Effects And Mechanism Of MiR-23b On Colon Cancer Through Targeting PDE7A

Background:Colon cancer is one of the most common malignant tumors of digestive tract.The incidence rate of colon cancerhas been high,in the global cancer incidence and third,and the improvement of people’s living standard,eating habits change,multi fatty meat and more food Rouge diet increased,the increasingly rapid pace of life,work pressure,environmental pollution and other factors,resulting in the incidence of colorectal cancer increased year by year.On the other hand,although medical technology continues to progress,and methods of diagnosis and treatment of colon cancer is more diverse,surgery,radiotherapy,chemotherapy and targeted therapy to improve comprehensive treatment,and the treatment of individual trends,but the therapeutic effect of colon cancer is still not significantly improved,early surgical intervention in colorectal cancer well,the five year survival rate was 90%,but the effect of advanced colorectal cancer patients with poor surgical intervention,late postoperative colon cancer combined with chemotherapy in the treatment of colon cancer patients,the 5-year survival rate for early co complex,involving the invasion and adhesion of cancer cells,epithelial mesenclorectal cancer is about 90%,and this data drops to 20%in metastatic colorectal cancer.Colon cancer mortality is still high,in the third global cancer mortality.The mechanism of tumor metastasis is complex,involving the invasion and adhesion of cancer cells,epithelial mesenchymal transition(EMT),extracellular matrix degradation,angiogenesis and microenvironmental chemotaxis.Which are affected by the abnormal activation or inactivation of many oncogenes or tumor suppressor genes and related signaling pathways.The activation or inactivation of oncogenes or tumor suppressor genes is regulated at transcriptional level,post-transcriptional and translational level,respectively.Studies have shown that miRNAs regulate the expression of at least 30%of the human protein-encoding genes and play a greater role than protein-encoding genes.In a variety of human tumors,miRNAs are characterized by abnormal expression due to gene muta tion or gene fragility sites,which in turn are associated with the occurrence and progression of tumors MiR-23b is currently known to be abnormally expressed in hepatocellular carcinoma cells,which reduce the proliferation and migration ability of hepatocellular carcinoma cells.The expression of microRNA-23b in various tumors is different,and it is increased in some tumors and decreased in others.Therefore,it is believed that in some tumors,microRNA-23b plays the role of proto-oncogene,while tumor suppressor genes in others.So the expression of miR-23b which is specific in tumor has dual specificity in the development of cancer.Additionally,Other studies have found that miR-23b can be used as a drug sensitive target due to it is associated with drug resistance and sensitivity in the treatment.And miR-23b can multi-directionally regulate target genes,which is beneficial to the diagnosis,treatment and prognosis.miRNA plays a specifically important role in being a tumor marker,and mir-23b can be regarded as a prognostic marker for a variety of tumors.The main function of phosphodiesterase(PDEs)is to hydrolyze the second messenger in the cell,the second messenger includes adenosine cyclophosphate,also known as cAMP and cyclophosphate,or cGMP.As a new therapeutic target,PDEs has attracted extensive attention from many scholars and has become a new research hotspot.PDE7A is a phosphodiesterase that regulates the cellular levels of cAMP and cGMP.Low level of cAMP and high level of PDE were found in multiple cancer cells,whereas PDE7A was found to be overexpressed in lymphocytic leukemia and endometrial cancer,indicating that PDE7A may also be involved in the development of cancer.this study investigate the miR-23b and PDE7A expression in colon cancer tissue and colon cancer cell lines.In addition,miR-23b target gene was searched and verified by target gene prediction software and dual luciferase reporter assay,analysis of miR-23b and its target genes in the role of colon cancer,to provide reference for new targets for the treatment of colon cancer,put forward theoretical basis for further in-depth study of colon cancer mechanism.Part Ⅰ The expression of miR-23b and PDE7A in colon cancer tissues and colon cancer cell linesObjective:To investigate the expression and clinical significance of miR-23b and PDE7A in colon cancer and colon cancer cell lines.Methods:1 30 cases of patients with colon cancer who were hospitalized in our department of general surgery between January 2016 and December 2016 were selected.Postoperative histopathological examination was used to identify tumor stage,type and degree of differentiation.2 SW620 and SW480 colon cancer cell lines were cultured in vitro.3 Real-time fluorescence quantitative PCR was used to detect the expression of miR-23b in fresh colon cancer tissues and human colon cancer cells.4 Real-time fluorescence quantitative PCR was used to detect the expression of PDE7A mRNA in fresh colon cancer tissues and human colon cancer cells.5 The expression of PDE7A protein in fresh colon cancer tissues and human colon cancer cells was analyzed by by Western blot.6 Pathological correlation analysis between miR-23b and colon cancer was analyzed.7Pathological correlation analysis between PDE7A and colon cancer was analyzed.8 Correlation analysis between miR-23 and PDE7A in colon cancer tissueswas analyzed.Results:1 Compared with the normal tissues adjacent to the carcinoma,the expression of miR-23b in the tissues of various pathological types of colon cancer showed a downward trend,and the difference between the two was statistically significant(P<0.01).2 In the highly differentiated colon cancer tissue,the expression of miR-23b was significantly reduced,and compared with the normal para cancerous tissue(P<0.05),the expression of miR-23b decreased with the decrease of differentiation.In TNM staging,the expression of miR-23b was further reduced with the increase of TNM staging level.The difference was statistically significant(P<0.05,P<0.01).The correlation between miR-23b expression and colon cancer clinical classification was analyzed.The decrease of miR-23b expression was positively correlated with the degree of colon cancer differentiation(r=0.625,P<0.05).and was negatively correlated with the stage of colon cancer(r=-0.816,P<0.05).3 Compared with the PDE7A expression in the normal tissues adjacent to the carcinoma,the expression of PDE7A in the tissues of various pathological types of colon cancer showed an upward trend,and the difference between the two groups was statistically significant(P<0.01).4 The expression of miR-23b in colon cancer cell lines SW620 and SW480 showed a downward trend compared with the expression of miR-23b in the normal tissues adjacent to the carcinoma,and the difference between the two groups was statistically significant(P<0.01).5 the expression of PDE7A mRNA and protein in the colon cancer cell lines SW620 and SW480 showed an upward trend compared with the expression of PDE7A in the normal tissues adjacent to the carcinoma,and the difference between the two groups was statistically significant(P<0.01).6 The expression of PDE7A was negatively correlated with the degree of colon cancer differentiation(P<0.05).The expression of PDE7A was positively correlated with the size of tumor(P<0.05),and was positively correlated with the stage of colon cancer(P<0.05).7 In colon cancer tissues,miR-23b was negatively correlated with PDE7A(P<0.05).Conclusion:1 The expression of miR-23b decreased in colon cancer and colon cancer cells.2 The expression of miR-23b was positively correlated with the differentiation of colon cancer.the expression of miR-23b was negatively correlated with the stage of colon cancer.3 The expression of PDE7A increased in colon cancer and colon cancer cells.4 The increased expression of PDE7A was negatively correlated with the differentiation degree of colon cancer.The expression of PDE7A was positively correlated with the size of tumor and positively correlated with the stage of colon cancer.5 In colon cancer tissues,miR-23b is negatively correlated with PDE7A.Part II Target regulatory relationship between miR-23b and PDE7A.Objective:To explore the target regulatory relationship between miR-23b and PDE7A in colon cancer cell lines.Methods:1.Over expression of miR-23b was constructed in SW620 and SW480 colon cancer cell lines.2.The effect of overexpression of miR-23b on PDE7A mRNA and protein expression was analyzed by Western blot.3.Dual luciferase reporter assay was used to verify the targeted relationship between miR-23b and PDE7A.Results:1 In SW620 cell line,compared with negative control group(NC group),miR-23b expression in miR-23b mimics group was significantly higher than that in negative control group(P<0.001).In SW480 colon cancer cells,after transfection of miR-23b minics,the expression of miR-23b in the miR-23b mimics group was significantly higher than that in the negative control group(NC group),with a statistically significant difference(P<0.001).2 In SW620 cell line,compared with the negative control group(NC group),the expression level of PDE7A mRNA in the miR-23b mimics group was significantly lower than that in the miR-23b mimics group(P<0.01).In SW480 colon cancer cells,the expression of PDE7A mRNA in the miR-23b mimics group was significantly lower than that of the negative control group(NC group)after transfection of miR-23b mimics(NC group),and there was a statistical difference(P<0.05).3 InSW620 cell line,compared with the negative control group(NC group),the expression level of PDE7A protein in miR-23b mimics group was significantly lower.In SW480 colon cancer cells,after transfection of miR-23b mimics,the expression of PDE7A protein in the miR-23b mimics group was significantly lower than that in the negative control group(NC group).4 miR-23b significantly inhibited the fluorescence activity of wild type PDE7A,but had no significant inhibitory effect on PDE7A mutant.Conclusion:1 PDE7A is the target gene of miR-23b.2 miR-23b can negatively regulate the expression of PDE7A mRNA and protein,and PDE7A is the downstream target gene of miR-23b.Part Ⅲ The effect and mechanism of over expression of miR-23b in colon cancer Objective:To explore the role and mechanism of over expression of miR-23b in colon cancer.Methods:1 Overexpression of miR-23b was established in colon cancer cell lines SW620 and SW480.2 The effect of overexpression of miR-23b on proliferation of SW620 and SW480 colon cancer cells was analyzed by MTT.3 The effect of overexpression of miR-23b on SW620 and SW480 colon cancer cells migration was analyzed by Transwell.4 The effect of overexpression of miR-23b on SW620 and SW480 colon cancer cells invasion was analyzed by Transwell.5 The effect of overexpression of miR-23b on SW620 and SW480 colon cancer cells EMT was analyzed by Western blot.Results:1 In SW620 cell line and the SW480 colon cancer cell,the expression of miR-23b in the miR-23b mimics group was significantly increased,resulting in the inhibition of the proliferation of two kinds of colon cancer cell lines.Compared with the NC group,there was a statistical difference(P<0.05).2 In SW620 cell line,compared with the negative control group(NC group),in the miR-23b mimics group,the miR-23b expression in the miR-23b mimics group was significantly increased,and the migration of W620 and SW480 colon cancer cells could be significantly inhibited(P<0.05;P<0.01).3 In SW620 cell line,compared with the negative control group(NC group),in the miR-23b mimics group,miR-23b expression in the miR-23b mimics group was significantly increased,which significantly inhibited the invasion of SW620 and SW480 colon cancer cells(P<0.05;P<0.01).4 In SW620 cell line,compared with the negative control group(NC group),the expression of EMT related protein E-cadherin E-cadherin was increased and the expression of Vimentin decreased when the expression of miR-23b was significantly increased in the miR-23b group.Conclusion:1 Overexpression of miR-23b can inhibit the proliferation of colon cancer cells.2 Overexpression of miR-23b can inhibit migration and invasion of colon cancer cells.3 Overexpression of miR-23b can inhibit colon cancer cell EMT,and then inhibit the progression of colon cancer.Part Ⅳ The role and mechanism of PDE7A knockout in colon cancerObjective:To explore the role and mechanism of PDE7A knockout in colon cancer.Methods:1 PDE7A was knock down in colon cancer cell line SW620 and SW480 cells.2 The effect of PDE7A knockout on proliferation of SW620 and SW480 colon cancer cells was analyzed by MTT.3 The effect of PDE7A knockout on migration of SW620 and SW480 colon cancer cells was analyzed by Transwell.4 The effect of PDE7A knockout on invasion of SW620 and SW480 colon cancer cellswas analyzed by Transwell.5 The effect of PDE7A knockout on SW620 and SW480 colon cancer cell EMT was analyzed by Western blot.Results:1 Knockout of PDE7A significantly reduced the expression of PDE7A mRNA,which was significantly different from NC group(P<0.05).2 After knockout of PDE7A,the expression of PDE7A mRNA in SW620 and SW480 colon cancer cells was significantly reduced.3 Knockout of PDE7A inhibited the expression of PDE7A,resulting in the inhibition of the proliferation of the two cell lines.There was a significant difference compared with NC group(P<0.05).4 In SW620 cell lines and SW480 colon cancer cells,the knockout of PDE7A could inhibit the expression of PDE7A and significantly inhibit the migration of W620 and SW480 colon cancer cells(P<0.05;P<0.01).5 In SW620 cell lines and SW480 colon cancer cells,the knockout of PDE7A could inhibit the expression of PDE7A and significantly inhibit the invasion of W620 and SW480 colon cancer cells(P<0.05;P<0.01).6 In SW620 cell lines and SW480 colon cancer cells,the knockout of PDE7A could inhibit the expression of PDE7A,the expression of EMT related protein E-cadherin increased and the expression of Vimentin decreased.Conclusion:1 Knockout PDE7A-1 inhibited the proliferation of colon cancer cells.2 Knockout of PDE7A-1 can inhibit migration and invasion of colon cancer cells.3 Knockout of PDE7A-1 can inhibit the EMT of colon cancer cells and inhibit the progression of colon cancer.