Down-regulation Of PARP1 By MiR-891b Sensitizes Human Breast Cancer Cells To Alkylating Chemotherapeutic Drugs

BackgroundsBreast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among females worldwide,with an estimated 1.67 million cases and 521,900 deaths in 2012.Breast cancer alone accounts for 25%of all cancer cases and 15%of all cancer deaths among females.The incidence of breast cancer is increasing all over the world,which might be due to rapid urbanization and changes in the lifestyle.In China,an estimated 268,600 women were diagnosed as breast cancer and 69,500 died of breast cancer in 2015,accounting for 15.1%of all new cancer cases and 6.9%of all cancer deaths in women,respectively.Drug resistance of tumor cells is a major obstacle in chemotherapy of breast cancer.Therefore,the research on the key factor of the breast cancer drug resistance,targets and regulation mechanism,is of great significance to improve the drug susceptibility.Poly-ADP-ribose polymerase（PARP）is found in the eukaryocyte and has a variety of hypotypes,such as PARP1,PARP2 and PARP3,etc.PARP1 is found to be the earliest and most studied which plays an important role in physiological activities and patho]ogical reactions in various cells,such as cancer development,genome stability and resistance to Systemic Therapy,etc.The previous studies indicated that PARP1 plays a key role in the drug resistance of tumor cells.Alkylating agents are widely used as anticancer drugs which attach an alkyl group to the tumor cell DNA.The fast growing cancer cells are susceptible to DNA damage and are destroyed quickly by apoptosis,PARP1 is an important DNA damage sensors and signal transducers.Damaged DNA can activate PARP1 and activated PARP1 participates in DNA repair.When the expression of PARP1 is inhibited,DNA repair that mediated by PARP1 is also inhibited.Therefore,drug resistance of tumor cells to alkylating agents is reduced or reversed.Hence,PARP1 is considered to be one of the most promising molecular targets in the development of novel anticancer drugs.MicroRNAs（miRNA）are a class of evolutionally conserved,single-stranded,small（18-25 nucleotides）and non-protein-coding RNAs.miRNAs act as post-transcriptional regulators of gene expression by binding to the 3’ untranslated region of the target mRNA through complementary base pairing,reducing the level of the target mRNA（degrading the mRNA）and the protein encoded by the target mRNA（inhibit mRNA translation）.Although miRNAs comprise only 3%of the total human genome,but they modulate the function of 20-30%mRNAs.The role of miRNAs in cancer has already been established and more than 50%of miRNAs are located in the fragile sites or in the cancer-related genomic regions.There is increase in evidence suggesting that miRNA plays a crucial role in cancer development and resistence to systemic therapy.In 2013,Riaz et al found the overexpression of miR-891b in BRCAl mutant cell lines,which indicatd that miR-891b may the involve in DNA damage response.Using the TargetScan database（http://www.targetscan.org/vert₇1/）,we found that PARP1 may contain the binding site of miR-891b,and we can conclude that miR-891b may be involved in the regulation of PARP1 expression,which may be related to the resistance of alkylating agents.In this study,we have explored the role of miR-891b on the sensitization of breast cancer cells HCC1806 to alkylating agent and we hope to find a new target for the therapy of breast cancer.ObjectivesThe study was aimed to explore the role of miR-891b on the sensitization of breast cancer cells to alkylating agent.This is consists of three parts.Firstly,we studied on the regulatory relationships between miR-891b and PARP1.Then,we studied the effect of miR-891b on sensitization of HCC1806 cells to alkylating chemotherapeutic drugs.Finally,we investigated effect of PARP1 on drug sensitization of HCC1806 cells.MethodsPart I:Using the TargetScan database,we sought the predicted consequential pairing of target region（the 30-UTR of PARP1）and miR-891b.The fragments from PARP1 containing the predicted miR-891b binding site were amplified by PCR and placed in a pmirGlO Dual-luciferase miRNA Target Expression Vector to form the reporter vector PARP-wild-type（MEG3-wt）.This vector was cotransfected with miR-891b mimics,or miR-NC into HCC1806 breast cancer cells.Then,we detected the targeted relationship between miR-891b and PARP1 by luciferase reporter gene.The miR-891b mimics and miR-891b NC were transfected into HCC1806 breast cancer cells.Finally,qRT-PCR was performed to detect the expression of miR-891b,and Western blot was used to monitor the protein expression of PARP1 in HCC1806 cells after miR-891b mimics and miR-891b NC transfection.Part Ⅱ:The effect of miR-891b on sensitization of HCC1806 cells to alkylating chemotherapeutic drugs:Firstly,human breast cancer HCC1806 cells were transfected with miR-891b mimics or miR-891b NC.Forty-eight hours after the transfection,the cells were exposed to 40 μM N-methyl-N-nitro-N-nitrosoguanidine（MNNG）for 1 hour and then incubated in drug-free medium for 4 days.Then,MTT assay was performed to detect the cell proliferation of miR-891b mimics + MNNG group,miR-891b group,miR-NC group and miR-NC + MNNG group.In addition,after 4 days of incubation,cell apoptosis was detected by flow cytometer.Part III:The effect of PARP1 on drug sensitization of HCC1806 cells:Firstly,stable expression of PARP1 plasmid was constructed.miR-891b mimics+pBABE and miR-891b mimics + PARP1 were then co-transfected into the HCC1806 cells,respectively.48 hours after the transfection,the cells were exposed to 40 μM MNNG for 1 hour and then incubated in drug-free medium for 4 days.Western blot was utilized to detect the protein expression of PARP1,and then the MNNG drug susceptibilities and apoptosis of HCC1806 cells were detected by MTT assay and flow cytometer,respectively.ResultsPart I:Using the TargetScan database,we found that PARP1 may contain the binding site of miR-891b.The luciferase report gene analysis showed that the percentage of luciferase activity in the miR-891b mimics group of cells were lower in comparison to the control group of cells（P<0.01）.Further research demonstrated the overexpression of miR-891b in these cells which were transfected with miR-891b mimics compared to control cells transfected with miR-NC.Western blot analysis revealed the decreased expression of PARP1 in the cells transfected with miR-891b mimics in comparison to the control cells transfected with miR-NC.The ratio of PARP1 to tubulin in cells also showed that the protein expression of PARPI in miR-891b overexpression cells was significantly decreased（P<0.01）.Part Ⅱ:The MTT assay was used to assess cellular proliferation.The results demonstrated that the cell viability in cells from miR-NC +MNNG group was lower than those from miR-NC group,and the cell viability of miR-891b mimics + MNNG group was lower than that of miR-891b group.Besides,the cell viability of miR-891b mimics + MNNG group cells was lower than that of miR-NC +MNNG group cells.Cell apoptosis was found to be maximum in miR-891b + MNNG group compared to other group of cells in miR-NC + MNNG group（P<0.01）and miR-891b group（P<0.01）.Part III:Western blot analysis showed that the protein expression of PARP1 was down-regulated when the miR-891b mimics was transfected into HCC1806 cells.However,the protein expression of PARP1 was restored in miR-891b mimics + PARP1 group cells compared to control cells and cells overexpressing miR-891b.MTT assay and flow cytometer analysis revealed that restoration of cell proliferation and decrease in the percentage of apoptotic cells occurred in miR-891b mimics + PARP1 group cells in comparison to miR-891b + MNNG group cells,respectively.ConclusionsIn this study,we found that miR-891b could combine with the 3’UTR target of PARP1.In addition,the protein expression of PARP1 was decreased,when the expression of miR-891 b was upregulated.This indicated that miR-891 b could negatively regulate the expression of PARP1.MNNG could decrease the HCC1806 cell proliferation.The overexpression of miR-891 b sensitized human breast cancer HCC1806 cells when the cells were treated with MNNG and promoted cell apoptosis.Furthermore,restoration of PARP1 expression reduced sensitization of HCC1806 cells to MNNG.In conclusion,miR-891b increased the susceptibility of alkylating chemotherapeutic agent MNNG in HCC1806 breast cancer cell line by suppressing PARP1 activity.The study demonstrate the beneficial role of miR-891b,which act as PARP inhibitors and may be utilized in the development of novel drug targets.