Study On The Target Regulation Of MiR-21 On TET1 And Its Effect On Biological Behavior Of Cancer Cells In Colorectal Cancer

Background:Colorectal cancer(CRC)remains an important global health problem and may lead to more than 1.2 million new cancer cases and over 600000 cancer-related deaths in 2008 worldwide.In recent years in China,with the development of economy,the improvement of people's living standards and the changes of diet structure,the incidence of colorectal cancer is on the rise year by year,and the incidence of colon cancer is hidden,usually the tumor is in the progress stage when it is found,which brings many difficulties and disadvantages to clinical treatment.So far,the prognosis of CRC patients has improved significantly due to advances in surgical techniques and the integration of radiotherapy and chemotherapy and targeted therapy.However,in advanced CRC patients and those with local recurrence or distant metastasis,the prognosis is still very poor.Exploring the causes and effects of CRC and improving its survival rate have been the goal of scientists.The occurrence and development of CRC is a complex process in which multiple factors,multiple stages,multiple steps and multiple genetic changes cooperate.The activation of oncogenes and the inactivation of tumor suppressor genes are the molecular basis for the occurrence and development of CRC.Current studies have shown that the occurrence,development,invasion and metastasis of CRC involve the changes of multiple related genes,but its mechanism is complex and not fully understood.Therefore,it is necessary to investigate the molecular mechanisms and key regulatory factors of CRC progression and metastasis to provide biomarkers to predict the risk of local recurrence and distant metastasis,so that we can choose best treatment strategies and ultimately improve patient prognosis.DNA methylation affects tumor progression by inhibiting the expression of certain oncogenes,while DNA demethylation can activate gene expression.DNA methylation is an active demethylation modification.Ten eleven translocation enzymes(TET1,TET2 and TET3)are a family of dioxygenase,which convert 5-methylcytosine(5-mC)to 5-hydroxymethylcytosine(5-hmC)and lead to CpG islands demethylation.The deficiency or mutation of TET proteins and subsequent down-regulation of 5-hmC content is common in various kinds of human cancers.Indeed,TET1 is often absent in breast,hepatic,pancreatic and prostate cancer.Moreover,it has been reported that TET1 was down-regulated in colon tumors from the initial stage and down-regulation of TET1 during colon cancer initiation led to repression of the promoters of WNT pathway inhibitors and resulted in a constitutive activation of the WNT pathway.However,further mechanisms leading to downregulation of this important tumor suppressor in CRC need to be elucidated.MicroRNA(miRNA),an endogenous non-coding single-stranded small RNA,exists extensively in the biological genome.It is able to pair with and bind to 3'untranslated regions(3'-UTRs)of the targeted mRNA incompletely to degrade mRNA or repress the translation of mRNA,and is involved in regulating more than half of all gene expression.In addition,miRNA is an important post-transcriptional regulatory factor and plays an extensive and important role in cell proliferation,differentiation,apoptosis,tissues development,oncogenesis and other physiological processes.Given the previous study that TET1 was involved in inhibiting CRC cells growth,we hypothesized that TET1 is regulated by miRNA at the post-transcriptional level.In our study,we found that the expression of miR-21 was upregulated in the CRC patient' s tissue samples compared with CRC patient' s adjacent-tissue samples,while the expression of TET1 showed a significant decrease.Furthermore,our findings demonstrated that miR-21 promoted proliferation of colorectal cancer cells by targeting the TET1.Part ?Expression of miR-21 and TET1 in colorectal cancer and their correlationObjective:To study the expression and correlation of miR-21 and TET1 in colorectal cancer tissues.Methods:The bioinformatics methods were used to predict that miR-21 could target TET1.Fifty patients with colorectal cancer diagnosed by the same surgical team in gastrointestinal surgery of Shandong provincial hospital affiliated to Shandong university during 2016-2017 were collected.The tumor tissue cells and the corresponding incised margin normal tissue cells that were negative for detection of tumor were taken,and the expression of miR-21 and TET1 were analyzed using fresh frozen samples.The expression of miR-21 and TET1 mRNA in clinical CRC and adjacent non-cancerous tissues was analyzed using qRT-PCR,and standard normalization was performed on them through endogenous control to analyze the expression differences between them,and further evaluated the correlation between miR-21 and TET1 expression.Results:At present,there are a lot of website software used to predict miRNA target genes,such as miRBase,TargetScan and starbase.Multiple bioinformatics prediction software were applied online to predict the target genes of miR-21,and the prediction results of 3 databases were intersected to obtain target genes with high reliability and accuracy.Combined with previous research results,a target gene most likely to be associated with tumor was selected for verification.The target gene we screened was TET1.Then,we used qRT-PCR to analyze the expression of TET1 mRNA in clinical CRC and adjacent non-cancerous tissues,and conducted standard normalization treatment with reference to endogenous control(GAPDH).The results showed that TET1 mRNA expression level was lower in tumor tissues of 50 CRC patients compared with corresponding adjacent tissues,and the difference was statistically significant(P<0.05).We also tested the expression level of miR-21 and found that the expression level of miR-21 in tumor tissues of CRC patients was higher than that of corresponding adjacent tissues,and the difference was also statistically significant(P<0.05).We then assessed the correlation between the relative expression levels of miR-21 and TET1 mRNA.As expected,we found a significant negative correlation between miR-21 level and TET1 mRNA level in CRC tissues(Pearson correlation coefficient r=-0.409,P=0.0032).Overall,our findings suggest that TET1 mRNA expression levels were lower in tumor tissues in 50 CRC patients compared to corresponding adjacent tissues,while miR-21 expression levels were higher.At the same time,we found a significant negative correlation between miR-21 level and TET1 mRNA level.Conclusion:Compared with the corresponding adjacent tissues,miR-21 was highly expressed and TET1 mRNA was low expressed in colorectal cancer tissues,and their expression levels were significantly negatively correlated.Part ?In vitro experiments confirmed that TET1 was the target gene of miR-21 and its regulatory relationshipObjective:In vitro experiments confirmed that mir-21 can target the 3'-UTRs of TET1 mRNA and inhibit the expression of TET1 in CRC cells.Methods:The 3'-UTRs sequence of TET1 mRNA or the mutation sequence that predicted the interaction with miR-21 were inserted into the pmirGLO vector(Promega,USA).They were respectively named pmirGLO-TET1-wt and pmirGLO-TET 1-mut.Hsa-miR-21 mimics(miR-21)/NC(miR-control)and pmirGLO-TET1-wt/pmirGLO-TET1-mut were co-transfected into colorectal cancer cell lines HCT15 and HT29.The luciferase activity of the transfected cells was detected using a luciferase detection device,and the binding between miR-21 and 3'-UTRs of TET1 mRNA was determined according to the test results.To further study the effect of miR-21 on TET1 expression,we performed transient transfection of miR-21 and anti-miR-21 in HCT15 and HT29 cells,and the expression of miR-21 and TET1 mRNA after transfection was analyzed using qRT-PCR.Meanwhile,the protein expression level of TET1 after transfection was detected by western blot.Results:Luciferase reporter gene analysis showed that the firefly luciferase activity of miR-21 and pmirGLO-TET 1-wt reaction groups in HCT15 cells was significantly lower than that of the other three groups(miR-21 and pmirGLO-TET 1-mut group,miR-control and pmirGLO-TET1-wt group,miR-control and pmirGLO-TET 1-mut group)(P<0.05),while there was no significant difference between the other three groups.The same results were obtained in HT29 cells.The experiment showed that miR-21 significantly inhibited the firefly luciferase activity of pmirGLO-TET 1-wt in a dose-dependent manner,while miR-21 could not play a role when the target site was mutated in HCT15 and HT29 cells,which confirmed that miR-21 could target the 3'-UTRs of TET1 mRNA.The data of transient transfection experiment showed that compared with miR-21 in HCT15 and HT29 cells of the control group,miR-21 expression was up-regulated in the experimental group after transfection with hsa-miR-21 mimics,which led to a significant decrease of TET1 mRNA level,while the expression of miR-21 was down-regulated after transfection with anti-miR-21,which led to a significant increase of TET1 mRNA level.When the expression of miR-21 in CRC cells was increased,the TET1 protein level was significantly reduced,while the TET1 protein level was significantly increased when the expression of miR-21 was inhibited in both CRC cells.In general,our experiments showed that miR-21 can target the 3'-UTRs of TET1 mRNA in CRC cells and induce the down-regulation of TET1 expression.Conclusion:TET1 is a target gene of miR-21 in CRC cells,which can induce the downregulation of TET1 expression in CRC cells.Part ?Effects of the expression of TET1 regulated by miR-21 on the biological behavior of colorectal cancer cellsObjective:To investigate the effects of regulation of TET1 expression by miR-21 on proliferation,metastasis and invasion of colorectal cancer cells.Methods:According to previous results,miR-21 can down-regulate the expression of TETI in CRC cells.However,recent studies have found that TET1 expression is significantly reduced in colorectal tumor tissue compared to normal tissue,and the proliferation of cancer cells is associated with the silencing of TET1.Therefore,we further studied the effects of miR-21 regulation of TET1 expression on proliferation,invasion and migration ability of colorectal cancer cells.Anti-miR-21,anti-miR-21+si-TET1 and the corresponding controls were transfected in two colorectal cancer cell lines HCT15 and HT29,and the expression levels of miR-21 and TET1 mRNA in each group after transfection were detected by qRT-PCR,and the expression levels of TET1 protein were detected by Western blot.The effect of miR-21 regulation of TET1 expression on the proliferation of colorectal cancer cells was confirmed by EdU detection and CCK-8 analysis,the effect of miR-21 regulation of TET1 expression on the invasion ability of colorectal cancer cells was verified by Transwell experiment,and the effect of miR-21 regulation of TET1 expression on the migration ability of colorectal cancer cells was verified by cell scratch experiment.Results:After transfection of anti-miR-21,anti-miR-21+ si-TETl and related control in two kinds of colorectal cancer cell lines HCT15 and HT29,we use qRT-PCR,Western blot test each miR-21 and TET1 expression levels and TET1 protein expression levels.Results show that in the two groups of CRC cells,miR-21 expression were down-regulated in anti-miR-21 groups,TET1 mRNA level increased significantly,TET1 protein expression were up-regulated;MiR-21 expression of the cells transfected anti-miR-21+si-TET1 were down-regulated compared with those of the control groups,and there was no significant difference in TET1 mRNA level and TET1 protein expression level.The results showed that anti-miR-21 transfection reduced the expression of miR-21 and increased the expression of TET1.At the same time,the expression of miR-21 was reduced after transfection of anti-miR-21+si-TET1,but the expression of TET1 was not significantly different from that of the control groups.Then,we further examined the changes in cell biological behavior after transfection.EdU detection showed that cell proliferation of anti-miR-21 groups of cells was significantly decreased than the control groups and anti-miR-21+si-TET1 groups(P<0.05),and cell proliferation ability of the control groups and anti-miR-21+si-TETl groups showed no statistical difference.The EdU assay manifested that cell proliferation was decreased when the cells were treated with anti-miR-21,but TET1 down expression could rescue inhibition of cell proliferation mediated by anti-miR-21 in HCT15 and HT29 cells treated with both anti-miR-21 and si-TET1.Results suggested that miR-21 modulated the proliferation of CRC cells relying on TET1 in part.To verify our conclusion,we also performed the CCK-8 assays.When treated with anti-miR-21 in HCT15 and HT29 cells,the proliferation capacity of CRC cells was significantly reduced other than treated with control groups or treated with both anti-miR-21 and si-TET1.Transwell experiments showed that the ability of cell invasion was reduced after anti-miR-21 treatment,while there was no statistical difference in the control groups or the groups treated with anti-miR-21 and si-TET1.The cell scratch test showed that the cell migration ability was reduced after the anti-miR-21 treatment,while there was no statistical difference in the control groups or the groups treated with anti-miR-21 and si-TET1.Conclusion:Mir-21 can promote cell proliferation,invasion and metastasis by targeting TET1 in CRC cells.