The Expression And Biological Function Of GRHL2in Colorectal Cancer

Background： Grainyhead like2(GRHL2)，as an important transcription factor，plays an important role in embryonic development， wound healing， skin barrierformation，tracheal epithelial development，neural tube closure and other life activities.Recent studies demonstrated that GRHL2plays an important role in the tumorigenesis andprogression of tumor，and regulates tumor cell proliferation，invasion and metastasis.However，the biological role of GRHL2in human colorectal cancer (CRC) has not beenexplored. The purpose of this study is to detecte the expression of GRHL2in CRCtissues，assess the relationship between GRHL2expression and clinical pathologicalfeatures. Furthermore， we investigated the biological effects of GRHL2on cellproliferation，invasion，metastasis in vitro and in vivo，and explored the relativemechanisms.Methods： Immunohistochemistry was performed to examine GRHL2in171CRCtissues. The relationship between GRHL2and clinical pathological features was assessedby Pearson’s Chi-square (2) test. Overall survival (OS) and recurrence-free survival (RFS)were evaluated with the Kaplan-Meier method. Cox’s proportional hazards modeling wasconducted to calculate hazard ratios (HR). GRHL2mRNA and protein levels in CRCtissues and cell lines were analyzed by qRT-PCR and western blot. GRHL2over-expression and knockdown stable CRC cells were generated by lentivirus-mediatedtransfection. The effect of GRHL2on cell growth in vitro and in vivo was examined byCCK-8，colony formation，cell cycle assay and tumorigenesis in mouse xenograft model. The role of GRHL2in tumor invasion and metastasis was investigated by scratch test，transwell migration/invasion assay，tail vein injection animal model. Regulations ofGRHL2on cell cycle proteins，epithelial mesenchymal transition （EMT） and ZEB1were assessed by qRT-PCR，Western blot and immunofluorescence.Results： GRHL2was over-expressed in CRC，and played a pivotal role in CRCtumorigenesis. GRHL2expression positively correlated with tumor size，pT status， TNMstage and Ki-67. Kaplan-Meier analysis showed that GRHL2was an independentprognostic factor for both OS and RFS. Ectopic over-expression of GRHL2in CRC cellline induced an increase of cell proliferation in vitro and promoting tumor growth in vivo.On the other hand，GRHL2knockdown inhibited cell proliferation. At the same time，GRHL2regulated cell cycle distribution and modulated the expression of cell cycleprotein. We then further demonstrated that GRHL2inhibited CRC cell invasion andmetastasis. Furthmore，GRHL2could regulate the EMT process. ZEB1was a target ofGRHL2in CRC cells. There existed a negative feedback loop between ZEB1and GRHL2.Conclusions： Together，our fndings revealed GRHL2is overexpression in CRC，and plays an important role in the tumorigenesis and progression of CRC. GRHL2promotes the proliferation of colorectal cancer cells，but inhibites tumor invasion andmetastasis. This dual role may be attributed in part to the regulating of cell cycle and EMT.The reciprocal feedback loop between ZEB1and GRHL2may be involved in theGRHL2-regulated EMT process.