MORC2 Promotes Development Of An Aggressive Colorectal Cancer Phenotype Through Inhibition Of NDRG1

ObjectiveColorectal cancer（CRC）is the third most common cancer and the fourth most common cause of cancer-related death in the world.Metastasis is the leading cause of death in CRC patients,but the molecular mechanisms of carcinogenesis and development of CRC remain elusive.MORC2（Microrchidia 2）protein is a member of the MORC protein family,consisting of a CW-type zinc finger domain and three coiled-coil domains.MORC2 is mainly localized in the nucleus.MORC2 regulates chromatin remodelling during DNA damage response,regulates gene transcription and promotes lipogenesis.MORC2 protein family consists of five members including MORC1,MORC2,MORC3,MORC4,and the divergent SMCHD1.MORC proteins have been classified into two subfamilies according to their CW（cysteine-tryptophan）-type zinc finger domains:MORC1 and MORC2 are assigned to sub-family I,whereas MORC3 and MORC4 belong to sub-family IX.Zinc finger doman is one of the most common DNA-binding domains in transcription factors.The first discovered zinc finger structure contains two cysteine（Cys,C）residues and two histidine（His,H）residues,which connect with the zinc,forming the C₂H₂ zinc finger.The recently discovered CW-type zinc finger is different from C₂H₂ zinc finger,consisting of four cysteine residues linked with zinc and 2-4 conserved tryptophan（Trp,W）residue,so called CW-type.Generally,proteins that contain CW-type zinc finger domain is nucleoprotein.MORC1 is specifically expressed in sperm cells.MORC2 and MORC3 are ubiquitously expressed.MORC4 is a potential biomarker as it is highly expressed in a lymphomas.MORC3 contains three distinct domains,namely nuclear matrix attachment region,RNA binding domain and coiled-coil region.Studies have shown that MORC3 can activate tumor suppressor p53 and induce cellular senescence,but the function and molecular mechanisms of MORC2 are poorly understood.NDRG1（N-myc down-regulated gene 1）is a member of the NDRG protein family.As a potential metastasis inhibitor,NDRG1 plays important roles in cell growth and development,differentiation,hypoxic response,tumorigenesis and metastasis.NDRG1 is not just a differentiation related factor,it also affects cell growth and apoptosis.Previous reports indicated that NDRG1 is necessary for the p53-mediated caspase-activation and apoptosis.Subsequent studies have shown that the inhibition of NDRG1 in liver cancer cells significantly inhibited cell proliferation,migration,and invasion and induced apoptosis.But the roles of NDRG1 in tumorigenesis and development of colorectal cancer is still controversial.The expression of NDRG1 is regulated by many factors.Iron chelator increases NDRG1 mRNA and protein expression levels.Hypoxia,a common feature of solid tumors,can induce the expression of NDRG1 through HIF-1α-dependent and independent pathways,so NDRG1 has been recognized as a hypoxia-inducible gene.Estrogens and androgens also modulate the expression of NDRG1.In addition,the expression of NDRG1 is regulated by myc,p53,PTEN,Egr-1,AP-1 and many other differentiation regulators.Myc recruits histone deacetylase to down-regulate the expression of NDRG1.CpG sites methylation in NDRG1 promoter is an important mechanism of NDRG1 gene inhibition.Although the regulation of NDRG1 expression has been well documented,the mechanism of NDRG1 expression regulation remain to be further studied.The aim of this dissertation is to study the mechanism of NDRG1 downregulation by MORC2 and the role of MORC2 and NDRG1 in aggressive colorectal cancer phenotype.Our findings provide evidence for the molecular mechanisms of tumorigenesis and development of colorectal cancer.Methods1.MORC2 down-regulates NDRG1 in colorectal cancer cellsTo detect the mRNA and protein expression of NDRG1 by real-time PCR and western blot2.MORC2 binds to NDRG1 promoter and inhibits NDRG1 promoter activity1)To detect the regions required for the repression function of MORC2 on NDRG1 transcription by luciferase assay.2)To detect whether MORC2 binds to the NDRG1 promoter by CHIP experiments3.MORC2 inhibits NDRG1 expression by decreasing the histone acetylation level of the NDRG1 promoter1)To detect the effect of Sirtinol on the mRNA and protein expression of NDRG1by real-time PCR and western blot.2)To detect the effect of SIRT1 on the mRNA and protein expression of NDRG1 by real-time PCR and western blot.3)To detect the effect of SIRT1 on the promoter activity of NDRG1 by luciferase assay.4)To detect the interaction between MORC2 and SIRT1 in vivo by IP.5)To detect the effect of MORC2 and SIRT1 on NDRG1 gene transcription regulation by luciferase assay.6)To detect the influence of MORC2 on the acetylation levelof histone H3 and H4 in the NDRG1 promoter by ChIP-qPCR.4.NDRG1 is essential in MORC2-mediated promotion of CRC cell migration and invasion1)To examine the effect of MORC2 and NDRG1 on the migration of colon cancer cells by transwell and wound healing assays.2)To examine the effect of MORC2 and NDRG1 on the invasion of colon cancer cells by transwell add matrix.5.NDRG1 is required for MORC2-mediated promotion of CRC cell pulmonic metastasisTo examine the effect of MORC2 and NDRG1 on aggressive colorectal cancer phenotype by nude mice assay.6.Negative correlation of MORC2 and NDRG1 expression in CRC samplesTo detect the expression of MORC2 and NDRG1 in colonrectal cancer samples and assay the correlation between MORC2 and NDRG1 in colonrectal cancer patients by immunohistochemistry.Results1.MORC2 down-regulated NDRG1 mRNA,protein levels and promoter activity in CRC cells.MORC2 bound to the–446^–213bp region of the NDRG1 promoter.2.Histone deacetylase SIRT1 was involved in NDRG1 transcriptional regulation.MORC2 was able to interact with SIRT1 and inhibit NDRG1 promoter activity cumulatively with SIRT1.3.NDRG1 was essential in MORC2-mediated promotion of CRC cell migration and invasion in vitro,as well as lung metastasis of CRC cells in vivo.4.Moreover,MORC2 expression correlated negatively with NDRG1 expression in CRC patients.High expression of MORC2 was significantly associated with lymph node metastasis（p=0.019）and poor pTNM stage（p=0.02）.Conclusions1.MORC2 down-regulates the mRNA,protein level and promoter activity of NDRG1.2.MORC2 was able to interact with SIRT1 and inhibit NDRG1 promoter activity cumulatively with SIRT1.3.MORC2 promoted the migration and invasion of CRC cells by inhibiting of NDRG1.4.MORC2 was overexpressed in CRC samples and MORC2 expression correlated negatively with NDRG1 expression in CRC patients.