhoA activity was detected by G-LISA; The nuclear and cytoplasmic protein extraction and immunofluorescence were used to detect the amount of β-catenin translocated into the nucleus; Western blot and Real Time PCR were used to detect the expression of c-Myc and CyclinD1, and MMP7 mRNA and protein levels.Results(1) MTT results showed that, the cell proliferation was increased when transfected with miR-223 mimics, while decreased with miR-223 inhibitor transfection, compared with the negative control.(2) Wound healing assay showed that:48h after the scratch, the cells transfected with miR-223 showed confluence between the scratch, while it was still wide in the miR-223 inhibitor group compared with the control group.(3) Transwell cell migration assay showed that, the number of migrated cells increased in the miR-223 mimics group, and reduced in the miR-223 inhibitor group, compared with the control group.(4) Matrigel invasion assay showed that, the number of invaded cells increased in the miR-223 mimics group, and reduced in the miR-223 inhibitor group, compared with the control group.(5) Western blot showed that, when transfected with miR-223 mimics, the expressions of p120 and E-cad were decreased, the expression of VIM increased; while transfected with miR-223 inhibitor, the expression of p120 increased, but the expressions of E-cad and VIM had no significant changes.(6) G-LISA results showed that, RhoA activity was significantly increased in transfection of miR-223 mimics group and the p120 siRNA group.(7) The nuclear and cytoplasmic protein extraction results showed that, transfection of miR-223 mimics increased the nuclear expression of β-catenin; immunofluorescence results confirmed the increased β-catenin translocating into the nucleus.(8) Western blot and Real Time PCR results showed that:the protein level of c-Myc and CyclinD1, and both the mRNA and protein levels of MMP7 increased in transfection of miR-223 mimics group.ConclusionsmiR-223 may promote the proliferation, migration and invasion of colon cancer cells by targeting p120 through reducing the intercellular adhesion, promoting the activity of RhoA and activating the β-catenin signaling pathway. |
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