MiR-216b Increases Cisplatin Sensitivity In Ovarian Cancer Cells By Targeting PARP-1

BackgroundOvarian cancer is a malignant tumor with the highest mortality rate in the female reproductive system,the incidence rate after cervical cancer and endometrial cancer.Ovarian cancer onset and to reach the pelvic or abdominal distension abdominal pain,the lesion has shifted,mostly late clinical stage.Main clinical treatment of ovarian cancer for ovarian cancer cytoreductive surgery and adjuvant platinum and paclitaxel based combination chemotherapy.Although combined with cisplatin based chemotherapy in patients with ovarian cancer,the first sensitive reaction rate is more than 80%,but prone to platinum resistance.Most patients eventually because of resistance to chemotherapy caused disease relapse and death.Therefore,reduce the recurrence rate of reversal of multidrug resistance in ovarian cancer chemotherapy,especially the mechanism and way of reversing platinum resistance has become the important research subject.In recent years,the great progress has been made in studies on Mechanism of tumor resistance,the resistance mechanism research including increased expression of resistant protein,cell detoxification function caused by the change of intracellular protease level enhancement,the enhanced DNA damage repair,target molecular level changes and so on.Poly adenosine phosphate ribose polymerase two-1(poly-ADPribosepolymerase-1,PARP-1)is a member of the PARP family,its content in the body is most specific,exists in eukaryotic cells,which belongs to the post-translational modification of enzyme.Recently,its role in tumorigenesis and invasion and metastasis in the molecular researchers pay great attention to the amount of.PARP-1 protein is 116KDa,contains 1014 amino acids,located on chromosome 1q41?42 region,including 23 exons.It can by the base excision repair of single strand DNA damage repair in methyl modification and transcription of DNA,cellular signal transduction,play an important role in the regulation of cell cycle and cell mitosis process.Many studies have found that a crucial role in maintaining genomic stability of PARP1,the activity of inhibition of PARP1 may make the body cells to DNA damage factors and tumor susceptibility.Our preliminary experiments also confirmed the increased expression of PARP1 in ovarian cancer,PARP1 is involved in the occurrence of ovarian cancer.Vimentin in ovarian cancer tissues,the expression of Snail was positively correlated with the expression of PARP-1,PARP-1 may be through regulating E-cadherin,vimentin,Snail expression in ovarian cancer metastasis and invasion process.The research showed that PARP1 in tumor cells gene repair,apoptosis,proliferation and differentiation and play an important role in invasion and metastasis,and by inhibiting the activity of DNA repair mechanisms can be inhibited by its mediated,enhanced tumor cell sensitivity to chemotherapy.Great progress has been made on the treatment of the PARP1 inhibitors have been used in clinical practice and in combination with chemoradiation,can enhanced sensitivity of some anticancer drugs.Small RNAs(microRNAs,miRNAs)is a class composed of 19-23 nucleotides,encoding endogenous small molecule non single stranded RNA.that can mediate the regulation of cell proliferation,apoptosis,metabolism,gene expression by post transcriptional physiological and pathological biological processes.Recent studies have proved that,miRNAs gene regulation of human protein encoding about 1/3,and the abnormal expression in many tumors,suggesting that miRNAs may mainly through direct regulation of target gene expression,involved in malignant tumor incidence,progression and invasion and metastasis process,has a close relationship with the diagnosis and prognosis of tumors.The single nucleotide polymorphism(SNP)is also associated with tumor drug resistance,so miRNAs could be a new target in tumor treatment.MiRNA-216b is a newly discovered miRNA,shown to play different roles in different malignant tumors.?miRNA-216b can effect directly on FoxMl to inhibit the glial cell's expression,proliferation and invasion.FoxMl in many malignant tumors increased,specific expression in proliferating cells,and is closely related to cell growth,transcription factor is a member of the Fox family.Mainly through the inhibition of cyclin dependent kinase inhibitor in cell proliferation and growth.?The guide So Suke miRNA-216b may be a tumor suppressor.PBK by regulating PBK(PDZ binding protein)is a regulatory protein in vivo drug transport,play a role in the mechanism of drug resistance of tumor.The expression of exogenous gene in breast cancer cell line miRNA-216b can inhibit SDCBP,thereby reducing the cell proliferation,migration and invasion.?MiRNA-216b can regulate the expression and activity of UDP xylitol transferase 2B(UGT2B)..UGT2B in liver of endogenous and exogenous compound solution of important coenzyme toxic,it can help the liver metabolism a variety of hormones,drugs and carcinogens.The latest research shows that the regulation of UGT2B is an important mechanism to induce liver cancer.The study showed that in hepatocellular carcinoma miRNA-216b,targeted inhibition of oncogene KRAS and its downstream AKT/ERK signaling pathway is the main way to play the role of tumor suppressor.MiRNA-216b can be used as liver cancer early diagnosis and judgment of an important molecular prognosis after operation.Although there is still no research confirmed that miR-216b with a specific tumor resistance has a clear correlation,but miRNA-216b may provide a new basis for predicting the efficacy of chemotherapy drugs and individuallized treatment options,and molecular targeted therapy of ovarian cancer to provide important new ideas.This paper made a further study.Part one.Effect of miRNA-216b on Platinum resistance of ovarian cancer cell line SKOV3 and drug-resistant cell line SKOV3/CDDPObjective:To study the expression of miRNA-216b in ovarian cancer cell line SKOV3 and its resistant strain SKOV3/CDDP and its influence on platinum resistanceMethods:1.Fluorescence quantitative real-time PCR was used to detect the expression of miRNA-216b in ovarian cancer cell line SKOV3 and its resistant strain SKOV3/CDDP;2.CCK-8 assay was used to detect the activity of ovarian cancer cell line SKOV3 and its drug-resistant cell line SKOV3/CDDP,and the relationship between the expression of miRNA-216b and the survival activity of ovarian cancer cell line was analyzed;3.CCK-8 was used to compared the proliferation of SKOV3/CDDP/miR-216b and control group cell line SKOV3/CDDP/miR-NC after transfection of miRNA216b stably;4.Flow cytometry was used to detecte difference of the apoptosis of miR-216b cell line SKOV3/CDDP/miR-216b and control group cell line SKOV3/CDDP/miR-NC.Results:1.The expression of miRNA-216b in SKOV3/CDDP cell line was significantly decreased compared with the cell line SKOV3;2.The activity of SKOV3/CDDP cell line was significantly higher than that of ovarian cancer cell line SKOV3 in different concentrations of cisplatin;3.Effect of miRNA-216b on chemosensitivity of ovarian cancer cell lines(1)After given different concentrations of cisplatin treatment,the proliferation of ovarian cancer cell line SKOV3/CDDP/miR-216b was decreased compared with the control group cell line SKOV3/CDDP/miR-NC,the difference was statistically significant.(2)After given different concentrations of cisplatin treatment,the late apoptosis of ovarian cancer cell line SKOV3/CDDP/miR-216b was increased,compared with the control group cell line SKOV3/CDDP/miR-NC,different concentrations of cisplatin treatment.Conclusions:MiR-216b is involved in the regulation of platinum resistance in ovarian cancer cells.Part two-miRNA-216b reverses platinum resistance of ovarian cancer cell line SKOV3/CDDP by modulating PARP-1 in vitro studyObjective:To study the regulation of miRNA-216b on PARP-1,and to investigate the molecular mechanism of miRNA regulating platinum resistance in ovarian cancer.Methods1.Bioinformatics analysis screened candidate genes related to ovarian cancer resistance;Real-time PCR identified candidate genes in ovarian cancer cell line SKOV3 and the drug-resistant cell line SKOV3/CDDP2.The expression of miRNA-216b and PARP-1 in ovarian cancer tissues was detected by real-time quantitative PCR,and then the correlation between the two expressions was analyzed;3.Double reported luciferase assay was used to detect the interaction of miRNA-216b and PARP-1;4.Transfected cells line SKOV3/CDDP/miR-216b and SKOV3/CDDP/miR-NC with PARP-1 plasmid.Weston blot was ued to observe the changes of the expression of PARP 1 in protein level.CCK-8 and flow cytometry were used to detect the changes of proliferation and apoptosis of cell line SKOV3/CDDP/miR-216b and control group cell line SKOV3/CDDP/miR-NC after transfection.Results:1.Bioinformatic analysis screened out four candidate genes of ACOX1,BIN1,RHOH and PARP-1;Real-time PCR showed that only PARP-1 was markedly reduced in miRNA-216b overexpressing cell lines SKOV3/CDDP/miR-216b;2.There is a negative correlation between the expression of PARP1 and miRNA-216b in epithelial ovarian cancer;3.Double report luciferase assay showed that miR-216b could inhibit transcription of PARP 1,but had no effect on the transcription of PARP 1 mutant;4.The protein expression of PARP1 increased in PARP1 plasmid transfected group,and cell activity increased and apoptosis decreased in PARP1 transfected group of SKOV3/CDDP/miR-216b cell line.Conclusions:MiR-216b can reverse the platinum resistance of ovarian cancer cell line SKOV3/CDDP by reducing the expression of PARP1 in vitro.Party three.miRNA-216b reverses platinum resistance of ovarian cancer cell line SKOV3/CDDP by modulating PARP1 in vivo studyObjective:To investigate the role of miR-216b in reversing cisplatin resistance in epithelial ovarian cancer by targeting PARP1 in a human ovarian cancer xenografted nude mice model.Methods1.Constructed a human platinum resistance ovarian cancer xenografted tumor nude mice model40 male cd-1/cd-1 nude mices aged 4-5 week were served as experimental subjects(BALB/cNude and CD-1 Nude are all available for oncology research and tumor transplantation.Relative to the BALB/C Nude,CD-1 Nude tumor cell inoculation quantity is relatively large,generally greater than 10?7 above,and into the tumor speed will be slower than BALB/cNude 1 to 2 weeks),20 rats in each group.Cells of SK0V3/CDDP/miR-216b and control group cells of SKOV3/CDDP/miR-NC in logarithmic growth with the density of 1X107/100ul,were subcutaneously injected into each side of posterior flank of the CD-1/CD-1 nude male mice.This process was approved by the Institutional Ethic Committee.The animals were monitored daily for the health conditions;2.Intraperitoneal injection of cisplatin in the treatment of subcutaneous transplanted tumor in nude mice and observation of the tumor growth;Nude mice were divided into 2 groups after injection:SKOV3/CDDP/miR-216b group and SKOV3/CDDP/miRLV-NC group.When subcutaneously transplanted tumor group to 5mm,cisplatin was injected by intraperitoneal way at 5mg/kg 2 times a week for 4 weeks.The size of the graft tumors was measured every 3Days,and the growth curve was measured by tumor volume.Volume(mm3)= 0.5*long(mm)*width 2(mm2).3.The difference of PARP1 expression between two groups of transplanted tumor was detected by Weston blotting.Three days after the end treatment,the mice were sacrificed.The tumor tissue was collected.The total protein was extracted,and the expression of PARP 1 in two groups of tumor tissues was detected by Weston and blotting.Results1.Successfully constructed the subcutaneous transplanted tumor modelin nudemice.Subcutaneous tumors grew about 7d after inoculation;The rate of tumor formation was 100%.2.miR-216b successfully revers cisplatin resistance of ovarian cancer cell line SKOV3/CDDP.Nude mice were randomly divided into SKOV3/CDDP/miR-216b group and SKOV3/CDDP/miR-NC group.When the tumors in the two groups grew to 5mm,intraperitoneal injection of cisplatin 5mg/kg was given 2 times a week for 4 weeks.Tumor volume of two group was measured after3,6,9,12d of cisplatin injection.Tumors grew slowly in SKOV3/CDDP/miR-216b group in the treatment of cisplatin.Tumors volum of nude mice in SKOV3/CDDP/miR-216b group is smaller and the weight is lighter.But tumors grew fastor in control group SKOV3/CDDP/miR-NC.Tumors volum of in SKOV3/CDDP/miR-NC group is bigger and the weight is heavier.21 days after cisplatin treatment,the volumes of tumors derived from SKOV3/DDP/miR-216b cells that were stably infected with miR-216b were significantly smaller than those from SKOV3/DDP/miR-216b cells(P<0.01).The average quality of subcutaneous tumor in SKOV3/CDDP/miR-216b group was significantly lower than that in group SKOV3/CDDP/miR-NC,and the difference between the two groups was statistically significant(P<0.01).3.The expression of PARP1 protein was decreased in subcutaneous tumor of SKOV3/CDDP/miR-216b group.The total protein was extracted from subcutaneous tumor tissue of the two groups SKOV3/CDDP/miR-216b and SKOV3/CDDP/miR-NC.The expression of PARP1 was detected by Weston blot.We found that the expression of PARP1 was significantly lower in the SKOV3/CDDP/miR-216b group than in the SKOV3/CDDP/miR-NC group.Conclusions:MiR-216b can reverse the platinum resistance of ovarian cancer cell line SKOV3/CDDP by reducing the expression of PARP1 in vitro.Summary:Lower expression of miR-216b was observed in cisplatin-resistant ovarian cancer cells.The present study demonstrates that miR-216b affects cisplatin sensitivity both in vivo and in vitro.We found that miR-216b was capable of reducing cell viability and enhancing apoptosis in cisplatin-resistant ovarian cancer cells.Its function may be exerted directly by binding to PARP1.We demonstrated that miR-216b could serve as a potential target in the treatment of ovarian cancer.