Construction Of Subtractive CDNA Library Of Esophageal Cancer And Observation The Clinic Significance Of ZNF14 Expressed In Esophageal Cancer

Esophageal cancer is the most common malignancy, and the Hebei Province Ci County is one of China's regions with a high incidence of esophageal cancer. According to a survey, the esophageal cancer has significantly positive family history and familial aggregation in a high incidence of esophageal cancer, positive family history can be as high as 60%. In our preliminary epidemiological survey, 70% of the Ci County esophageal cancer patients have a clear family history. Although nearly 10 years, people have done a lot of work looking for the esophageal cancer-related genes, so far not oncogenes or tumor suppressor genes are found with closely related to the esophageal cancer.Suppression Subtractive Hybridization (SSH) was a differential genes cloning technology invented by Diatchenk etc in 1996. SSH are widely used in cancer research for its low false positive rate, high specificity and cloning effectively to low abundance of differentially expressed genes. SSH method of this study, we selected esophageal cancer and matching normal esophageal mucosa from 10 cases of esophageal cancer with a clear family history of cancer in the surgical resection specimens, and separated differentially expressed fragments of esophageal cancer. These differentially expressed fragments were cloned to the T vector, after Blue-white colony screening, the positive clones were selected by polymerase chain reaction (PCR). Eventually the subtracted cDNA library of esophageal squamous cell carcinoma in Cixian was constructed. In addition, we have analysised and identified a part of differentially expressed genes, selected eight genes related to esophageal cancer, and analysised the expression of tumor- associated gene ZNF14 in esophageal cancer and matched normal esophageal mucosa using in situ hybridization to discussed the relationship between ZNF14 expression and clinical pathological features of esophageal cancer.Partâ… Construction of subtractive cDNA library of esophageal cancer and screening the gene of ZNF14Objective:Construct the differentially expressed genes cDNA library of carcino-household esophageal cancer tissue and normal esophageal mucosa for Hebei Province Ci County where was high morbidity area of esophageal cancer. For further screening the differentially expressed genes of esophagus, and provide the earlier stage basis research for discussing whether there are familial esophageal heredity genes,or whether exists a definitely correlative mutational gene locus of esophageal cancer and whether the variety of this mutation is related to the occurrence of the esophageal cancer. Methods: The esophageal cancer tissue and normal esophageal mucosa specimens for construction of subtractive cDNA library in our experiment came from 10 patients with esophageal cancer of the different cancer households in Hebei Province Ci County, male 6 and female 4. The patients' father generation or female generation have an explicitly cancer history of esophageal cancer or liver cancer etc. All the tissue specimens were fresh after the surgery and preserved in the liquid nitrogen preparing for construction of subtractive cDNA library. The pathologic diagnosis of patients after the operation was confirmed to be different stages of esophageal squamous cell carcinoma (SCC) together by pathology department of Cixian People's Hospital and the Hebei Provincial People's Hospital.The differentially expressed genes of the 10 carcino-household esophageal cancer and normal esophageal mucosas were subtracted, cloned, blast analysised and identified by SSH. The mRNA of the tissue specimens were extracted and reverse transcription to the cDNA. Using SSH for subtractive hybridization reaction,the subtractive cDNA library could be obtained through the twice PCR relying on the nested primers. Cloned the productions, measured the sequences of clones, analysised the gene homology, and then selected the closely-related genes to the tumor.Results: Using SSH to construct the subtractive cDNA library of esophageal cancer, further selected 20 differentially expressed gene fragments of esophageal cancer to measure the sequences of clones and analysis gene homology. There were eight closely-related genes to the the period of tumorigenesis and progression: ZNF14(zinc finger protein 14), DSC3(desmocollin 3), EEF1A1(eukaryotic translation elongation factor 1 alpha 1), ZNF 66(zinc finger protein 66), TLR10 (toll-like receptor 10),CYP4F8 (cytochrome P450, family 4, subfamily F, polypeptide 8), SLC31A1(solute carrier family 31, member 1), FMNL2(Homo sapiens formin- like 2), MTMR6(myotubularin related protein 6 MTMR6). 4 homo sapiens chromosome open reading frames : C6orf208,C14orf153,C1orf96,C20orf177.Conclusions: The subtractive cDNA library of esophageal cancer was constructed successfully by using SSH. We have obtained eight genes associated with the period of esophageal tumorigenesis and progression, and conducted a preliminary study to ZNF14.Partâ…¡The clinic significance of ZNF14 expressed in esophageal cancerObjective:Detected the ZNF14 expression in esophageal cancer tissue and normal esophageal mucosa, discussed the clinical significance of the ZNF14 in esophageal SCC, and analysised the relationship of ZNF14 expression and the esophageal cancer clinical pathological features.Methods: 34 cases of samples were taken from the patients with esophageal cancer after surgery of the Hebei Provincial People's Hospital Thoracic Surgery in 2005~2006 years. Each sample had the explicitly pathology diagnosis and staging. The expression of ZNF14 in esophageal cancer tissues and normal esophageal mucosa were analysised by means of in situ hybridization(ISH). The positive cells count informations were analysised according to staining results of ISH and the data was analyzed by statistics software SPSS10.0, p <0.05 hints the significant.Results: The ZNF14 was expressed different level in 32 samples of esophageal cancer from total 34 cases(94.3%),But only 4 were observed in the normal mucosa(11.8%), there were significant statistics significance (P<0.01) compare with the two result. The ZNF14 expression level was relevant to the tumors differentiation and metastasis (p<0.05), but not significant both the different tumor sizes and tumors invading depth.Conclusions: The results that ZNF14 was specificitily expression in esophageal SCC suggest that it were relevant obviously to esophageal tumorigenesis and differentiation of esophageal cancer. It is speculated that The ZNF14 expression might plays roles during canceration proceeding of esophageal SCC. Our results confirmed further the accuracy of the cDNA library in the first part of thesis.