| Colon cancer is one of common and high-risk malignant tumors in China with the third of cancer mortality.Epidemiological statistics shows that nearly one million cases of colorectal cancer have been found in 2010 worldwide,of which more than 200,000 cases of deaths.Especially in recent years,with the development of economy and living standards,dietary structure of domestic residents has already significantly changed,while also affected by negative factors,such as population aging and environmental problems,therefore resulting in the increase of incidence of colorectal cancer every year.Now colorectal cancer has become the focus of medical research.Although,with the advances in technology and in-depth research,we have made significant progress in early diagnosis and treatment of colorectal cancer,including the development of endoscopic techniques and chemotherapy drugs,but the tumor diagnosis and treatment still faces many challenges.The main difficulty is that the mechanisms are not yet fully clear.The clinical treatment of colorectal cancer is radical surgery combined with chemotherapy,but the 5-year survival rate of local tumor is still not ideal,and tumor metastasis has been found in more than 50% of patients at the time of diagnosis,resulting in high recurrence rate and death in patients with colon cancer.Therefore,in-depth exploration of the mechanism regulating the expression of genes related to the development of colon cancer,play crucial roles in the development of new therapeutic strategies and discover new drug targets.miRNA is a class of 19-24 length nucleotides of newly identified non-coding single-stranded RNA.miRNA was found to have extensive and important physiological functions,mainly through direct or indirect regulation of messenger RNA of the target gene’s 3 ’ binding untranslated region(3’-UTR).The present studies have already confirmed that abnormal miRNA expression has played a very important role in tumor development.Research on the relationship between cancer cells and miRNA expression has revealed that miRNA participated in the regulation of cell function,cell proliferation and apoptosis,migration and invasion,and thus affecting the individual growth,body functions and other aspects.The abnormal expression of miRNA is identified as one of the key factors in tumor development.It is worth noting that the effects and mechanisms of different miRNA in tumorigenesis have their specificity.In addition,downstream of miRNA regulatory mechanisms invarious cancer cells tends to be more complex.In order to clarify the role of miRNA in the development and mechanisms of tumor,the networks of gene regulation and protein targets need to be further studied.Recent studies have found that miRNA-93 plays an important role in multiple cancers.Abnormal expression and function of miRNA-93 has been demonstrated in a variety of tumors.Recently,it is reported that abnormally elevated miR-93 expression was observed in patients with colon cancer in vivo.Moreover,the level of miR-93 expression is significantly related with all stages of colon cancer and prognosis.In addition,abnormally elevated miR-93 expression is associated with cell invasion and metastasis of colon cancer.The reduction of miR-93 expression by specific inhibitors can increase the sensitivity of chemotherapy drugs on animal models.Although the research now confirmed the occurrence and prognosis of miR-93 in colon cancer,the regulatory mechanisms is still not clear.Tumor suppressor gene PTEN,namely phosphatase and tensin homolog that deleted on chromosome ten,is located on chromosome 10q23.3 people phosphatase and tension homolog gene.Now present studies demonstrate that PTEN as a tumor suppressor gene shows a dual specificity phosphatase activity,thus this anticancer target protein is rapidly becoming a hot research field.PTEN protein as an important member of the protein family PTP(protein tyrosine phosphatases),is one of the core protein with the ability of tumor suppression.Now it is reported that its main role is achieved by the regulation of the cell division cycle,inhibition of cell proliferation and promote apoptosis.Further research indicates that,PTEN protein plays an important role in the regulation of cell migration,malignant biological behavior of new angiogenesis.It has been confirmed expression levels of PTEN protein functions as a tumor prognostic evaluation,so further to clarify the mechanism of PTEN has important value in diagnostic and prognostic drug targets for cancer research.Our paper first detected miR-93 expression in colon cancer and adjacent normal tissues by real-time PCR technology.We aimed to study the impact of miR-93 on a variety of biological behavior of colon cancer cells in vitro model.We have predicted the downstream target genes of miR-93 by forecasting software and to clarify its role in the molecular mechanism of colon cancer.Our study will deepen the understanding of the pathogenesis of colon cancer,thus providing the new clues for early diagnosis and treatment of colon cancer.1.Materials and Methods:Clinical samples were collected from September 2012 to September 2015 in Affiliated Hospital of Jilin University,including 25 cases of colon cancer surgery,23 cases of adjacent tissues as a control group.Real-time PCR method was used to detect miR-93 in colon carcinoma and adjacent tissues,HCT116 cells and SW480 human colon cancer cells;in situ hybridization was used to confirm miR-93’s expression in colon cancer.According to the expression of cell lines,HCT116 cell was used for further study.Using LNA anti-miR-93 inhibitor,the expression of miR-93 was inhibited in transfected-HCT116 cells.MTT cell viability assay and cell clone were used to detect cell proliferation and apoptosis state.PTEN may be the downstream target of miR-93 by the prediction of bioinformatics.The dual luciferase reporter gene system was used to confirm the results of prediction of bioinformatics.The miR-93 mimics was transfected into human colon cancer cell lines HCT116 cells to enhance expression of miR-93,and LNA anti-miR-93 inhibitor was used to inhibit the expression of miR-93.On the basis of the above-described cell model,we detected the expression of PI3 K / Akt signaling pathway to confirm the mechanism for the regulation of miR-93 in human colon cancer cell.2.Results:2.1 Real-time PCR showed that miR-93 in human colon tissue and human colon cancer cell lines was significantly upregulated.Compared with normal colon tissue,miR-93 expression in tumor tissue was significantly increased.A significant increase in the expression of miR-93 was found,90.5%(22/25)of colon cancer(p <0.01),the average expression of colon cancer group was 2.09 times(compared with the control group);in situ hybridization experiments confirmed the increased expression of miR-93 in colon cancer.To further verify the abnormal expression of miR-93,human colon cancer cells SW480 and HCT116 in miR-93 expression levels were detected.The results showed that miR-93 expression level was 3.08 times higher in SW480 cells compared with normal tissues(p <0.01);3.79 times higher in HCT116 cell(as compared to adjacent tissues,p <0.01);therefore the subsequent experiments,we chose HCT116 cell line for miR-93 function and mechanism studies.2.2 Inhibition of the expression of miR-93 can affect the viability of colon cancer cells and cloning proliferation and promote cancer cell apoptosis.In order to examine the role of miR-93 in colon cancer development,we apply a locked nucleic acid antisense inhibition of miR-93 expression in HCT116 cells of miR-93.MTT assay demonstrated that 24 h after transfection,a significant decrease in cell viability was reduced to 0.64 ± 0.07,and with increasing time of transfection,cell viability continued to decline,compared with the control group and negative control group(p <0.01),with statistical difference.The results of soft agar experiment showed that soft agar colony formation was also suppressed after inhibition of miR-93.On the basis of miR-93 down-regulated expression of the above cell HCT116 model,we examined the effects of miR-93 expression on cell apoptosis.The flow cytometry results showed that after transfection for 24 h,the percentage of apoptotic cells in control group was 8.7 ± 1.5%,the proportion of apoptotic cells in the negative control group(LNA control)was 12.6 ± 1.9%.After transfection HCT116 apoptosis was significantly increased to 25.7 ± 2.9%,compared with the negative control group(p <0.01),with significant difference.2.3 PTEN is target proteins of miR-93.By using the online database miRNA target gene prediction,we screened the PTEN protein as a potential target of miR-93.To verify the regulation of miR-93 on PTEN,we constructed a luciferase reporter plasmid containing PTEN ’UTR sequence(wild type-PTEN)or delete miR-93 binding site(mutant-PTEN)sequence.We found that significantly lower PTEN luciferase activity in the wild-type colon carcinoma cell compared to control miRNA transfected cells,but transfected with mutant PTEN plasmid within colon cancer cell fluorescence There was no significant difference in luciferase activity in the experimental group compared with the control group.To further verify the predictions,we used western blot with transfected LNA anti-miR-93 inhibitor,or miR-93 mimics,to confirm that after miR-93 upregulated,downregulated expression of PTEN in colon cancer cells in HCT116,and the down-regulated expression of miR-93 increases the expression of PTEN in colon cancer cells.2.4 miR-93 regulates of PI3 K / Akt signaling pathway.In order to study miR-93 in colon cancer cells HCT116 gene regulatory networks,we examined the effect of miR-93 on PI3K-Akt signaling pathway.The miR-93 expression was inhibited in colon cancer cells by transfection LNA miR-93 inhibitor in HCT116 cells,subsequently mRNA levels of PI3K-Akt pathway was detected by real-time quantitative PCR.After miR-93 was downregulated,PI3 K,Akt and mTOR’s mRNA levels were declined significantly,which PI3 K mRNA decreased to 0.54 ± 0.04(compared with the control group,p <0.01),Akt mRNA decreased to 0.51 ± 0.05(control compared group,p <0.01),mTOR mRNA decreased to 0.72 ± 0.06(compared with the control group,p <0.05).To further verify the impact of miR-93 in PI3K-Akt pathway in colon cancer cells,we suppressed miR-93 expression in colon cancer cells by transfection HCT116 LNA miR-93 inhibitor,subsequently PI3K-Akt protein expression was measured by western blot.The results are consistent with the PCR results.Namely,after downregulation of miR-93,PI3 K,Akt and mTOR protein levels were significant decreased,which PI3 K protein decreased to 0.49 ± 0.05(compared with the control group,p <0.01),Akt protein decreased to 0.53 ± 0.05(compared with the control group,p <0.01),mTOR protein decreased to 0.75 ± 0.08(compared with the control group,p <0.05).3.Conclusion:3.1.miR-93 was significantly higher expression in colon cancer and colon cancer cell lines.3.2.After miR-93 expression was inhibited,cell viability and proliferation of HCT116 cells were inhibited,the proportion of apoptosis was increased,indicating that miR-93 may act as a potential target for gene therapy.3.3.The bioinformatics combined dual luciferase method confirmed PTEN might be the target proteins of miR-93.PTEN expression was inhibited miR-93.3.4.After inhibition of miR-93 expression,the expression of PI3 K,Akt and mTOR were decreased in HCT116 cells,suggesting that miR-93 may be involved in the process of developing colon cancer by regulation of PI3 K / Akt signaling pathway. |
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