Research On The Mechanisms Of MiR-34a Inhibiting Ovarian Cancer Cell Piroliferation And Cisplatin Resistance By Targeting HDAC1

BackgroundEpithelial ovarian cancer(EOC)is one of the most prevalent gynecologic malignancies and represents the first leading cause of death among them.Almost 75%of EOC patients are usually diagnosed at advanced stages.EOC patients have a poor prognosis,with only 40%having a 5-year survival rate.More than 60%of EOC patients relapse in two years.The acquired resistance of cancer cells to chemotherapy drugs is an important cause of treatment failure.Cell proliferation and drug resistance are the biggest challenges for EOC treatment.MiR-34a belongs to the family of mir-34ac/34bc-5p/449abc/449c-5p,located at the 1p3 6 site of chromosome,and plays an important role in the imbalance of cell proliferation and apoptosis.It has been reported that microRNA-34a acts as an "anti-oncogene " in cancer cells,and can inhibit the proliferation,invasion,metastasis and drug resistance of cancer cells.Acetylation and deacetylationare are important components ofpost-translational modification of histones.The imbalance of histone acetylation may lead to changes in chromatin structure and dystranscription of genes involved in cell cycle progression,differentiation and apoptosis.Like many genes required for normal development,dysfunction of HD AC can lead to cancer.Therefore,overexpression of HD AC has been observed in many cancers.High expression of HDAC isoenzymes and low acetylation of histones are commonly found in cancer cells.However,no experiments have yet been done to elucidate thecritical roles of miR-34a in EOC.The expressions and drug resistance of miR-34a and HD AC 1 were detectedby RT-qPCR in ovarian cancer tissues and cell lines.This study further verified the target effect of HD AC 1 on m i R-3 4 a,which is great significance to the study of EOC proliferation,reversal of drug resistance and new targeted therapy.It will basis for the treatment of EOC and will provide a new way of treatment for EOC.Part I MiR-34a expression in ovarian cancer and clinical significanceObjective:Expression of miR-34a in ovarian cancer tissues was detected.And then we analyzed its relationship with clinicopathological features and progression,invasion and metastasis of EOC.Method:From July 2011 to December 2013,54 cases of EOC in the affiliated hospital of Qingdao University were collected.The age ranged from 38 to 70 years with medium age of 5 4.2 ± 7.5.Among them,48 cases were serous OC and 6 cases were mucinous EOC;12 cases were borderline ovarian tumors,the age ranged from 3 1 to 60 years with medium age of 46.9 ± 8.1,including 8 cases of serous ovari an tumor,4 cases of mucinous ovarian tumor;10 cases of normal ovarian tissue,the age ranged from 48 to 59 years old with medium age was 52.3± 3.4,all of them were ovariectomized because of uterine fibroids.All patients signed an informed consent approved by the Ethics Committee of the affiliated hospital of Qingdao University.The expression of miR-34a in different ovarian tissues was compared by RT-qPCR,and the relationship between the expression and clinicopathological characteristics of ovarian cancer was further analyzed.The close relationship between miR-34a expression and the progress,invasion and metastasis of ovarian cancer was verified.Results:1.Expression of miR-34 a in ovarian tumors and normal ovarian tissues:The dissolution curves of miR-34a expression in different ovarian tissues by RT-qPCR showed a single peak,no non-specific fluorescence was produced,the quantification was accurate.The relative expression of miR-34a in EOC tissues(0.547 1±0.1 526)was significantly lower than that in borderline ovarian tumors(0.9426±0.0849)and normal ovarian tissues(1.0375±0.0817)(p<0.001),while there was no difference between the latter two(p=0.998).2.T h e relationship between the expression of miR-34a and various clinicopathological features in ovarian cancer:2.1 The relationship between expression of miR-34a and FI GO stages in ovarian cancer tissues:The relative expression of miR-34a in EOC stage Ⅰ and Ⅱ patients was 0.7715±0.0813,while that in stageⅢ and Ⅳ patients was0.4608 ± 0.050 1,the difference has statistical significant(p=0.022).2.2 The relationship between expression of miR－34a and EOC lymphatic metastasis in ovarian cancer tissues:The relative expression of m i R-3 4 a i n 9 patients with lymph node metastasis was 0.4511 ± 0.0610,while that in patients without lymph node metastasis was 0.5 6 6 3±0.1 5 8 4;The expression level of miR-34a in EOC tissues was negatively correlated with lymph node metastasis,and the difference has statistical significant.(p=0.002).2.3 The relationship between expression of mi R-34 a and degree of EOC differentiation in ovarian cancer tissues:The relative expression of m i R-3 4 a in high-grade adenocarcinoma tissues was 0.523 6±0.1314,while that in low-grade adenocarcinoma tissues was 0.8413 ± 0.0668.There was no significant difference(p=0.1 9 3).2.4 The relationship between expression of miR-34 a and age in ovarian cancer tissues:The relative expression of miR-34a in EOC patients≤50 years of age was 0.5642 ±0.1715 whilein patients>50 years of age was 0.547 1 ±0.1 526.There was no significant difference between the two groups(P=0.1 94).2.5 The relationship between expression of miR-34a and histological classification in ovarian cancer tissues:the expression of miR-34a in mucinous adenocarcinoma was 0.6 1 28±0.2033 whilethat in serous adenocarcinoma was 0.53 89±0.1457.There was no significant difference between the two groups(p=0.112).2.6 The relationship between expression of miR-34a and level of CA125 in ovarian cancer tissues:I n patients with CA125≤200U/ml,the relative expression of miR-34a was 0.7770±0.0946,while in patients with C A 1 2 5>200U/ml,the relative expression of miR-34a was 0.5071 ± 0.1 225.There was no significant difference between the two groups(p=0.5 8 3).Conclusions:The low expression of miR-34a may be closely related to the progressionof EOC.Part Ⅱ:Proliferation ability and cisplatin resistance of OC cells induced by miR-34aObjective:The aim of our study was to uncover the biological effects of miR-34a on the proliferation and chemoresistance.Moreover,the possible molecular mechanisms are also discussedMethods:Cell viability and cisplatin sensitivity of OC cell lines(SKOV3 and OVCA433)were measured by MTT and soft agar colony formation assay,and the relative expression of miR-34a in different OC cells was detected by RT-qPCR.The OC cell lines SKOV3 and OVCA433 were resuscitated and cultured,the cisplatin-resistant cell line(SKO V3 cp)was established from SKO V 3 cells through continuous cisplatin administration for 6 months.S KOV3 cell lines were transfected with mimics and inhibitors of miR-34a.RT-qPCR was used to detect the expression of miR-34a in SKOV3 cancer cell lines and drug-resistant SKO V3 cp cell lines,short-term(48h+4μg/ml)cisplatin intervention in SKOV3 cancer cell lines was also detected.SKOV3,SKOV3 cp,SKOV3cp+miR-34a and SKOV3+miR-34a inhibitorwere treated with different concentrations of cisplatin(0,0.5,1,2,4,or 8 ug/mL).MTT was used to determine the survival rates of these cells at different concentrations of cisplatin.Results:1.Effect of miR-34a mimics on survival of ovarian cancer cells:SKOV3 and OVCA43 3 were transfected with miR-34a mimics,respectively,they were transfected with miRNAs in random sequence as NCgroup.Cell growth inhibition was evaluated 1-5 days after transfection.S KO V 3 growth inhibition rates were 0.075 ± 0.005,0.138±-0.011,0.245±0.026,0.467± 0.03 8 and 1.045±0.136.OVAC433 growth inhibition rates were 0.05 1±0.007,0.13 8±0.013,0.245±0.023,0.467±0.035 and 1.045±0.1 1 3.There are significantly different between the two groups(p<0.001).With the extension of time,S KO V3 and OVCA433 cells were continuously proliferating whether they were transfected with mi R-34a,and the relative survival rate of the two lines transfected with miR-34a mimics was significantly lower than that of the NC group((p<0.00 1))2.Effect of miR-34a mimics on colony formation of ovarian cancer cells:Result of soft agar colony formation assay:The colony count of NC group in SKOV3 cell was 210.3 ± 19.5,and that in miR-34a inhibitor group was 1 46.5±9.6.The difference was statistically significant(p=0.0022).The colony count of the NC group in OVCA433 cell was 200 ± 35.2,while that in m i R-3 4 a inhibitor group was 118.25 ± 7.1.The difference was statistically significant(p=0.0076).Compared with the NC group,the average colony count of SKOV3 and OVCA43 3 cells in miR-34a inhibitor group decreased about 2g%and 42%.3.Effect of miR-34a inhibitor on survival of ovarian cancer cells:SKOV3 and OVCA433 were transfected with miR-34a inhibitor,respectively,they were transfected with miRNAs in random sequence as NCgroup.Cell growth inhibition was evaluated 1-5 days after transfection.SKOV3 growth inhibition rates were 0.075±0.007,0.1 87 ±0.023,0.502 ± 0.034,0.987 ± 0.047 and 1.965 ± 0.146.OVAC43 3 growth inhibition rates were 0.075±0.007,0.1 57±0.028,0.432±0.024,0.83 7±0.077 and 1.7 6 5±0.0 9 6.W i th the extension of time,SKOV3 and OVCA43 3 cells were continuously proliferating whether they were transfected with miR-34a inhibitor,and the relative survival rate of the two lines transfected with miR-34a inhibitor was significantly higher than that of the NC group((p<0.00 1))4.Effect of miR-34 a inhibitor on colony formation of ovarian cancer cells:Result of soft agar colony formation assay shows the colony count of NC group in SKOV3 cell was 17 6.5±34.6,and that in miR-34a inhibitor group was 242.0±11.9.The difference was statistically significant(p=0.02).The colony count of the NC group in OVCA433 cell was 173.5± 14.2,while that in miR-34a inhibitor group was 2 2 8.5±9.8.The difference was statistically significant(p=0.0015).Compared with the NC group,the average colony count of SKOV3 and OVCA43 3 cells in miR-34a inhibitor group increased about 40%and 36%.5.Expression of miR-34 a in SKO V3 and SKOV3cp cells:The expression levels of mi R-34a in SKOV3 and SKO V3 cp cells were analyzed by RT-qPCR.The result showed that the relative expression level of miR-34a in SKOV3 cells(1.0 0±0.2 3)was t wi c e mor e than that i n SKO V cp(0.38±0.1 1).The expression of m i R-34a in SKO V3 cp cell was significantly down-regulated(p<0.0 1).6.Effect of short-term cisplatin(4 μg/ml+48h)treatment on miR-34a expression:SKO V3 cells were treated with short-term cisplatin at the same concentration as SKOV3 cp cell.The result shows that the relative expression level of miR-34a in SKOV3 cells(1.00 ± 0.15)was twice more thanthat in SKO V 3 cells receiving short-term cisplatin treatment(0.45±0.08).The expression of miR-34a in SKOV3 cell lines receiving short-term cisplatin treatment was significantly down-regulated(p<0.01).7.Effects of miR-34a o verexpr ession on cisplatin resistance:SKO V3 cp cells were transfected with miR-34a to simulate the effect of miR-34a overexpression on chemotherapy in cisplatin resistant cells.24h after transfection,the cells were treated with different concentrations of cisplatin(0,0.5,1,2,4,8 μg/ml).Results of MTT showed that the survival rate of thre groups decreased significantly when cisplatin concentration increased.The survival rate of S KO V 3 cp cells was significantly higher than that ofSKOV3 cells When exposed to the same concentration of cisplatin(p<0.001).Compared to SKOV3 cp cells without miR-34a,the survival rate of SKO V3 cp cells transfected with mir-34a was decreased(p<0.001).8.Effect of miR-34a inhibitor on cisplatin resistance:Two types of cells were still used in the experiment:S KO V3 and SKO V3 cp cells.However,different from the above experiment,SKOV3 cell was transfected with mi R-34a inhibitor to simulate the corresponding response of ovarian cancer cells to chemotherapy when mi R-34a expression was inhibited in S KO V 3 cells.24h after transfection,the cells were treated with different concentrations of cisplatin(0,0.5,1,2,4,8 μg/ml).Results of MTT showed that the survival rate of the cells decreased significantly when the concentration of cisplatin increased.Compared to S KO V 3 cells transfected without miR-34ainhibitor,the proliferation ability of SKOV3 cells transfected with mi R-34a inhibitor was significantly enhanced,and the inhibitory ability of cisplatin at different concentrations on cancer cells was weakened(p<0.001)Conclusions1.miR-34a restrained OC cell proliferation.2.Overexpression of miR-34a can reverse chemoresistance,which provides a theoretical basis for targeted therapy of microRN A in OC.Part III:Target function of HDAC1 on miR-34a and its effects on cell proliferation and cisplatin resistanceObjective:1.We explored whether HDAC1 was a direct target of m i R-3 4 a,and the role of HD AC 1 gene in ovarian cancer was discussed;2.Whether mi R-3 4 a can suppress the proliferation and reverse chemoresistance of O C cells by targeting HD AC I was verified.Methods:The 751 potential targets of miR-34a were predicted by informatic analysis with Targets can(www.targetscan.org),and 14 cancer-related genes were selected for further study.Then,we constructed reporter vectors containing the luciferase coding sequence followed by the 3 ’-UTRs of the above potential miR-34a targets.The dual luciferase reporter assay was conducted in SKOV3 cells.Western-Blot was verified the expression of HD AC 1 in ovarian cancer cells and its regulatory relationship with miR-34a.E ffect of HD AC 1 gene expression on ovarian cancer cell viability was detected by MTT assay and soft agar colony formation assay.RT-qPCR,Western-Blot and MTT were applied to verify the effect of HD AC 1 gene expression on cisplatin resistance to ovarian cancer cells.A plasmid overexpressing HD AC 1 was c o n s true t ed.M TT and soft agar colony formation assay were used to detect the effect of HDAC1 overexpression on the proliferation of S KO V 3 and SKOV3 cp cells transfected by miR-34a.Results:1.Screening and analysis of suspected miR-34 a genes:14 cancer-related genes were selected for further study by informatics analysis with T argetScan.Results showed that transfection with the miR-34a mimic down-regulated the expressions of 6 cancer-related genes in SKO V 3 and OVCA433 cells,such as Sox4,HDAC1,ITGB8,CDH4,TCF12,and WNT1.The dual luciferase reporter assay showed significant differences in luciferase activity between TCF12 and HD AC 1,while miR-34a strongly inhibited the luciferase activity of HDAC1(p<0.00 1).2.Inhibition of miR-34a on HDACI expression:miR-34a mimic were transfected into the 3’-UTR complementary site of HD AC 1 gene.In the wild-type group,the luciferase activity was strongly inhibited(p<0.001),while in the mutant group,the mutation of the complementary site in the HDAC1 3’-UTR abolished the inhibitive effect by interrupting the interaction between miR-34a and HDAC1.3.Expression of HDAC1 in OC cells and its relationship with miR-34a:The results of electrophoresis showed that the transfection of mimic RN A-3 4 a could inhibit the expression of HDAC1 in SKOV3,OVCA43 3 and SKOV3cp cells.4.Effect of HDAC1 gene expression on viability of OC c ell:S KO V3 cells were transfected with HD AC 1 overexpressed plasmid or HD AC 1 siRNA as the experimental group and the untransfected group as the NC group.MTT assay was used to evaluate cell growth 1-5 days after transfection.The growth rate of SKO V 3 in the HDAC1 overexpressed group was 0.079 ± 0.005、0.398 ±0.010、0.690±0.009、1.132 ± 0.121、1.893 ± 0.089.The growth rate of SKOV3 in the HDAC1 siRNA group was 0.071 ± 0.005,0.31 2 ± 0.014,0.45 6±0.007,0.5 17± 0.1 14,1.236 ± 0.089.The difference was statistically significant compared to the untransfected group(p<0.001).The results of soft ager colony formation test showed that Compared tothe NC group(colony counts 228.3± 8.8),the HD AC 1 overexpression group(colony count 296.3 ± 24.5)significantly promoted colony formation of SKOV3 cells.When HD AC 1 was silenced(colony count 153.8 ± 15.0),SKOV3 cell colony formation was significantly inhibited compared to the NC group(colony count 2 1 1.3±9.7).5.Effect of HDAC1 gene expression on viability of OC cell:Real-time PCR and immunoblot analysis revealed that the mRNA and protein level of HDAC1 in SKOV3 cells were much less than those in S KO V3 cp cell s(p<0.01),overexpression of HDAC1 significantly improved the survival rate of SKOV3 cells that were treated with cisplatin(p<0.001);6.Effect of overexpression of HDAC1 on proliferation of SKOV3 or SKOV3cp cells transfected by miR-34a:MTT assay and soft agar colony formation showed that miR-34a dramatically suppressed the viability and colony formation of SKOV3 cells,However,HDACI overexpression markedly abolished the inhibiti ve effect.Treatment with cisplatin led to a significant(approximately 60%)decrease in the viability of S KO V 3 cells,whereas cisplatin treatment had little(approximately 15%)effect on SKOV3 cp cell viability.miR-34a dramatically increased the sensitivity of SKO V3 cp cells to cisplatin,cell viability was reduced to approximately 50%in SKR3 cp cells.In contrast,S KO V3 cp cells that were transfected with miR-34a were de-sensitized to cisplatin via HD AC 1 overexpression,the cell viability was reduced to only about 20%(p<0.001).Conculsions:1.HD AC 1 was a direct target of miR-34a.HDAC1 decreased cisplatin sensitivity and promoted proliferation in OC cells.2.MiR-34a suppressed the proliferation and chemoresis-tance of OC cells by directly targeting HD AC 1...