Study On The Molecular Mechanisms Of MicroRNA-200b Negatively Regulated By HDAC1/4 Involved In Chemoresistance Of Human Lung Adenocarcinoma

Background and ObjectiveLung cancer,a predominant public health problem worldwide,is responsible for more cancer-related morbidity and mortality than any other cancer in both women and men.Lung adenocarcinoma(LAD)is the most common histological form of lung cancer and chemotherapy is a significant component of the current fist-line therapies for LAD.However,chemoresistance is the most significant obstacle towards LAD treatment,and this process involves genetic and epigenetic regulation of chemoresistance-related genes.Histone acetylation is one of the most important epigenetic mechanisms controlling gene expression,and it can regulate various physiological and pathological processes by regulation of gene expression.Our previous study showed that microRNA(miRNA,miR)-200b was significantly downregulated in c he mores istant LAD cells and its ove rexpress ion could reverse the chemoresistance of human LAD.And,we found that inhibition of histone deacetyltransferase(HDAC)significantly upregulated miR-200b expression.On the previous basis of docetaxel-resistant LAD cell lines establishment and functional and bio informational analysis of miRNA-200b,the aim of this study is to investigate the molecular mechanisms of HDACs induced-negative regulation of miR-200b expression by co-immunoprecipitation,chromatin immunoprecipitation,site-specific mutation and luciferase activity assay,to illustrate the association of histone-acetylation regulation of miR-200b expression with chemoresistance of LAD.It will provide a novel strategy for individualized treatment and reversing chemoresistance of LAD in clinic.Materials and Methods1.Real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR)analysis was perfermed to detect the miR-200b level of human LAD cell lines(SPC-A1 and H1299)and their corresponding docetaxel resistant cell lines(SPC-A1/DTX and H1299/DTX)treated with(or without)DNA methyltransferase inhibitor 5-Aza-2’-deoxycytidine(5-Aza-CdR).Pyrosequencing technology was used to measure promoter methylation level of miR-200b gene in docetaxel resistant LAD cells treated with 5-Aza-CdR.2.Western blot was performed to detect protein expression of acetyl-histone H3 in docetaxel resistant LAD cells treated with histone deacetylase inhibitors[(tricho statin A,TSA)and(valproic acid,VPA)].qRT-PCR analysis was perfermed to detect the miR-200b level of human docetaxel resistant LAD cell lines(SPC-A1/DTX and H1299/DTX)treated with(or without)histone deacetylase inhibitors(TSA and VPA).3.MTT assay was performed to detect the 50%inhibitory concentration(IC50)values of DTX and PTX to SPC-A1/DTX and H1299/DTX cells treated with different concentration of histone deacetylase inhibitors(TSA and VPA).4.The synthetic siRNAs directed against HDAC1-11 were transfected to SPC-A1/DTX and H1299/DTX cells.Western blot was performed to confirm the transfective efficiency of the above siRNAs.qRT-PCR analysis was perfermed to detect the miR-200b level of human docetaxel resistant LAD cells transfected with the siRNAs against HDAC1-11.qRT-PCR and Western blot analysis were performed to detect the mRNA and protein expression of HDAC1/4 in parental LAD cells and docetaxel resistant LAD cells,respectively.5.The promoter regions of miR-200b were then analyzed.Luciferase reporter plasmids[p GL3-promoter-1,pGL3-promoter-2,pGL3-promoter-1(Sp1-1mut),pGL3-promoter-1(Sp 1-2mut),pGL3-promoter-1(Sp1-1/2mut),pGL3-promoter-2,pGL3-promoter-2(Sp1 mut)]containing Spl binding sites were constructed.The RNA interference plasmids against HDAC1/4(sh-HDAC1/4)were constructed respectively.The above Luciferase reporter plasmids were transfected or cotransfected with sh-HDAC 1/4 into SPC-A1/DTX and H1299/DTX cells.Dual luciferase reporter assay was used to detect the luciferase activity.qRT-PCR analysis was perfermed to detect the miR-200b level of SPC-A1/DTX and H1299/DTX cells cotransfected with sh-HDAC1/4 and siRNA-Sp1 vectors(or nothing).6.Co-Immunoprecipitation(Co-IP)assay was performed to determine whether HDAC1 or HDAC4 interacted with Spl in vivo.Chromatin immunoprecipitation(ChIP)assay was conducted to determine whether Spl and HD AC 1/4 could bind with the promoters of miR-200b in vivo.ChIP assay was used to detect the effects of sh-HDAC1/4 on regulation of histone-H3 acetylation level of the miR-200b promoters at Spl-binding sites.7.A total of 68 cases of clinical LAD tissues were gathered from patients at advanced stage and divided into "sensitive" and "insensitive" groups based on the response to the docetaxel-based chemotherapies.qRT-PCR analysis was perfermed to detect the HD AC 1/4 and miR-200b levels of clinical LAD tissues.The relationship between HD AC 1/4 and miR-200b levels and the correlation between the expression level of HDAC1/4 and clinic prognosis were then analyzed in the clinical LAD tissues.8.The single chain inhibitor(miR-200b inhibitor)against miR-200b and the mimics of miR-200b were synthesized,respectively.The pcDNA3.1-HDAC1 and pcDNA3.1-HDAC4 plasmids were kindly provided by Prof Jun Lu(Northeast Normal University,China)and Professor Shu-han Sun(Second Military Medical University,China).Then,the above mentioned plasmids and vectors and sh-HDAC1/4 vectors were transfected(or co-transfected)into SPC-A1/DTX and H1299/DTX cells.MTT assay was used to detect IC50 values of DTX and PTX to SPC-A1/DTX and H1299/DTX cells treated with the indicated vectors.9.Sh-HDAC1/4 and miR-200b inhibitor were transfected(or co-transfected)into SPC-A1/DTX and H1299/DTX cells.Colony formation assay was performed to detect cell proliferation activity in vitro.Flow cytometric analysis was used to analyze cell cycle and apoptosis.Western blot was used to measure the protein expression of cleaved-Caspase 3 and Caspase 3.10.Pri-miR-200b gene expression vector(pPG/miR-200b)was constructed and stably transfected(or co-transfected with sh-HDAC1/4 vector)into H1299/DTX cells which were then used to form xenograft transplantation in nude mice.After xenograft was successfully transplanted,docetaxel(DTX)was administered by intraperitoneal injection.Tumor growth curve was performed and all nude mice were sacrificed at 6 weeks.The tumor tissues were then gathered for the following experiment.qRT-PCR analysis was perfermed to detect the miR-200b levels in the tumor tissues.Immunostaining was perfermed to detect expression level of Ki67 and PCNA in the tumor tissues.Tunel staining was performed to measure the apoptotisis level of the tumor tissues.11.Sh-HDAC1/4 and miR-200b inhibitor were transfected(or co-transfected)into SPC-A1/DTX and H1299/DTX cells.qRT-PCR analysis was perfermed to detect the mRNA expression levels of E2F3、Aurora-A and Survivin in SPC-A1/DTX and H1299/DTX cells.Western blot was used to detect the protein expression of E2F3、Aurora-A and Survivin in SPC-A1/DTX and H1299/DTX cells.Western blot was used to detect protein expression of HDAC1/4、E2F3、Aurora-A and Survivin in the tumor tissues of nude mice.Immunostaining was perfermed to detect expression of HDAC1/4、E2F3、Aurora-A and Survivin in the tumor tissues of nude mice.12.The promoter regions of Aurora-A and Survivin were then analyzed.Luciferase reporter plasmids[pGL3-Aurora-A,pGL3-Aurora-A(E2F3-1 mut),pGL3-Aurora-A(E2F3-2 mut),pGL3-Aurora-A(E2F3-1+2 mut),pGL3-Survivin,pGL3-Survivin(E2F3 mut)]containing E2F3 binding sites were constructed and transfected or cotransfected with sh-HDAC1/4、sh-control and miR-200b inhibitor into SPC-A1/DTX cells.Dual luciferase reporter assay was used to detect the luciferase activity.ChIP assay was performed to detect the effects of sh-HDACl/4 on the amounts of E2F3 binding with the promoters of Aurora-A and Survivin in SPC-A1/DTX cells transfected with sh-HDAC1/4 vectors.Results1.The mRNA expression level of miR-200b was significantly lower in chemoresistant SPC-A1/DTX and H1299/DTX cells compared with that in parental SPC-A1 and H1299 cells(p<0.01).The mRNA expression level of miR-200b didn’t change obviously in SPC-A1/DTX and H1299/DTX cells treated with enough concentration of 5-Aza-CdR(p>0.05).Pyrosequencing technology indicated that the promoter regions of miR-200b gene were hypermethylated in SPC-A1/DTX and H1299/DTX cells.The methylation level of miR-200b promoter regions didn’t change obviously in SPC-A1/DTX and H1299/DTX cells after administration of enough concentration of 5-Aza-CdR(20μmol/L).2.The protein expression of acetyl-histone H3 significantly upregulated in docetaxel resistant LAD cells treated with histone deacetylase inhibitors(TSA and VPA).The mRNA expression level of miR-200b significantly elevated in a concentration-and time-dependent manner in SPC-A1/DtX and H1299/DTX cells treated with TSA and VPA(p<0.01).3.Histone deacetylase inhibitors(TSA and VPA)could decrease IC50 values of DTX and PTX to SPC-A1/DTX and H1299/DTX cells in a dose-dependent manner(p<0.01).4.All of the synthesized siRNAs against HD AC1-11 could downregulate their protein expression,respectively.Silencing of HDAC1/4 could significantly upregulate the mRNA level of miR-200b in chemoresistant LAD cells(p<0.01).The mRNA and protein expression of HD AC 1/4 was significantly higher in chemoresistant SPC-A1/DTX and H1299/DTX cells compared with that in parental SPC-A1 and H1299 cells.5.It was found that there were Spl binding sites in both of the two promoter regions of miR-200b.The two promoters of miR-200b had luciferase activity in the two chemoresistant LAD cells(p<0.01).Suppression of HDAC1/4 elevated the promoter activities of the two promoters of miR-200b(p<0.01).The effects of silencing of HDAC1/4 on the two promoter activities of miR-200b weakened or disappeared while Spl binding sites in the promoters were partially or completedly mutated(p<0.05 or p>0.05).Inhibition of HD AC 1/4 could significantly upregulate the mRNA level of miR-200b in chemoresistant LAD cells(p<0.01).Silencing of Spl could partially reverse the elevating effects of silencing of HDAC1/4 on the mRNA level of miR-200b(p<0.05).6.Spl could combine with both HDAC1 and HDAC4 in vivo.Sp1、HDAC1 and HDAC4 could combine with promoter regions of miR-200b gene at Spl binding sites in vivo.HDAC1/4 repression increased the histone-H3-acetylation level of the miR-200b promoters at Spl-binding sites.7.The mRNA level of HDAC1 and HDAC4 was significantly up regulated in the docetaxel-insensitive group compared with the docetaxel-sensitive group(p<0.01).miR-200b was statistically significant inverse correlation with HDAC1 in tumor tissues(rho=-0.799,P<0.01).miR-200b was also statistically significant inverse correlation with HDAC4 in tumor tissues(rho=-0.781,P<0.01).Patients witha higher HDAC1 level had a significantly shorter progression free survival(PFS)than did those with a lower HDAC1 level(p<0.05).Also,patients with a higher level of HDAC4 had a significantly shorter PFS than did those with a lower level of HDAC4(p<0.01).8.Inhibition of HD AC 1/4 significantly decreased the IC50 values of DTX and PTX to chemoresistant SPC-A1/DTX and H1299/DTX cells(P<0.01).Nevertheless,the inhibition effects could be partially abrogated by miR-200b inhibitor(P<0.05).Upregulation of HD AC 1/4 significantly increased the IC50 values of DTX and PTX to chemoresistant SPC-A1/DTX and H1299/DTX cells(P<0.05).The effects of upregulation of HDAC1/4 on the IC50 values could be abrogated by upregulation of miR-200b(p>0.05).9.In the presence of DTX,downregulation of HDAC1/4 significantly reduced the colony formation capacities of H1299/DTX and SPC-A1/DTX cell lines(p<0.01),and these effects were partially reversed by miR-200b inhibitor(p<0.05).When the cells were treated with docetaxel,downregulation of HDAC4 dramatically increased the percentage of cells in G2/M phase in H1299/DTX and SPC-A1/DTX cells(P<0.01),which could be partially reversed by miR-200b inhibitor(P<0.05).In the presence of DTX,downregulation of HDAC1/4 significantly increased early stage apoptosis rate of H1299/DTX and SPC-A1/DTX cells(P<0.01),and apoptosis-promoting effect was partially reversed by miR-200b inhibitor(p<0.05).Downregulation of HD AC 1/4 significantly increased protein expression of cleaved-Caspase 3 in H1299/DTX and SPC-A1/DTX cells,which could be partially reversed by miR-200b inhibitor.10.After administration of DTX,tumors in nude mice derived from H1299/DTX cells stably transfected with sh-HDAC1/4 or miR-200b-expression vector grew more slowly than those in control groups(p<0.05).The expression of miR-200b was upregulated in sh-HDAC1/4 and miR-200b-expression vector groups compared with that in the control groups(p<0.01).The positive rate of Ki67 and PCNA was significantly decreased and the positive rate of apoptosis cells was significantly increased in tumor tissues of nude mice,while HDAC1 was supressed.The positive rate of Ki67 and PCNA was significantly decreased and the positive rate of apoptosis cells was significantly increased,while HDAC4 was downregulated or miR-200b was upregulated in tumor tissues of nude mice.11.In chemoresistant SPC-A1/DTX and H1299/DTX cells,silencing of HDAC1 led to the decreased mRNA and protein levels of E2F3 and Survivin(p<0.01);inhibition of HDAC4 led to the decreased mRNA and protein levels of E2F3,Aurora-A and Survivin(p<0.01);and the downregulating effects of HDAC1/4 on expression of E2F3、Survivin or Aurora-A could be partially rescued by miR-200b inhibitor(p<0.05).The protein level of E2F3 and Survivin was significantly decreased and the positive rate of E2F3 and Survivin was significantly decreased,while HDAC1 was suppressed in tumor tissues of nude mice.The protein level of E2F3、Survivin and Aurora-A was significantly decreased and the positive rate of E2F3、Survivin and Aurora-A was significantly decreased,while HDAC4 was downregulated or miR-200b was upregulated in tumor tissues of nude mice.12.There were multiple binding sites for the E2F3 transcription factor in promoter regions of Aurora-A and Survivin.The promoters of Aurora-A and survivin had lucife rase activity in c he mo res istant SPC-A1/DTX cells(p<0.01).HDAC1 suppression significantly decreased the luciferase activity of the Survivin promoter(P<0.01),however,the effect of HDAC1 inhibition on Survivin promoter was almost abolished in constructs in which the E2F3 binding sites were mutated(p>0.05).HDAC4 suppression significantly decreased the luciferase activity of the Aurora-A and Survivin promoters(P<0.01),however,the effects of HDAC4 inhibition on Aurora-A and Survivin promoters were almost abolished in constructs in which the E2F3 binding sites were mutated(p>0.05).The effects of HDAC4 suppression on regulation of the promoter activity of Aurora-A were weakened while E2F3 binding site was partially mutated at the Aurora-A promoter(p<0.05).Inhibition of HDAC1 significantly reduced the amount of E2F3 binding with the promoter of Survivin in SPC-A1/DTX cells(P<0.01),however,HDAC1 repression had no effect on the amount of E2F3 binding with the promoter of Aurora-A(p>0.05).Downregulation of HDAC4 significantly reduced the amount of E2F3 binding with the promoters of Aurora-A and Survivin(P<0.01).Conclusions1.Histone acetylation instead of DNA methylation was involved in regulation of miR-200b in human chemoresistant LAD SPC-A1/DTX and H1299/DTX cells,which was responsible for chemoresistance of human LAD.2.In HDAC1-11 iso types,it was HD AC 1/4 which was engaged in regulation of miR-200b in human chemoresistant LAD cells.The expression levels of HDAC1/4 and miR-200b were closely related to the prognosis of LAD patients.3.The HDAC1/4/miR-200b signaling pathway might be involved in the regulation of chemoresistance of human LAD both in vivo and in vitro.4.HDAC 1/4/Spl/miR-200b signaling axis regulating the expression of E2F3/Aurora-A and E2F3/Survivin might be the key mechanisms by which HDAC 1/4/miR-200b signaling pathway regulated chemoresistance of human LAD.5.To the best of our knowledge,the present study was the first to report that HDAC1/4/Spl/miR-200b/E2F3/Aurora-A/Survivin signaling axis was involved in the regulation of chemoresistance of human LAD.This study indicated that the expression level of HDAC1/4 was closely correlated with miR-200b in clinical tissues.Also,the expression levels of HDAC1/4 were closely related to the prognosis of LAD patients.This study revealed that abnormal histone acetylation was the reason that miR-200b was downregulated in human LAD cells.The present study would provide new molecular targets for individualized treatment and reversing c he mo resistance of human LAD in clinic.