Epigenetic Study Of Biliary Tract Carcinoma

Bile duct carcinoma is a malignancy originating from the biliary epithelium.To datethere is no imaging techniques or molecular markers that can aid in the accurate diagnosisand evaluation of bile duct carcinoma in the early stage of the disease.So many patientspresented with unresectable diseases when the diagnosis was definite.And also Bile ductcarcinoma is an aggressive malignancy typified by and unresponsiveness to the existingchemotherapy and radiotherapy regimes in the vast majority of cases.So,it is importantto explore new drugs for the treatment of bile duct carcinoma.Epigenetics is a new science that studies the change of gene expression caused bynon-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 morescholars' attention and is becoming one of the major study fields for the function ofgenome.Epigenetics provides a brand-new research direction for the study of the tumortherapy.Epigenetics refers to alternate phenotypic states that are not based on differencesin genotype,and are potentially reversible,which provides the theoretical basis for the useof epigenetie therapy for tumors.Nowadays,the fact that subsets of many cancers lack chromosomal or microsatelliteinstability argues against the hypothesis that genomic instability plays an essential role inthe initiation and maintenance of oncogenesis.Cancer-specific,epigenetically-basedchanges in gene expression caused by abnormalities in DNA methylation,histonemodifications,and nucleosome positioning are gaining recognition as driving events intumorigenesis,in which the epigenetic gene silencing is considered to be a key event.Soour research focuses on reversing the abnormal epigenetic pattern of tumor cells,especiallyreactivating the silent tumor suppressor gene by epigenetic drugs.In our previous studies,we observed the cell cycle alteration of human biliary tractcarcinoma cell line QBC₉₃₉ after treatment with DNMTs inhibitor 5-Aza-CdR and foundthat 5-Aza-CdR could suppress the growth and proliferation of QBC₉₃₉ in vivo and in vitro and induce apoptosis,we also found that using antisense RNA technology to down-regulateDNMT1 and DNMT3b expression levels in human biliary tract carcinoma cell line QBC₉₃₉could make the promoter region of the RASSF1A gene to be demethylated and couldreactivate it.These experimental results suggested that DNA methyltransferases might playan important role in re-expressing tumor suppressor gene of QBC₉₃₉ cells.In recent years,advances in research of chromatin structure,histone modification,transcriptional activityand DNA methylation have resulted in an increasingly integrated view of epigenetics.It isconfirmed that aberrant gene transcription resulting from epigenetic changes are frequentevents in the molecular pathogenesis of malignant transformation.DNA hypermethylationand histone deacetylation are critical for determining a closed chromatin structureresponsible for or related with aberrant gene transcription in malignancies.Based on suchobservations,in this research we focus on the effect of DNA methylationinhibitors(hydralazine) and histone deacetylase inhibitors(valproate) on the proliferationand invasiveness of the QBC₉₃₉ cells and the expression of tumor suppressor gene of thecells.In addition,it is reported that SIRT1,a member of silentin formation regulator 2family,plays an important role in the remodeling of chromatin and tumorigenesis.And inour research,we also found that using hydralazine and valproate in combination coulddown-regulate the SIRT1 expression of QBC939 cells,which was insensitive to histonedeacetylase inhibitors.So we further explored the mechanism of the effect of hydralazineand valproate on SIRT1 gene,and the role of SIRT1 gene in re-expressing RASSF1A byusing hydralazine and valproate in combination.This research may lay a foundation for theepigenetic therapy of bile duct carcinoma and clarify the mechanism of the effect of DNAmethylation inhibitors and histone deacetylase inhibitors on tumor cells. Partâ… Intervention effect of DNMTi and HDACi on cells of humanbile duct carcinoma in vitroPaper 1 The effect of DNMTi and HDACi on the expression of tumorsuppressor genes of QBC₉₃₉ cells in vitroObjective To investigate the effects of hydralazine and valproate on P16,RASSIF1A andE-cardherin gene of QBC₉₃₉ cells in vitro.Methods The QBC₉₃₉ cells were divided into four groups.The experimental groups weretreated separately with hydralazine and valproate either alone or combined(the finalconcentration was 10mmol/L),and the control group was added with RPIM-1640 culturemedium.After 48hours,the expression of the mRNA and the protein of the three genes wasevaluated by reverse transcription-polymerase chain reaction and Western blot assay,andthe methylation status of promoter region of the three genes was detected byMSP(mehtylation specific PCR).Results It was found that the promoter regions of P16,RASSIF 1A and E-cardherin gene ofQBC₉₃₉ cells were hypermethylated;valproate alone could not contribute to demethyaltionof the three genes,while hydralazine could make them to be partially demethylated.However,the methylation status of the three genes could be thoroughly reversed by usingvalproate and hydralazine in combination.It was comfirmed that the three genes of QBC₉₃₉cells could not be transcriptionally reactivated by valproate alone,while hydralazine alonecould induce minimal re-expression of the three genes.However,using valproate andhydralazine in combination ccould result in robust re-expression of the threegenes(P＜0.01).Conclusion The two drugs can synergistically reactivate the P16,RASSF1A andE-cardherin gene of QBC₉₃₉ cells and make the promoter region of the three genes to bedemethylated. Paper 2 The effect of DNMTi and HDACi on the growth and invasion ofQBC₉₃₉ cells in vitroObjective To investigate the effect of hydralazine and valproate on the growth and invasionof QBC₉₃₉ cells in vitroMethods The QBC₉₃₉ cells were divided into four groups.The experimental groups weretreated separately with hydralazine and valproate either alone or combined(the finalconcentration was 10mmol/L),and the control group was added with RPIM-1640 culturemedium.After 48hours,the apoptosis of treated cells was detected by flow cytometry withAnnexin V/PI staining,and the in vitro invassiveness of the treated cells was detected byTranswell permeable assays.Results 1.The flow cytometry assays showed that the treated cells were induced toapoptosis in different degree,but the number of apoptotic cells in both drugs group wasmuch higher than that of the other two groups(P＜0.01).It also showed that usinghydralazine alone could induce more cells to apoptosis than valproate could(P＜0.01).2.The Transwell assays demonstrated that the invasiveness of treated cells wasreduced in different extent,but a combination of two drugs worked better than either drugalone(P＜0.01).It was also found that hydralazine worked better than valproate on reducingthe invasiveness of QBC₉₃₉ cells.Conclusion The two drugs have synergy effect on reducing the growth and invasiveness ofthe QBC₉₃₉ cells. Partâ…¡Mechanism of the effect of hydralazine and valproate onSIRT1 gene of QBC₉₃₉ cellsPaper3 DNMTi and HDACi decrease the expression of SIRT1 viare-expressing the HIC 1 geneObjective To investigate the mechanism of the effect of hydralazine and valproate onSIRT1 gene of QBC₉₃₉.Methods 1.The QBC₉₃₉ cells were divided into four groups,and HIBEC-HR cells weretreated identically.Both of them were treated separately with hydralazine and valproateeither alone or combined(the final concentration was 10mmol/L),and the control groupwas added with RPIM-1640 culture medium.After 48hours,the expression of SIRT1 andHIC1 gene of the treated cells and cells in the contral group were detected with RT-PCRand Western blot.2.One pair of HIC1 target sequence-specific small interfering RNA (siRNA) was designedand synthesized,then siRNA/liposome complex was used to transfect QBC cells.Aftertreating the transfected cells with hydralazine and valproate inhibitors for 48 hours,theexpression of SIRT1 and HIC1 gene of these cells was detected with RT-PCR and Westernblot.Results The expression of SIRT1 gene was detected to be decreased only when the cellswere treated with hydralazine and valproate in combination(P＜0.01);the inhibition ofhydralazine and valproate on SIRT1 gene was weakened when HIC1 gene wasslieneed(P＜0.01).Conclusion Hydralazine and valproate decrease the expression of SIRT1 via re-expressingthe HIC1 gene. Partâ…¢Using hydralazine and valproate in combination toreactivate the RASSF1A gene by repressing the SIRT1 gene ofQBC₉₃₉ cellsPaper4 Using hydralazine and valproate in combination to reactivate theRASSF1A gene by repressing the SIRT1 gene of QBC₉₃₉ cellsObjective To investigate the role of SIRT1 gene in re-expressing RASSF1A of QBC₉₃₉cells by using hydralazine and valproate in combination.Methods a batch of QBC₉₃₉ cells were divided into three groups,then siRNA/liposomecomplex was used to transfect one group of QBC₉₃₉ cells,the other two groups of cellswere transfected with pHK or none.After treating the transfected cells with hydralazine andvalproate for 48 hours,the expression of RASSF1A was evaluated by reversetranscription-PCR and western blot.Another batch of QBC₉₃₉ cells were divided into twogroups,After siRNA/liposome complex was used to transfect these cells,hydralazine andvalproate were added into the cells.Then one group of cells were added with nicotinamide,the other group were added with RPIM-1640.48h later,the expression of RASSF1A wasevaluated by reverse transcription-PCR and western blot.Finally the promoter regionmethylation status of RASSF1A of the five groups of cells was detected withMSP(mehtylation specific PCR).Results In the first batch of QBC₉₃₉ cells,the expression of RASSF1A of the group whichwas transfected with siRNA was much weaker than that of the other two groups(P＜0.01).In the other batch of QBC₉₃₉ cells the expression of RASSF1A of the group which wastreated with nicotinamide was much higher than that of the other group(P＜0.01).The statusof the promoter region of RASSF1A of the five groups was demethlated.Conclusion Hydralazine and valproate reactivate the RASSF1A gene of QBC₉₃₉ cells insome extent via repressing the expression of SIRT1. Our research focused on the the effect of DNMTi and HDACi on the expression oftumor suppressor genes of QBC₉₃₉ cells in vitro.Then we studied the mechanism of theeffect of hydralazine and valproate on SIRT1 gene of QBC₉₃₉ cells by utilizing siRNAtechnology to knockdown HIC 1 expression.Finally we explored the the role of SIRT 1 genein re-expressing RASSF1A of QBC₉₃₉ cells by using hydralazine and valproate incombination.According to the experimental results,we summarized the conclusions andingenuities as follows:1.It is the first research that explored the effect of DNMTi and HDACi on QBC₉₃₉cells lines in vitro.The results of the experiment demonstrated that the two drugs cansynergistically reactivate the P16,RASSF1A and E-cardherin gene of QBC₉₃₉ cells andmake the promoter region of the three genes to be demethylated,and the two drugs havesynergy effect on reducing the growth and invasiveness of the QBC₉₃₉ cells.2.Human bile duct carcinoma cell line QBC₉₃₉ cells and human normal biliaryepithelial cell line HIBEC-HR cells were both treated with hydralazine and valproate.Theresult showed that the expression of SIRT1 of QBC₉₃₉ cells was suppressed,while that ofHIBEC-HR cells did not change,which suggested that the expression of SIRT1 in QBC₉₃₉cells was abnormal.3.QBC₉₃₉ cells were transfected with siRNA/liposome complex.After treating thetransfected cells with hydralazine and valproate inhibitors for 48 hours,the expression ofSIRT1 and HIC1 gene of these cells was detected.Then it was found that hydralazine andvalproate decreased the expression of SIRT1 via re-expressing the HIC 1 gene.4.QBC₉₃₉ cells were transfected with siRNA/liposome complex.After treating thetransfected cells with hydralazine and valproate inhibitors and SIRTl-specific inhibitor for48 hours,the expression of RASSF1A gene of these cells was detected.The result of theexperiment demonstrated that hydralazine and valproate reactivated the RASSF1A gene ofQBC₉₃₉ cells in some extent via repressing the expression of SIRT1. 5.This research revealed that DNA methylation and histone deacetylase inhibitorscould regulate the SIRT1 abnormal expression in QBC939 cells.At the same time,SIRT1was found to be the effector of DNMTi and HDACi,which would provide the fundamentalfor the further exploring new factors,and for epigenetic-therapy.