| Since 1993,Ambro et al first discovered the miRNA in nematode and published the results in authoritative magazines Cell.There are more and more researches on the functions and roles of miRNA.There is no doubt that miRNA,as a small molecule that regulates the expression of genes,participates in the regulation of the development of various tumors.It has been pointed out that about 50% of the miRNA in human genome is located in the sequences of chromosome fragile sites,which means that miRNA transcription is prone to abnormal expression,deletion or amplification,resulting in essential correlation in the process of cell carcinogenesis.Colorectal cancer(CRC)is one of the most common malignant tumors in the rectum or colon.It has become the most common malignant tumor of digestive system in the world.The process of its occurrence and development is also regulated by many genes.At the same time,there are different kinds and intensity of miRNA involved in each stage of the disease,forming a long-term and complex regulation process.It is a prerequisite for individualized treatment to explore the difference of individual gene expression in colorectal cancer.In recent years,a large number of studies have revealed that there are many kinds of specific miRNA in tumor tissues of colorectal cancer patients.They are of great values in the diagnosis,prognosis and targeted therapy of colorectal cancer.It has been demonstrated that the level of serum miRNA-141 can be used as a diagnosis of colorectal cancer early evaluation of liver metastasis,and abnormal expression of elevated serum miRNA-141 was significantly associated with the high metastasis and prognosis in colon cancer.The clinical studies have demonstrated that miRNA-141 is closely related with colorectal cancer process.In this study,the expression of mi R-141 in colorectal cancer was measured by the quantitative analysis of real-time quantitative PCR.The human colon cancer HT29 cells were used to established the stable expression of mi R-141 in vitro model,so as to reveal the role of mi R-141 in colorectal cancer.The target gene of mi R-141 was predicted by bioinformatics,and the molecular mechanism involved in the regulation of colorectal cancer was illustrated by experimental verification.1 Experimental methodIn the first part of the study,we used real-time fluorescence quantitative PCR to detect the expression of miRNA-141,ZEB1 and E-cadherin in colorectal cancer tissues and human colorectal cancer cell lines.In the second part,we used the over expression and transfection technology to establish miRNA-141 overexpressing human colon cancer HT29 cells.The proliferation of human colon cancer HT29 cells was detected by CCK-8,the migration of colon cancer HT29 cells was detected by cell scratch test,the invasion of human colon cancer HT29 cells was detected by Transwell,and the cell apoptosis of HT29 colon cancer cells was detected by flow cytometry.In the third part,we predicted the target genes of miRNA-141 by bioinformatics technology,and verified by luciferase gene reporter system,real-time fluorescence quantitative PCR and Western blot.In the fourth part,we studied the downstream regulation of ZEB1 gene expression regulated by miRNA-141.We used real-time fluorescence quantitative PCR and Western blot to reveal the molecular mechanism of miRNA-141 regulation.This study used statistical software SSPS18.0 to analyze the raw data.The data were all expressed in the form of MEAN + SD.The difference level of each group was analyzed by t test(student ’s t text)and chi square test,P < 0.05 was considered as statistically significant.2 Results2.1 Reduction of mi R-141 in colorectal cancer and human colon cancer cells The expression level of mi R-141 in colorectal cancer tissues and adjacent tissues of 30 patients was detected,and the mi R-141 content in colorectal cancer decreased.And the expression level of mi R-141 in human colon cancer HT29 cells was 0.35 + 0.01 times that of adjacent tissues.The expression level of mi R-141 in SW480 cells was 0.71 + 0.06 times that of adjacent tissues.It showed that the expression of mi R-141 in colorectal cancer tissue and human colon cancer cells was abnormally reduced.2.2 The relationship between the expression level of mi R-141 and the clinicopathological features of colorectal cancer patients According to the statistics,the clinical features of patients with miRNA-141 showed that the expression level and the age and sex of patients and tumor site mi R-141 had no significant correlation(P>0.05),but with colorectal cancer tumor infiltration depth,differentiation degree,clinical stage,lymph node metastasis index has significant correlation(P<0.05).2.3 The effect of over expression of mi R-141 on the cell proliferation,migration,invasion and apoptosis of HT29 cells The expression level of mi R-141 in HT29 cells transfected with over expressed plasmid increased significantly,indicating that miRNA-141 overexpression plasmid can stably express after transfection into HT29 cells,which can be used for subsequent functional studies.Based on the expression of HT29 cell model constructed by mi R-141,we examined the expression of mi R-141 on HT29 cell proliferation ability,test results showed that in cultured 48 h,human colon cancer HT29 cell proliferation,migration and invasion was inhibited and the apoptosis ratio increased significantly.2.4 Target gene prediction and verification of miRNA-141 First,science and technology on miRNA-141 target genes were predicted by bioinformatics,combined with the target substrate in the widely used Target Scan,mi RBase online database and mi RGen Targets prediction of mi R-141 because of ZEB1,combined with Blast database preliminary verification,found that the structure of the sequence 3 ’UTR region ZEB1 deposit and interact with mi R-141 in the 5’ end.In order to verify the ZEB1 is the target gene regulated by mi R-141,we were constructed including wild type ZEB1 and mutant 3’UTR luciferase reporter plasmid by dual luciferase reporter assay showed that ZEB1 of wild type 3’UTR gene plasmid and mi R-141 were transfected into HT29 cells,visible luciferase activity was significantly decreased(P<0.01),and ZEB1 mutant 3’UTR luciferase reporter gene plasmid and mi R-141 were transfected into HT29 cells after the luciferase activity had no significant effect.This result shows that the 3’UTR region of the ZEB1 gene can be associated with mi R-141,and its expression is directly regulated by mi R-141.In order to further confirm the effect of mi R-141 on ZEB1 gene expression,we detected the m RNA and protein of ZEB1 in miRNA-141 overexpressing human colon cancer HT29 cells,and confirmed that ZEB1 and m RNA levels in miRNA-141 overexpressing HT29 cells decreased significantly.2.5 The role of ZEB1 in the regulation of malignant biological behavior of HT29 cells by miRNA-141 After inhibiting the expression of ZEB1 in human colon cancer cell line HT29 by si RNA,the results showed that it could further enhance the inhibition of miRNA-141 induced proliferation,migration and invasion of HT29 cells and promote apoptosis.These results confirm that ZEB1 is the target protein for the regulatory role of mi R-141 on the malignant biological behavior of colon cancer cells.2.6 Mi R-141 regulates downstream signal pathway molecules in ZEB1 In order to further study the downstream molecular mechanism of mi R-141 regulating the malignant phenotype of colorectal cancer,we detected the expression of E-cadherin in downstream signaling pathway of ZEB1.Downregulation of ZEB1 or HT29 cells after overexpression of mi R-141,compared with the negative control group,the expression level of ZEB1 protein in the cells in the experimental group were significantly lower(p<0.05),while the expression of E-cadherin was significantly increased(p<0.05),confirmed that mi R-141 regulates ZEB1 expression at the same time,also had a regulating effect on the expression of E-cadherin.Transfection and expression of ZEB1 protein in si RNA interference miRNA-141 expression in HT29 cells,enhance the inhibitory effect of ZEB1 protein on the synthesis of E-cadherin protein in a certain extent,the increased expression of E-cadherin protein in HT29 cells(p<0.01,compared with the negative control group),thereby inhibiting HT29 cell invasion and metastasis.At this point,we have demonstrated the important role of the mi R-141-ZEB1-E-cadherin axis in regulating the malignant phenotype of colorectal cancer at the level of cells in vitro.3 Conclusion3.1 The expression level of miRNA-141 in colorectal cancer and colorectal cancer cell lines was decreased.The expression level was correlated with tumor differentiation,TNM staging and lymph node metastasis.3.2 Enhanced expression of miRNA-141 can inhibit the proliferation,invasion and metastasis of human colon cancer cells,and can promote the apoptosis of cancer cells.It is confirmed that miRNA-141 may be a potential gene target for colorectal cancer treatment.3.3 ZEB1 is the target protein that miRNA-141 inhibited the malignant biological behavior of colon cancer cells,and its regulation may be realized by ZEB1-E-cadherin. |
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