The Molecular Mechanism Of DNMT3A Regulates Colorectal Cancer Progression Via Targeting DAB2IP

Objective Colorectal cancer(CRC)is the third most frequently diagnosed cancer and the second leading cause of cancer-related death worldwide.On account of the lack of peculiar incipient symptoms and effective treatment,most CRC patients are diagnosed at a distant stage and have a five-year survival rate of only 14%.It is urgently required to investigate the underlying molecular mechanisms of CRC progression and identify additional therapeutic strategies for this fatal disease.Aberrant epigenomic reprogramming is a driver of almost every type of human malignant tumor.DNMT3A(DNA methyltransferase 3A)is responsible for epigenetic regulation of genes involved in oncogenesis such as liver cancer and breast cancer.However,the molecular mechanism of DNMT3 A in CRC progression has not been well-defined.DAB2IP(Disabled homologue 2 interaction protein)is a putative tumor suppressor,which is frequently downregulated in aggressive cancers by EZH2-mediated H3K27 methylation.However,whether DNMT3 A affects the aberrant expression of DAB2 IP has not yet been reported.In this study,we aimed to examine the endogenous expression of DNMT3 A and DAB2 IP in CRC,and determine whether DNMT3 A can epigenetically obstruct the transcription of DAB2 IP in CRC cells.Methods The expression of DNMT3 A in CRC tissues and cells was detected by q RT-PCR,Western Blot and IHC(immunohistochemistry),and then verified by the TCGA database.The survival probability of DNMT3 A in CRC patients was obtained by the Kaplan-Meier method.Subsequently,the expression of DNMT3 A was silenced or overexpressed by si RNA or plasmids in HT29 and HCT116 cells,respectively.CCK8 assay,clone formation assay and flow cytometry were performed to evaluate the proliferative capacity of DNMT3 A in CRC cells.Next,the expression of DAB2 IP in CRC tissues was detected and the correlation between DNMT3 A and DAB2 IP was analyzed by Pearson correlation coefficient.To determine whether DNMT3 A can epigenetically obstruct the transcription of DAB2 IP in CRC,we then measured the effect of DNMT3 A on the methylation status of the DAB2 IP promoter by MSP(Methylation-Specific PCR)and BSP(Bisulfite sequencing PCR).Furthermore,dual-luciferase reporter assay and Ch IP-PCR were performed to verify the interaction between DNMT3 A and DAB2 IP promoter.In addition,we explored whether DNMT3 A exert tumor promoting function through repressing DAB2 IP expression in CRC by a rescue study.Previous studies have reported that DAB2 IP loss is involved in the progression of multiple malignant tumors via the activation of MEK-ERK cascade.Therefore,we postulated that DNMT3 A may regulate the MEK/ERK signaling pathway through repressing DAB2 IP in CRC cells.MEK/ERK inhibitor was used to verify whether DNMT3 A drove the growth of CRC cells by regulating this pathway.Results q RT-PCR,Western Blot and IHC showed that the expression of DNMT3 A was elevated,while the expression of DAB2 IP was decreased in CRC tissues,compared to the adjacent normal tissues.Correlation analysis showed that the expression of DNMT3 A and DAB2 IP was negatively correlated.Kaplan-Meier curve showed that increased expression of DNMT3 A was associated with poor survival in CRC patients.Compared to the negative control,knockdown of DNMT3 A in HT29 and HCT116 cells dramatically inhibited cell viability and clonal formation ability,induced G1-phase arrest,and upregulated the level of DAB2 IP.Whereas,DNMT3 A overexpression had an opposite effect.Application of demethylation drug Decitabine neutralized the inhibitory effect of DNMT3 A on the expression of DAB2 IP.MSP and BSP proved that the expression of DAB2 IP was epigenetically suppressed by DNMT3A-mediated promoter methylation in CRC cells.Using dual-luciferase reporter assay and Ch IP-PCR assay,we further confirmed that DNMT3 A restrained the transcriptional activity of DAB2 IP promoter through directly binging to it.Furthermore,DNMT3 A silence significantly reduced the proliferation of CRC cells,while downregulation of DAB2 IP recovered the proliferative ability inhibited by DNMT3 A knockdown.Conversely,DNMT3 A overexpression highly enhanced the growth of CRC cells.Meanwhile,restoration of DAB2 IP attenuated the cell growth mediated by DNMT3 A overexpression.In addition,our data indicated that DNMT3 A activated the MEK/ERK signaling cascade via restraining the expression of DAB2 IP,and the pharmacological inhibition of the MEK/ERK pathway could weaken the DNMT3A-dependent tumor cell growth.Conclusion Our findings showed that the expression of DNMT3 A was highly elevated in CRC and was negatively correlated with DAB2 IP expression,which correlated with poor prognosis.DNMT3 A could actuate the malignancy of CRC through epigenetically silencing DAB2 IP transcription and consequently activating the MEK/ERK pathway.In view of the proliferative effects of DNMT3 A in CRC cells,inhibition of DNMT3 A may be a promising target for the prevention and therapy for CRC.