Correlation Study Between Transcription Factor FOXF1, NOTCH2, HNF6 And Human Liver Cancer

Mechanism of carcinogenesis has always been an important research area in tumor molecular biology.Cloning and identification of tumor related gene not only has theoretical value in tumor mechanism investigation,but also has potential practical value in tumor diagnosis and therapy.Liver cancer are the most common malignant tumors. Mechanism of carcinogenesis of liver cancer has not been clearly defined.Multi step and multi factor action may result in their occurrence.Oncogene activation and tumor suppressive gene inactivation may be associated with their development.Identification of differently expressed genes between normal liver tissue and liver cancer,can provide valuable candidate genes for prevention,diagnosis and therapy of liver cancerBased on phenotype analysis after gene knockout,endoblast transcription factor HNF6 may regulate mesoblast transcription factor Foxfl.Notch2 is a downstream gene of Foxf1.HNF6 may regulate Notch2 through Foxfl and regulate cystic development and growth.Since HNF6 expression was upregulated in surgical resected hepatocytes and bile duct epithelial cells of obstructive jaundice models,and liver cancer and cholangiocarcinoma are all proliferative diseases,we speculate that abnormal expression of above transcription factors may exist.Based on this concern,correlation study between transcription factor FOXF1,Notch2,HNF6 and human liver cancer was conducted.Forkhead box(FOX) proteins are evolutionary conserved transcription factor superfamily characterized by Forkhead DNA binding domain.It can regulate multiple biological processes.Operative dissected normal human hepatic tissue,hepatic carcinoma tissue,normal bile duct tissue and cholangiocarcinoma fresh samples were collected.Tissue total RNA was extracted by Trizole method.RNA concentration was measured by OD260 obsorbance.The same amount RNA was reverse transcribed into cDNA by M-MLV reverse transcriptase primed by random primer.Partial sequence of FOXF1 were amplified by PCR from cDNA template,mRNA level of transcription factor FOXF1 in hepatic carcinoma was found significantly lower than that in normal liver tissue.Homogenization with tissue lysis buffer containing detergent NP-40 obtained total protein.After protein concentration measurement with Coomassie brilliant blue G-250,the same amount proteins were loaded onto SDS-PAGE. Electrophoresis separated proteins were transferred onto Nitrocellulose membranes. After blocking with blocking solution containing bovine serum albamin(BSA), membranes were hybridized with diluted rabbit polyclonal antibody to human FOXF1. Membranes were incubated with HRP labeled second antibody.After chemical illuminance,develop and fixation,protein level of transcription factor FOXF1 in hepatic carcinoma was found significantly lower than that in normal liver tissue. Immunohistochemical detection confirmed that above results.Notch was found expressed in multiple species from non vertebral to vertebral animals.There is high conservation among Notch family members which play key roles in cell differentiation and development.Notch2 can be amplified from normal liver tissue, hepatic carcinoma,normal bile duct tissue and cholangiocarcinoma fresh samples.PCR products were confirmed by sequencing.No significant difference of Notch2 expression was observed between normal liver tissue and hepatic carcinoma.Western blot failed to detect Notch2 protein expression in all tissues.HNF6 is a new member of liver enriched transcription factor family.It can regulate liver specific gene expression.By action on HNF3β,HNF6 play some role in endoblast differentiation.Reverse transcription PCR can detect HNF6 gene expression in all tissues. Western blot can detect HNF6 protein expression in all tissues.No significant difference of HNF6 gene and protein expression was observed between normal liver tissue and hepatic carcinoma.Immunohistochemistry,reverse transcription PCR and Western blot were combined in this research.Expression of transcription factor FOXF1,Notch2 and HNF6 in operative dissected normal liver tissue and liver cancer were investigated.FOXF1 mRNA and protein expression was found significantly downregulated in hepatic carcinoma.This result laid foundations for further research concerning its roles in occurrence and development of liver cancer.The interrelationship of these transcription factors in carcinogenesis of liver cancer was explored.Clues were given to search new upstream and downstream factors.Detection methods of gene level and protein level were combined overcoming their limitations and exerting their advantages.Overall perspective ofgene expression were investigated.