Epigenetic Study Of Biliary Tract Carcinoma

Epigenetics is a new science that studies the change of gene expression caused by non-gene sequence alteration. It decides gene transcription and the way of gene expression. Since human being enters the post-genome era, epigeneitcs has attracted more and more scholars' attention and is becoming one of the major study fields for the function of genome. DNA methylation is one of the key parts of epigenetics and is one of the main ways to regulate gene expression. DNA methylation can make some genes that are not essential to some tissues and cells silence and inactivation by methylation of the promoter region and cytosine in the near CpG islands. DNA methyltransferases (DNMTs) are the essential enzymes to catalyze and maintain DNA methylation and play an important role in DNA methylation.Tumor is a pathologic process that involves multiple factors, many genes and pathways, and tumorigenesis is related to the activation of oncogene and inactivation of tumor suppressor gene. Many studies demonstrate that inactivation of tumor suppressor gene is closely related to hypermethylation of its promoter region. Over-expression and/or increased activity of DNMTs are considered to be one of the main causes of hypermethylation in the promoter region of tumor suppressor gene.In our previous studies, we observed the cell cycle alteration of human biliary tract carcinoma cell line QBC₉₃₉ after treatment with DNMTs inhibitor 5-Aza-CdR and found that 5-Aza-CdR could suppress the growth and proliferation of QBC₉₃₉ in vivo and in vitro and induce apoptosis. These experimental results suggested that DNMTs may play an important role in pathogenesis and development of biliary tract carcinoma and that down-regulation of DNMTs expression may has potential significance for reversing the hypermethylation status of tumor suppressor genes and for treating bile duct cancer. We also found the expression loss rate of RASSF1A (a tumor suppressor gene) in extrahepatic biliary tract carcinoma tissues is 68.75%. It is much higher than that in adjacent non-cancerous tissues. The aberrant expression loss of RASSF1A was related with lymph node metastasis and TNM stages. By using methylation specific PCR and DNA sequencing in the previous studies, we also confirmed that hypermethylation of CpG loci in promoter region was the key mechanism of the inactivation of tumor suppressor gene RASSF1A.To further explore the relationship among DNA methylation, gene expression and tumorigenesis, we took DNMT1, DNMT3b (two DNA methyltransferase genes) and RASSF1A (a tumor surpressor gene) as target genes and used antisense RNA technology to down-regulate DNMT1 and DNMT3b expression levels in human biliary tract carcinoma cell line QBC₉₃₉ in order to study the roles of DNMT1 and DNMT3b in regulation of RASSF1A gene expression and the methylation status in the promoter region of RASSF1A gene and to study the biological effects of down-regulation DNMT1 and DNMT3b expression in human biliary tract carcinoma cell line QBC₉₃₉. This study may provide a new fundamental and experimental data for exploring the pathogenesis mechanism of biliary tract carcinoma in epeigenetic pathway. Part â… : The Expression and Significance of DNMT1 and DNMT3b Gene in Human Biliary Tract CarcinomaPaper 1: The Expression and Significance of DNMT1 and DNMT3b Gene in Human Biliary Tract Carcinoma Tissues and CellsObjective To study the expression of DNA methyltransferase 1 (DNMT1) and DNA methyltransferase 3b (DNMT3b) in human biliary tract carcinoma tissues and cell line QBC₉₃₉, to analyze the relationship between the expression levels of DNMT1 and DNMT3b and the clinicopathologic features of biliary tract carcinoma and to explore the roles of DNMT1 and DNMT3b during the tumorigenesis of biliary tract carcinoma.Methods1. The expression of DNMT1 protein and DNMT3b protein were detected in 24 biliary tract carcinoma specimens and in 20 chronic cholangeitis tissues specimens by immunohistochemistry method. The results of DNMT1 or DNMT3b in the two kinds of tissues were compared and analyzed the relationship with the clinicopatholigic features of biliary tract carcinoma.2. RT-PCR and Western Blot were used to detect the expression of DNMT1 and DNMT3b gene at the transcriptional level and protein level in human biliary tract carcinoma cell line QBC₉₃₉.Results1. The rates of positive stain cells of DNMT1 protein were (37.57±11.24)% and (10.73±6.61)% in 24 biliary tract carcinoma specimens and in 20 chronic cholangeitis specimens respectively. There were very significant difference in the expression level of DNMT1 protein between the two groups(P =0.005); The rates of positive stain cells of DNMT3b protein were (46.56±12.12)% and (3.34±2.73)% in 24 biliary tract carcinoma specimens and in 20 chronic cholangeitis specimens respectively. There were also very significant difference in the expression level of DNMT3b protein between the two groups (P =0.000).2. The increased expression of DNMT1 and DNMT3b protein were correlated with differentiation grade of carcinoma. The protein levels of DNMT1 and DNMT3b increased progressively from well to moderately to poorly differentiated adenocarcinomas. The increased expression of DNMT1 and DNMT3b protein were not correlated with gender, age, size of tumor, location of tumor, metastasis of lymph node and clinical TNM stage.3. The mRNA expression and protein expression of DNMT1 and DNMT3b were both positive in human biliary tract carcinoma cell line QBC939.Conclusions1. The expression levels of DNMT1 and DNMT3b protein in biliary tract carcinoma are significantly higher than in chronic cholangeitis tissues. The increased DNMT1 and DNMT3b protein levels are both correlated with the differentiation grade of tumor and are not correlated with gender, age, size of tumor, location of tumor, metastasis of lymph node and clinical TNM stage. This result suggests that the over-expression of DNMT1 and DNMT3b may be related to the pathogesis of biliary tract carcinoma and may be an early molecular event during tumorigenesis of biliary tract carcinoma.2. Both DNMT1 and DNMT3b gene express in the human biliary tract carcinoma cell line QBC939. Part â…¡: Construction and Identification of Eukaryotic Expression Plasmids of Antisense DNMT1 and DNMT3b GenePaper 2: Identification of Eukaryotic Expression Plasmid of Antisense DNA methylferase 1 GeneObjective To identify the antisense DNA methylferase 1 (DNMT1) gene eukaryotic expression plasmid that was constructed in National Cancer Insitute (USA) and obtained as a gift from Prof. Fang Jing-yuan, and to provide a tool to study the function of DNMT1 gene.Methods According to the construction method and the map of antisense DNMT1 gene eukaryotic expression plasmid, Xba â…  and BamH â…  were used to identify the constructed recombinant plasmid for double enzyme digestion. The recombinant plasmid was used as PCR template for PCR confirmation and the empty plasmid pCMV was used as control template.Results1. After Xba â…  and BamH â…  double digestion, pCMV-THM produced 4 DNA fragments which length were 1.1, 1.2, 3.9 and 4.7kb respectively. This result was accordance with the map of this recombinant construction and corresponding references.2. A 442bp specific DNA band which represented DNMT1 gene was obtained by PCR which took pCMV-THM as PCR template, meanwhile, no corresponding DNA band was observed in control group which took pCMV as PCR template. This result indicated that the recombinant plasmid contained the coding sequence of human DNMT1 gene. Conclusion The constructed eukaryotic expression plasmid of antisense DNMT1 gene was confirmed by using double enzyme digestion and PCR method. It can be used for further studies of DNMTl gene function. Paper 3: Construction and Identification of Eukaryotic Expression Plasmid of Antisense DNA Methylferase 3b GeneObjective To construct the eukaryotic expression plasmid of antisense DNA methylferase 3b (DNMT3b) gene, and to provide a tool to study DNMT3b gene function.Methods PCR primers were designed according to the coding sequence of human DNMT3b gene, and BamH â…  and EcoR â…  recognition sequence and cutting sites were added to the 5' end of the sense and antisense primer respectively. The 485bp specific DNA fragment was obtained from the cDNA of human biliary tract carcinoma cell line QBC₉₃₉ by using RT-PCR, the purified DNA fragment was then subcloned reversedly into the multiple cloning site of eukaryotic expression vector pEGFP-C1. The constructed recombinant plasmid was identified by PCR confirmation, EcoR â…  and BamH â…  double enzyme digestion and DNA sequencing.Results1. In PCR comfirmation, a 467bp specific DNA band was observed by PCR which was taken the recombinant plasmid as PCR template, and no corresponding DNA band was detected by PCR which was taken pEGFP-C1 as PCR template. This result suggests that the recombinant plasmid pEGFP-DNMT3b(AS) contains part of coding sequence of human DNMT3b gene.2. Xba â…  and Kpn â…  double digestion produced a 471bp and a 5.0kb DNA fragment which represented the inserted DNA fragment and the vector respectively. This result indicated that the DNMT3b gene fragment was subcloned reversedly into the multiple cloning site of pEGFP-C1 successfully.3. The DNA sequencing results showed that the sequence of the 471bp inserted segment was identical with the reverse complemented sequence of 1651～2117bp cDNA of DNMT3b gene. The results of nucleotide-nucleotide BLAST in NCBI also showed the sequence alignment is completely correct. It confirmed that the antisense DNMT3b gene eukaryotic expression plasmid was constructed successfully.Conclusion The eukaryotic expression plasmid of antisense DNMT3b gene was constructed successfully by using gene cloning technique. Part â…¢: Regulation of RASSF1A Gene Expression and the Biological Effects of Transfection with Antisense DNMT1 and DNMT3b Gene in Biliary Tract Carcinoma CellsPaper 4: Demethylation in the promoter region of RASSF1A gene and its re-expression induced by down-regulating DNMT1 and DNMT3b gene expression in human biliary tract carcinoma cellsObjective To study the effects of co-transfection of antisense DNMT1 gene and antisense DNMT3b gene eukaryotic expression plasmids on the methylation status in the promoter region of RASSF1A gene and on its expression in human biliary tract carcinoma cell line QBC939, and to explore the function of DNMT1 and DNMT3b gene in DNA methylation and regulation of gene expression.Methods1. The test of the suitable concentration of G418 for QBC939 cells was performed at different concentration from 0μg/ml to 1000μg/ml. The lowest concentration that could cause all the QBC939 cells to die was taken to decide the suitable concentration of G418.2. The constructed antisense DNMT1 gene eukaryotic expression plasmid and the antisense DNMT3b gene eukaryotic expression plasmid were both transfected into QBC₉₃₉ cells using lipofectamine transfection reagents. The stable transfection cell lines were obtained by selection of fluorescent cell clone under fluorescent inverted microscope after G418 selection.3. The expression levels of DNMT1 and DNMT3b protein were detected by Western Blot analysis after transfection. The methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression of RASSF1A gene mRNA and protein was observed by semi-quantitative RT-PCR and Western Blot respectively.Results1. After the test of the suitable concentration of G418, 800(μg/ml was the best concentration for G418 selection in QBC₉₃₉.2. The result of Western Blot demonstrated the protein level(s) of DNMT1 and/or DNMT3b decreased significantly after transfection of the antisense DNMT1 and/or DNMT3b gene eukaryotic expression plasmids. Transfection of pCMV and pEGFP-C1 plasmids could not affect the protein level(s) of DNMTl and/or DNMT3b in QBC₉₃₉.3. The methylaiton status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation after co-transfection of antisense DNMT1 gene and antisense DNMT3b gene and single transfection of antisense DNMT1 gene, meanwhile, single transfection of antisense DNMT3b gene or co-transfection pCMV and pEGFP-C1 could not induce demethylation in the promoter region of RASSF1A gene.4. The result of semi-quantitative RT-PCR demonstrated that the mRNA expression level of RASSFIA gene was increased from 0.000 to 0.714±0.063 and 0.409±0.026 (P=0.000) after co-transfection of antisense DNMT1 gene and antisense DNMT3b gene and single transfection of antisense DNMT1 gene respectively, and single transfection of antisense DNMT3b gene or co-transfection pCMV and pEGFP-C1 could not induce RASSF1A gene mRNA re-expression.5. Western Blot analysis showed that co-transfection of antisense DNMT1 gene and antisense DNMT3b gene and single transfection of antisense DNMT1 gene could induce RASSFIA protein re-expression. The effect of re-expressing RASSF1A protein of co-transfection is more obvious than single transfection of antisense DNMTl gene. Single transfection of antisense DNMT3b gene or co-transfection pCMV and pEGFP-C1 could not induce RASSF1A protein re-expression. Conclusions1. Transfection of antisense DNMT1 gene and/or antisense DNMT3b gene eukaryotic expression plasmid can decrease the expression level(s) of DNMT1 and/or DNMT3b protein in QBC₉₃₉.2. Down-regulating the expression of DNMT1 and DNMT3b or single down-regulating the expression of DNMT1 can cause demethylation in the promoter region of RASSF1A gene and induce its re-expression in QBC₉₃₉. The former is more effective than the later. Single down-regulating the expression of DNMT3b can not affect the methylation status in the promoter region of RASSF1A gene and can not induce its re-expression in QBC₉₃₉.3. DNMT1 gene may play a key role and DNMT3b gene may act as an accessory in DNA methylation and in regulation of gene expression. The combination of these two DNA methlytransferases genes can enhance their functions significantly. Paper 5: Biological effects of down-regulating DNMT1 and DNMT3b gene expression in human biliary tract carcinoma cellsObjective To study the effects of co-transfection of antisense DNMT1 gene and antisense DNMT3b gene eukaryotic expression plasmids on the growth and proliferation ability of human biliary tract carcinoma cell line QBC939 in vivo and in vitro, and to explore the role of DNMT1 and DNMT3b gene in tumorigenesis of biliary tract carcinoma.Methods1. The constructed antisense DNMT1 gene eukaryotic expression plasmid and the antisense DNMT3b gene eukaryotic expression plasmid were co-transfected into QBC939 cells using lipofectamine transfection reagents. After stable transfection, the expression levels of DNMT1 and DNMT3b protein were detected by Western Blot.2. The proliferation ability of different transfection cell groups was observed by MTT method and the experiment of colony formation in soft agar. Flow cytometry was used to detect the change of the cell cycle and the apoptosis rate. Transfected cells were injected into the subcutaneous tissue of nude mouse and the weight and volume of tumors in different groups were measured 3 weeks later respectivly.Results1. The protein levels of DNMT1 and DNMT3b decreased significantly after stable transfection.2. Both co-transfection group and single transfection of antisense DNMT1 gene group affected the cell growth curve. The former delayed the cell growth more than the later.3.The cell colony formation rate of co-transfection group and single transfection of antisense DNMT1 gene were (6.78±0.89)% and (14.86±2.13)%. They were less than that of control group significantly (P=0.000).4. Both co-transfection group and single transfection of antisense DNMT1 gene group blocked the cell cycle at G1 phase and increased the apoptosis rate from (1.63±0.27)% to (18.47±1.46)% and (6.19±0.78)% respectively. There were very significant difference between the co-transfection group and single transfection of antisense DNMT1 gene group (P=0.000).5. The tumor in the subcutaneous tissue of nude mouse of co-transfection group was the smallest, and the tumor of antisense DNMT1 gene transfection group was the second smallest comparing to the control group and other experiment groups. The tumor control rate of co-transfection group (84.31%) was significantly higher than that in single transfection of antisense DNMT1 gene group (49.17%) (P=0.000).6. Co-transfection of the plasmid pCMV and pEGFP-C1 or single transfection of the antisense DNMT3b gene did not suppress the growth and proliferation of QBC₉₃₉.Conclusions1. Co-transfection of antisense DNMT1 gene and antisense DNMT3b gene eukaryotic expression plasmid can down-regulate the expression levels of DNMT1 and DNMT3b, and can suppress the growth of human biliary tract carcinoma cell line QBC₉₃₉ and induce apoptosis. It has more effect on the cell growth than single transfection of the antisense DNMT1 gene.2. Our study also suggests that DNMT1 gene plays a key role in DNA methylation and DNMT3b gene may act as an accessory to support the function in QBC₉₃₉ cells. These genes may be related to the tumorigenesis of biliary tract carcinoma via DNA methylation pathway. In this study, DNA methylation, a major part of epigenetics, was taken as the main research focus, and DNMT1, DNMT3b (two DNA methyltransferase genes) and RASSF1A (a tumor surpressor gene) were taken as target genes to study. We observed the expression levels of DNMT1 and DNMT3b in human biliary tract carcinoma tissues and in human biliary tract carcinoma cell line QBC₉₃₉, and used antisense RNA technology to down-regulate their expression levels in QBC₉₃₉. We also explored the mechanism of regulation of RASSF1A gene expression and the methylation status in the promoter region of RASSF1A gene by down-regulating the expression of DNMT1 and DNMT3b, and studied the biological effects of down-regulating the expression of DNMT1 and DNMT3b on the human biliary tract carcinoma cell line QBC₉₃₉. According to the experimental results, we summarized the conclusions and some new findings as follows:1. At the present time, to our knowledge, there are no corresponding reports on the expression of DNMT1 and DNMT3b in biliary tract carcinoma. This is the first study to explore the expression of DNMT1 and DNMT3b in human biliary tract carcinoma tissues and cell line QBC₉₃₉ by using immunohistochemical method and to analyze the relationship between the expression of DNMT1 and/or DNMT3b and the clinicopathologic features of biliary tract carcinoma. The experimental results demonstrated that the expression levels of DNMT1 and DNMT3b protein in biliary tract carcinoma are significantly higher than in chronic cholangeitis tissues. The increased DNMT1 and DNMT3b protein levels are correlated with the differentiation grade of tumor and are not correlated with metastasis of lymph node and clinical TNM stage. Both DNMT1 and DNMT3b gene express in the human biliary tract carcinoma cell line QBC₉₃₉. This result suggests that the overexpression of DNMT1 and DNMT3b may be related to the pathogesis of biliary tract carcinoma and may be an early molecular event during tumorigenesis of biliary tract carcinoma.2. The eukaryotic expression plasmid of antisense DNMT3b gene was constructed successfully by using gene cloning technique. It is the first recombinant plasmid for DNMT3b gene research. The eukaryotic expression plasmid of antisense DNMT3b gene will be a useful tool to study DNMT3b gene function in DNA methylation and the regulation of tumor related gene expression.3. Co-transfection of antisense DNMT1 and antisense DNMT3b gene eukaryotic expression plasmids can decrease the expression levels of DNMT1 and DNMT3b in QBC₉₃₉. The result also indicats that antisense RNA technology is very useful for down-regulation of gene expression.4. Co-transfection of antisense DNMT1 and antisense DNMT3b gene eukaryotic expression plasmids can induce demethylation of the promoter region of RASSFIA gene and its re-expression, and can also suppress the growth of human biliary tract carcinoma cell line QBC₉₃₉ in vitro and in vivo and induce apoptosis. The effects of co-transfection of antisense DNMT1 and antisense DNMT3b gene are more profound than single transfection of antisense DNMT1 gene. Single transfection of antisense DNMT3b gene has not the above biological effects. The results described above indicat us some new viewpoints:1) DNMT1 gene plays a key role and DNMT3b gene may act as an accessory in DNA methylation and in regulation of gene expression. The combination of these two DNA methlytransferases genes can enhance their functions significantly.2) These two DNA methyltransferases, DNMT1 and DNMT3b, may participate in the regulation of gene expression and tumorigenesis via DNA methylation pathway.3) Down-regulating the expression of DNMT1 and DNMT3b in tumor cells can induce demethylation of the promoter region of tumor related genes and make their re-expression to suppress the growth of tumor. This research may provide a new epigenetic method for biliary tract carcinoma gene therapy.