Preliminary Study On The Role And Mechanism Of HNF3? In Proliferation,Invasion And Metastasis Of Colorectal Cancer Cells

Colorectal cancer is a common malignant tumor and still represents the third leading cause of cancer-related death in China^[1].The prognosis of colorectal cancer directly correlated with the extent of tumor invasion and metastases.Despite appreciable progressions in the management and treatment of colorectal cancer on several fronts,such as earlier diagnosis,radical surgery,radiotherapy and neoadjuvant chemotherapy,there is still no satisfactory therapy because the mortality of colorectal cancer patients have increased as a results of recurrence or metastases^[2].The diagnosis and prevention of recurrence and early tumor metastasis are critical and there is a clear need to explore the indicator moleculars for colorectal cancer aggravation.However,to date,the regulatory mechanisms underlying colorectal cancer occurrence and recurrence or metastasis remain largely unknown and few novel prognostic biomarkers have been identified.Hepatocyte nuclear factor 3??HNF3??,also known as the transcription factor forkhead box A2?FOXA2?,a key member of the hepatocyte nuclear factor?HNF?family,which consists of HNF1,HNF3,HNF4,HNF6 and CCAAT/enhancer binding proteins?CEBPs?,play a critical role in the development^[3].Previous studies have shown that the HNF3transcription factors,especially HNF3?/FOXA1,HNF3?/FOXA2 and HNF3?/FOXA3,play important roles in the gastro-intestinal tract differentiation^[4].HNF3?mRNA first expressed during embryogenesis,and was observed during gastrulation in the anterior primitive streak and node with subsequent expression in gut^[5-7].HNF3?deficiency was embryonic lethal due to failed node and notochord development^[8].It plays crucial role not only in embryonic development,including the formation of node,nervous system and endoderm-derived structures,but also in postnatal development via regulation of normal bile acid and lipid homeostasis^[9].Many studies have shown that HNF3?suppression was closely related to tumorigenesis,progression and metastasis,which was documented in several kinds of tumors,including hepatocellular carcinoma,lung cancer,pancreatic cancer,gastric cancer and thyroid cancer^[10-14].However,the role of HNF3?in colorectal cancer has not been well established and the function of HNF3?in colorectal cancer's aggravation remains obscure.In this study,we analyzed the HNF3?expression level and its prognostic role in 174 colorectal cancer patients as well as the tumor biological functions in colorectal cancer cells.Part? The expression and clinical significance of HNF3?in tissues of colorectal cancerObjectives:The aim of the first part of this study was to explore the expression of HNF3?in tissues of colorectal cancer in order to analyse the relationship between the expression of HNF3?and clinical parameters in patients with colorectal cancer,and to find the effect of HNF3?on the outcome of colorectal cancer patients.Methods:Immunohistochemistry assay was used to detect the expression of HNF3?protein in different kinds of colorectal tissues,including 8 cases from rectal inflammatory tissues,14 cases from low grade intraepithelial neoplasia of colorectum,18 cases from high grade intraepithelial neoplasia of colorectum,174 cases form the resected specimen of colorectal neoplasia and 3 cases from normal colorectal tissues.All the above tissue samples were obtained from the patients collected from the Department of gastrointestinal surgery in Xijing Hospital from the First affiliated Hospital of the Military Medical University from October 2010 to September 2011.None of these patients received neoadjuvant chemotherapy or radiotherapy prior to surgery,and the patients with stage?,?and?after surgery were treated with Xelox chemotherapy regimens?oxaliplatin and capecitabine?.Clinical data and follow-up data were collected from patient's electronic medical records and telephone.Yates correction or Fisher's exact probability chi-square test and Spearman rank correlation analysis were used to compare HNF3?with patients'clinical data,Kaplan-Meier method was used to draw the survival curve of colorectal cancer patients,and Cox proportional hazards regression model was used for multivariate analysis to evaluate whether each index was an independent prognostic factor for colorectal cancer patients.Result:HNF3?protein was highly expressed in 3 cases from normal colorectal epithelial tissue,8 cases from colorectal inflammatory tissue,14 cases from rectal low grade intraepithelial neoplasia,and 18 cases from high grade intraepithelial neoplasia,while it was low in 174 cases of colorectal cancer tissues.Fifty-six percent of the 174 specimens form the resected specimen of colorectal neoplasia were in low expression“+”or no expression”-”,and there was significant difference in the staining of HNF3?in colorectal cancer and non-cancer tissues?P<0.0001?,which demonstrated that HNF3?is lowly expressed or not expressed in colorectal cancer.The low expression of HNF3?was closely related to the clinical stage,T stage,the number of lymph node metastases,with or without distant metastasis?P<0.05?,and was not related to age,gender,histological type,degree of tumor differentiation,location of tumor?P>0.05?.Univariate analysis showed that low expression of HNF3?,clinical stage of UICC,degree of invasion,lymph mode metastasis,and distant metastasis were closely related prognosis?P<0.001?.Multivariate analysis by the Cox proportional hazards regression model showed that low expression of HNF3??HR:0.601,95%CI:0.387-0.933,P=0.023?,distant metastasis?HR:5.890,95%CI:1.028-33.772,P=0.046?and clinical stage of UICC?HR:3.737,95%CI:1.611-8.668,P=0.002?were independent predictors of overall survival for patients with colorectal cancer,while the low expression of HNF3??HR:0.606,95%CI:0.392-0.937,P=0.024?,lymph node metastasis?HR:0.171,95%CI:0.032-0.899,P=0.037?and clinical stage of UICC?HR:9.935,95%CI:2.214-44.574,P=0.003?were independent predictors of progression free survival for colorectal cancer patients.Conclusion:HNF3?was confirmed in protein levels to be low or not expressed in colorectal cancer tissues,but high expression in non-cancer tissues,including normal colorectal epithelium,colorectal inflammatory tissue,low grade intraepithelial neoplasia and high grade intraepithelial neoplasia.The expression of HNF3?was highly relevant to clinical stage of UICC,T stage,lymph node metastasis,distant metastasis and the prognosis of patients with colorectal cancer,and colorectal cancer patients with low expression of HNF3?had extremely poor survival.Therefore,the expression of HNF3?is an independent predictor of prognosis in patients with colorectal cancer.Part?The effect of HNF3?on proliferation,invasion and metastasis of colorectal cancer SW480 cell line and its possible mechanismObjectives:The aim of the second part of this study was to explore the effect of HNF3?on proliferation,invasion and metastasis of colorectal cancer SW480 cell line,and to observe the efficiency of nude mice oncogenicity of HNF3?in vitro on tumor weight,and study its possible mechanism initially.Methods:HNF3?Lentivirus and no-loaded virus were constructed and transfected into SW480 cells respectively,then SW480 cell line with HNF3?overexpression and SW480 cell line with no-load virus were established.Western blot was used to detect in HNF3?in transfected cells in order to confirm the successful construction of cell model in protein level.SW480 cell line with HNF3?overexpression and SW480 cell line with no-load virus were established.MTT cell proliferation assay was used to detect the change of cell proliferation after HNF3?overexpression.The cell migration ability of HNF3?after overexpression was studied by cell scratch assay and Transwell cell migration assay.The nude mice tumorigenesis assay was used to examine the effect of HNF3?overexpression on tumor growth in colorectal cancer.HNF3?over-expression colorectal cancer SW480 cell line and no-load virus SW480 cell line were established,and the expression of key proteins such as IL6,JAK1 and STAT3 in JAK/STA signaling pathway was detected by Western blot.Result:The SW480 cell line with overexpress HNF3?had significantly decreased cell proliferation compared with on-load virus SW480 cell line and normal SW480 cell line,especially on the 4th,5th and 6th day?P<0.05?.There was a similar phenomenon in colon cancer SW480 cell proliferation,invasion and migration experiments?P<0.05?.Colorectal cancer SW480 cell line overexpression in nude mice oncogenicity assay showed that tumor weight in the overexpression group was significantly lower than that in the normal group and on-load virus group?P<0.05?.The key proteins including IL6,JAK1 and STAT3 in JAK/STA signaling pathway were found decrease in expression after SW480 cell line with overexpress HNF3?in vitroConclusion:The low expression of HNF3?is relevant to the proliferation,invasion and metastasis of colorectal cancer cells,and the nude mice xenograft tumor model in vivo also confirmed that nude mice with overexpression HNF3?were lighter,which suggests that HNF3?may be an anti-oncogene of colorectal cancer.HNF3?inhibits the proliferation,invasion and metastasis of SW480 cells through JAK/STA signal pathway,suggesting that HNF3?may be an anti-oncogene of colorectal cancer,which provides a possible direction for clinical targeted therapy of colorectal cancer.