Research Of Histone Deacetylation Regulation On Cell Cycle Of Breast Cancer Cell

[Background and Objective]Breast cancer is one of most common malignant tumors for woman in the world.In most countries,the incidence and the mortality rate of breast cancer is rising,there was about 1,300,000 people being diagnosed of breast cancer each year in the whole world, Accounting for 21%of the complete female malignant tumor total,and there was about 400,000 people died of breast cancer each year.So we can say that breast cancer is threatening woman's life and the health seriously.In view of breast cancer prevention, diagnosis and treatment,although the diagnosis technology,the chemotherapy,the surgery,the radiotherapy,the endocrine treatment and the biological treatment has yielded encouraging result,the current method of prevention,diagnosis and treatment were still limited,this was still enormous exploration space for the breast cancer research.With the development of molecular biology,researchers found that histone deacetylation regulation is a kind of epigene modification for cells,it invoves in the occurrence and development of many malignant tumors(including neurocytoma, melanoma,leukemia,prostate cancer,lung cancer,ovarian cancer and colon cancer and so on) by adjusting the balance of histone acetylase(HAT) and histone deacetylase(HDAC).As to breast cancer,some researchers studied in the field of the estrogen receptor (ER) signaling passway,the BRCA1 gene,the HER-2 gene,the cell differentiation,the cell apoptosis and stem cell and so on,they confirmed that histone deacetylation regulation is also involving in breast cancer.In this research,we will construct histone deacetylase 1(HDAC1) plasimid,screen the highly effective histone deacetylase inhibitor,which will serve as double-way intervention.Under the double-way intervention,we will observe the change of breast cancer cell cycle in different estrogen receptor condition,the influence on CyclinDl and CyclinA2,we will discuss the mechanism of histone deacetylation regulation on breast cancer cell,then we will evaluate the curative effect and side effect of histone deacetylase inhibitor alone and combined with chemotherapy drugs by animal model.Through this research,we hope that we can establish the breast cancer cell model transfected by HDAC1 plasimid and breast cancer animal model,which provides the experimental model for the correlation research;in theory,we can discuss the mechanism of histone deacetylation regulation on breast cancer cell in the view of CyclinDl and CyclinA2 expression.More importantly in the clinical practice aspect,the research will helps us to know the the quantity-effect relation and time-effect relation of histone deacetylase inhibitor for breast cancer cell,the animal model study will also provide experimental data for seeking new way to treat breast cancer and provide useful experimental reference for the histone-deacetylase-targeting-drug research and development of breast cancer.[Methods]1.Effect of histone deacetylase 1(HDAC1) on breast cancer cell cycle (Positive intervention)1.1 To construct histone deacetylase 1(HDAC1) plasimid with HA tag (pcDNA3.1-HA- hdac1) by subcloning technique;1.2 To make competence bacterium(E.coli) by CaCl₂ method;1.3 To transform pcDNA3.1-HA-hdacl plasimid into competence bacterium (E.coli),harvest large number of pcDNA3.1-HA-hdacl plasimid,purify and check it by the enzyme digestion and DNA sequence;1.4 To transfect the breast cancer cell with pcDNA3.1-HA-hdacl plasimid,detect the protein expression level with the Western bloting;1.5 To examine the change of breast cancer cell cycle after transfection of pcDNA3.1-HA-hdac1 plasimid by using Flow cytometry(FCM).2.Effect of histone deacetylase inhibitor(HDACi) on breast cancer cell cycle (Negative intervention) 2.1 To observe the change of breast cancer cell cycle after treated respectively by four kind of histone deacetylase inhibitor TSA,SAHA,CS055,MS-275 by MTT methods;(Screening inhibitor)2.2 To examine the change of breast cancer cell cycle after treatment of different concentration of histone deacetylase inhibitor by using Flow cytometry(FCM).(Determination of inhibitor concentration)3.Study of the influence of double-way histone deacetylation regulation on CyclinDl and CyclinA2 gene.3.1 To transfect the breast cancer cell with pcDNA3.1 -HA-hdac1 plasimid; (Positive intervention)3.2 To treat breast cancer cell by using histone deacetylase inhibitor; (Negative intervention)3.3 To detect the Cyclin D1 and Cyclin A2 protein expression level with the Western bloting;3.4 To examine the cell number which has Cyclin Dl and Cyclin A2 protein expression by using Flow cytometry(FCM).4.Animal mode research of histone deacetylase inhibitor alone and combined with chemotherapy drugs on breast cancer cell.4.1 To feed Balb/c-nu/nu(Crl:NU-Foxnlnu) female nude mouse(SPF grade),to construct transplanted tumor nude mouse model by injection of breast cancer cell suspension;4.2 To design the histone deacetylase inhibitor concention gradient based on the above in vitro experiment dosage once more,gives the medicine through the tail intravenous injection,screen the best effective dose of this inhibitor in nude mouse,then conduct the next step experiment;4.3 To divide nude mouse with transplanted tumor into 6 groups:control group, inhibitor group,taxol group,adriamycin group,taxol plus inhibitor group,adriamycin plus inhibitor group.To give related treatment factor separately.After 1 week,to get blood from the tail vein to carry on the blood cell analysis,and examine the items of the blood fat,the liver function and the kidney function.4 weeks later,to get the body weight,then pull the neck to execute the mouse,take the tumor out by surgery,get the wet weight of tumor.To count tumor growth inhibition rate and tumor volume.[Results]1.Effect of histone deacetylase 1(HDAC1) on breast cancer cell cycle (Positive intervention)1.1 Construction and identification of histone deacetylase 1(HDAC1) plasimid with HA tag(pcDNA3.1-HA- hdac1):After successfully constructing of histone deacetylase 1(HDAC1) plasimid with HA tag(pcDNA3.1-HA- hdac1),we confirmed that hdacl DNA fragment is 1449bp(cutting position spot is BamH I/EcoR I)by the enzyme digestion and DNA sequence checking;Western bloting showed HDAC1 protein expression in both estrogen receptor positive and negative cell lines after transfection of HDAC1 plasimid;1.2 Effect of histone deacetylase 1(HDAC1) on breast cancer cell cycle:after transfection of HDAC1 plasimid into cells,Flow cytometry(FCM)showed:for estrogen receptor positive cell line MCF-7,G0/G1 phase cell proportion dropped(40.31±2.15)%,S-phase cell proportion increased(17.98±1.87) %,P<0.05;for estrogen receptor negative cell line MDA-MB-435s,G0/G1 phase cell proportion dropped(41.22±2.27)%,S- phase cell proportion increased(16.70±2.33)%,P<0.05.Compared between the two cell lines, P>0.05。2.Effect of histone deacetylase inhibitor(HDACi) on breast cancer cell cycle (Negative intervention)2.1 Screening inhibitor:24h after treatment of TSA,SAHA,CS055 and MS-275,the cell demonstrated growth inhibition,the greatest inhibition happened at 48h after treatment;within four inhibitors,SAHA demonstrated strong inhibition ability (at the 48h time point,comparing SAHA group with TSA,CS055 and MS-275 group separately,P<0.05),for MCF-7 cell line,the inhibition of SAHA is (70.31±2.35)%;for MDA-MB-435s cell line,the inhibition of SAHA is (78.36±2.01)%。Compared between the two cell lines,P>0.05。2.2 Inhibitor concentration:After 1.0,2.0,4.0,8.0,16.0μmol/L concentration gradient SAHA treating cell,4.0umol/L showed S-phase cell proportion dropping obviously(P<0.05),when the density further increased,the S-phase cell proportion did not continue dropping.3.Study of the influence of double-way histone deacetylation regulation on CyclinDl and CyclinA2 gene.3.1 Transfection of HDAC1 plasimid(Positive intervention):As to CyclinA2,cell line MCF-7 did not show change of cell number of CyclinA2 protein expression, for cell line MDA-MB-435S,cell number of CyclinA2 protein expression increased about 8%;Regarding to cyclinD1,cell number of CyclinD1 protein expression decresaed 10%in both MCF-7 and MDA-MB-435S cell lines.3.2 Treatment of inhibitor SAHA(Negative intervention):In MCF-7 and MDA-MB-435S cell lines,cell number of CyclinA2 protein expression expression decresaed about 40%,cell number of CyclinD1 protein expression expression incresaed about 40%.4.Animal mode research of histone deacetylase inhibitor alone and combined with chemotherapy drugs on breast cancer cell.4.1 Screening the suitable dosage of SAHA in vivo:different dosage of SAHA was given to nude mouse with transplanted tumor,curative effect was observed as to tumor growth inhibition rate and tumor volume within 0.10～0.42 mg/kg dosage scope,the SAHA treatment effect presented the dosage-dependence relations.When the dosage rised from 0.42 mg/kg to 0.84 mg/kg,no further curative effect was observed.4.2 Treatment factors were given to mouse:4.2.1 Curative effects:tumor growth inhibition rate and tumor volume:compared with the control group,P<0.05,the difference has statistics significance; compared with each other,the taxol plus SAHA group showed superiority;4.2.2 Side effects:â‘ Body weight:the body weight dropped in the group containing taxol or adriamycin;â‘¡Blood cell analysis:the white blood cell number dropped in the group containing taxol or adriamycin,compared between SAHA alone and SAHA combined with chemotherapy drugs group,P>0.05;â‘¢Cholesterol,urea nitrogen and creatinine:Compared with the control group, P>0.05;â‘£Blood serum glutamic-pyruvic transaminase:Compared with the control group,P<0.05;compared between SAHA alone and SAHA combined with taxol group,P<0.05;⑤Blood serum bilirubin:Compared with the control group,the level rised in the group containing taxol or adriamycin,P<0.05,but compared between SAHA alone and SAHA combined with chemotherapy drugs group,P>0.05.[Conclusions]1.The histone deacetylase 1(HDAC1) gene express in both estrogen receptor positive and negative breast cancer cell lines,its expression can promote cancer cell growth;2.In vitro experiment,all of the four kind of histone deacetylase inhibitor(TSA,SAHA, CS055 and MS-275)can inhibite cell growth,in which SAHA is the strongest one, its inhibitory action is most obvious at 48h,suitable concentration is 4.0μmol/L;3.Histone deacetylase inhibitor SAHA may regulate the expression of Cyclin A2 and Cyclin D1 gene,eventurally resulting of reduce of S-phase and M-phase cells, suppressing DNA duplication,synthesis and cell mitosis,causing the cell to be detained in the G1-phase,finally towards death,achieving the inhibition of breast cancer cell growth;4.Histone deacetylation regulation was influenced by the hormone receptor condition slightly;5.In breast cancer nude mouse animal model experiment,SAHA showed curitive effect,within 0.10 -0.42 mg/kg dosage scope,presenting the dosage dependence relations,0.42 mg/kg were most obvious,which is higher than in vitro experiment;6.The curitive effect of SAHA alone is lower than chemotherapy drugs taxol or adriamycin,but combined with taxol or adriamycin may show synergistic effect;7.SAHA leads little side effect to kidney function,blood cell and cholesterol,but has certain influence to the blood serum glutamic-pyruvic transaminase,this kind of influence does not increase while combined with chemotherapy drugs;8.SAHA combined with chemotherapy drags may be good choice for breast cancer treatment.