Expression Of GRHL2 In Pancreatic Ductal Adenocarcinoma And Its Mechanism Of Inhibiting Metastasis Of Pancreatic Cancer

Pancreatic cancer is one of the most common malignant tumors in the world,with five-year survival rate less than 5%.The incidence of pancreatic cancer is higher in developed countries.Among all cancers types,pancreatic cancer death rate ranks fourth in developed countries.In recent years,nearly 100,000 patients die of the disease each year in our courntry.Although its incidence is lower than that of liver cancer,lung cancer,colorectal cancer and other malignancies,its degree of malignancy is high.The incidence and mortality of pancreatic cancer are almost equal.The epidemiological causes of pancreatic cancer include genetic factors,obesity,alcohol consumption,smoking,chronic pancreatitis and so on.The commonly diagnostic marker of pancreatic cancer is CA19-9 which is lack of specificity and sensitivity.The location of the pancreas is relatively concealed,which causes the early symptoms of pancreatic cancer hard to be found,and consequently most patients miss their best period of treatment.Patients who have pancreatic cancer is not sensitive to radiotherapy and chemotherapy.It lacks of specific diagnostic markers and effective treatment.Surgical resection is the most effective way with five-year survival rate only 20%.In recent years,some progress has been made in the treatment of pancreatic cancer and the targeted drug is gradually being used in clinical treatment.However,these drugs did not increase the overall survival time of pancreatic cancer signigicantly.Therefore,the etiology of pancreatic cancer,diagnostic markers and new therapeutic targets for pancreatic cancer is an urgent problem to be solved.Grainy head-like 2(GRHL2)gene is one of the homologous genes of Drosophila grainyhead(GRH)in mammals,which is acted as a transcription to regulate the differentiation of intestine endoderm and epidermal epithelial cells.GRHL2 is not only related to neural tube closure,epithelial integrity,epidermal damage repair and auditory impairment during development,but also can enhance the integrity of alveolar epithelium by regulating tight junctions in humans.GRHL2 shows different expression patterns and functionsin different cancers.As an oncogene,GRHL2 can promote resistance to anoikis apoptosis in breast cancer cells by inhibiting the occurrence of death receptors.It can also activate ErbB3 and other oncogenes to induce cell proliferation.In oral squamous cell carcinoma,GRHL2 induce cell proliferation by inhibiting the activation of human telomere reverse transcriptase.But when GRHL2 is acting as a tumor suppressor,it can reverse EMT by inhibiting TGF-β signaling pathway in gastric cancer cells.In ovarian cancer cells,the expression of ZEB1 is inhibited indirectly through miR-200b/a to maintain the epithelial phenotype of cells.Inhibition of EMT occurs in breast cancer cells by inhibiting ZEB1 promoter.The mechanism of GRHL2 in different tumors is not the same.There is a lack of studies related to GRHL2 and pancreatic cancer in the literature.Given the background above,the first part of this study intended to detect the expression level of GRHL2 in clinical samples of pancreatic cancer and analyze the clinical signigicance.The second part we intended to detect the biological function of GRHL2 in pancreatic cancer cells in vitro and in vivo.In the third part,the RNA sequencing method was used to evaluate the target molecules related to GRHL2 and to provide new ideas for further research.In the fourth part,the methylation frequency of the CpG island region of the GRHL2 gene was detected by pyrosequencing in pancreatic cancer.And we analyzed the relationship between methylation and GRHL2 expression in pancreatic cancer cells.OBJECTIVE: To detect the expression level of GRHL2 in pancreatic cancer and analyze the relationship between GRHL2 and clinicopathological parameters.To detect the effect of GRHL2 on the biological function of pancreatic cancer cells in vivo and in vitro.To filter targeted gene affecting biological function of pancreatic cancer cells after overexpression of GRHL2.To detect the methylation status of GRHL2 gene CpG island in pancreatic cancer,and to analyze the relationship between the degree of methylation and GRHL2 in pancreatic cancer cells.METHODS:(1)The tumor tissues and corresponding paracancerous tissues were collected from pancreatic resection specimens.The expression of GRHL2 was detected by qRT-PCR,Western Blot and immunohistochemistry from the mRNA and protein levels in the samples.(2)Construct GRHL2 overxpression and interference lentivirus vectors and transfect it into pancreatic cancer cells.Verify from mRNA and protein levels of GRHL2 in stable overexpression and interfernce cell lines.CCK-8 and colony formation assay were used to detect the proliferation of transfected cells.Transwell chamber and scratches were used to test the migration and invasion ability of transfected cells.Flow cytometry was used to detect the distribution of transfected cell.Western Blot was used to detect the expression of EMT marker in transfected cells.Orthotopic implantation technique in nude mice was used to detect proliferation and metastasis of transfected cells.The transfected cancer cells were injected into the spleen and tail veins to detect the ability of metastasis and invasion after transfection.(3)Transcriptome sequencing was performed on PANC-1+GRHL2 overexpressing cells and its control group and bioinformatics analysis was performed.The analysis included: analysis of differentially expressed genes,analysis of GO enrichment,analysis of KEGG enrichment,alternative splice analysis,and SNP/INDEL analysis.Screened the differentially expressed genes by qRT-PCR.(4)Pyrosequencing was used to detect the methylation frequency in 19 cases of pancreatic cancer tissue and corresponding paracancerous tissues.The pancreatic cancer cells were treated with different concentrations of methylase inhibitor 5-Aza-dC to detect the changes of GRHL2 expression levels.RESULTS:(1)qRT-PCR,Western Blot results showed that the expression level of GRHL2 in pancreatic cancer tissues was lower than that of its adjacent control tissue.Immunohistochemistry showed that the expression of GRHL2 correlated with the tumor size and depth of invasion.Patients with high GRHL2 expression had a longer overall survival time than patients with low expression.GRHL2 was associated with the location of tumor,histological differentiation and the expression level of Ki67 in pancreatic cancer.(2)Comparion between cell line transtected with GRHL2 overexpression and its empty vector showed: overexpression of GRHL2 inhibit cell proliferation,colony-formation ability,migration and caused cell S phase arrest.Overexpression of GRHL2 can significantly increase the expression of E-cadherin and decrease the expression of N-cad and Zeb1 which considered as EMT realational protein.Overexpression of GRHL2 inhibit cell proliferation and disseminationin in orthotopic implantation in nude mice pancreas.Overexpression of GRHL2 inhibit metastasis in spontaneous liver metastasis nude mice model and tail vein metastasis nude mice model.(3)By RNAseq of overexpressing GRHL2 PANC-1 cells and its negative control group,364 differentially expressed genes were screened,including the expression of 313 genes increased and that of 51 relevant genes decreased.GO analysis showed that more genes are involved in epithelial regulation and intercellular adhesion.In terms of molecular function,genes are mainly involved in regulation of enzyme and molecular signal transduction.KEGG analysis showed that more genes were involved in sphingolipid biosynthesis,tight junctions,and focal adhesions.In the overexpression of GRHL2 and its negative control PANC-1 cells,the differential expression patterns of LAMB3,LAMC2,OVOL1,PRSS8,PVRL4,RAB25,TJP3,ErbB3,and CDH1 genes were consistent with the transcriptome sequencing results.(4)The methylation frequency of GRHL2 promoter region in pancreatic cancer tissues was significantly higher than that in adjacent tissues.Treated PANC-1 cells with 5-Aza-dC increase the expression of GRHL2.CONCLUSION:(1)The expression level of GRHL2 in pancreatic cancer tissues was lower than that of its adjacent control tissue.Patients with low expression of GRHL2 had shorter overall survival and poor prognosis which is related to the pathogenesis and differentiation of pancreatic cancer.(2)Overexpression of GRHL2 in PANC-1 cells can inhibit the proliferation and migration in vivo and in vitro.Overexpression of GRHL2 promotes the up-regulated epithelial phenotype protein and the down-regulation of mesenchymal phenotype.(3)The expression of GRHL2 is the most closely related to epithelial phenotype maintenance,cell intercellular adhesion,intercellular adhesion and other related genes in pancreatic cancer cells.(4)The reduction of GRHL2 in pancreatic cancer is closely related to the methylation of GRHL2 promoter region.