Preliminary Study On The Role Of MicroRNA-199a-3p In Development And Cisplatin Resistance Of EOC

Ovarian cancer is one of the most common and highest mortality rate malignant tumors in gynecology.According to the WHO histological type,Epithelial Ovarian Cancer(epithelia ovarian cancer,EOC)is the most common histological type of ovarian cancer,the standard treatment of EOC is “the first ideal cytoreductive surgery with chemotherapy based platinum”,but in clinical work,we found that although chemotherapy is the most important means of adjunctive therapy,the effect of chemotherapy is not ideal because of many patients with primary or secondary resistance to chemotherapy in the treatment process,the recurrence rate and mortality increased.In recently,many researchers paid more attention to find some bio-molecules related to occurrence and development of ovarian cancer,through the exploration of the biological and metabolic mechanism of ovarian cancer,we hope to find out the possible bio-molecules targeted therapy of ovariancancer.It had been found that miRNA plays an important role in the regulation of cells,which can act on the growth and apoptosis of cells.Mi RNA can control the proliferation,invasion and apoptosis of cancer cell,and miRNA can also increase or decrease the chemotherapy resistance of ovarian cancer cells through promote apoptosis or inhibit tumor cells as influence factors of upstream or downstream signaling pathway which closely related to drug resistance of ovarian cancer cells.The abnormal expression of miR-199a-3p occurred in some cancers according to some researches,such as liver cancer,prostate cancer or breast cancer,which can inhibit the growth,migration and invasion of tumor cells,promote tumor cell apoptosis,regulate the sensitivity of tumor cells to chemotherapy through the interaction with target gene of upstream or downstream,however,there are few reports about the role of miR-199 a in ovarian cancer.In our previous work,we found that the low level of expression of miR-199a-3p in ovarian cancer chemoresistant cells through gene chips and PCR,suggesting that miR-199a-3p may play an important role in the malignant biological behavior of ovarian cancer.What clinical significance is miR-199a-3p in EOC? What roles is miR-199a-3p to malignant biological behavior of ovarian cancer? What regulatory mechanism of the roles of miR-199a-3p in ovarian cancer? In order to answer these questions,we choose miR-199a-3p as the ovarian cancer research target,we hope to reveal the role and possible mechanism of miR-199a-3p in the development of ovarian cancer and platinum chemotherapy in tissue and cell level.Integrin is a kind of cell surface adhesion molecule,which is composed of two subunits of alpha and beta.Integrin linked matrix proteins or adjacent cells in the extracellular and connected to cytoskeletal system in the intracellular,which constitute the important survival and development link of tumor cell;on the other hand,integrin as a receptor can be combined with a variety of cytokines,transmit biological information between intracellular and extracellular,and a tumor cell reacted accordingly the information hub.Integrin is involved in tumor migration,metastasis and chemoresistance.In the tumor resistant strains,upregulated expression of various integrin isoforms have been found,integrins may be involved in the apoptosis of tumor cells induced by chemotherapeutic drugs.Target gene of miR-199a-3p is ITGB8(Integrin beta 8)according to prediction of biological information analysis software and verified by dual luciferase reporter,through further experience demonstrated that the regulation of miR-199a-3p targeted ITGB8 in the development of EOC and platinum resistance play a role,and study the mechanism of miR-199a-3p/ITGB8 signaling pathway in ovarian cancer development and platinum resistance.This study will provide strong evidence for the involvement of miR-199a-3p in platinum resistance of ovarian cancer,and provide a theoretical basis for further elucidation of the role of miR-199a-3p gene in EOC development and drug resistance.OBJECTIVES Preliminary study on the clinical significance of micro RNA-199a-3p in EOC and the roles of miR-199a-3p target ITGB8 to malignant biological behavior of ovarian cancer,and reveal the role and possible mechanism of miR-199a-3p in the development and cisplatin chemoresistance of ovarian cancer in clinical tissues,vitro and vivo.The study results will supply candidate target and theoretical supports to control the development and chemoresistance of EOC.METHODS1.The objects of study were the EOC platinum resistance and platinum sensitivity pathological section and clinical data,the q RT-PCR method was used to test the expression of different groups of miR-199a-3p,and to explore the relationship between miR-199a-3p gene expression level and different clinical stage or other factors and the platinum resistance of EOC.2.Culturing ovarian cancer SKOV3 Cell,cisplatin(DDP)resistant of SKOV3 cell line was established by progressive increasing concentration method.Methods including drug resistance assay(MTT),Wound healing assay and transwell cell invasion assay were used to assess the difference functions between SKOV3 and SKOV3/DDP,and using quantitative real-time polymerase chain assay(q RT-PCR)to assess the different level of miR-199a-3p in different cells.3.Mi R-199a-3p mimic and inhibitor transfected SKOV3/DDP and SKOV3 cells.The different effects of SKOV3/DDP and SKOV3 after transfection in cell migration and invasion and resistance index was detected by MTT,Transwell and Wound healing,then analysis the rule of miR-199a-3p on biological behavior of SKOV3/DDP and SKOV3 cells.4.Find miR-199a-3p possible target genes using biological information prediction software predicted and summary of miR-199a-3p target genes from literature.q RT-PCR and WB methods detected expression of m RNA and protein level of these target genes in SKOV3/DDP and SKOV3 cells;according to analysis of biological information prediction software,the luciferase reporter vector was constructed by amplifying ITGB8 3'-UTR fragment containing miR-199a-3p targeting sites,the PCR product was digested with Xho I and Not I,which was ligated with the same enzyme-digested psi CHECK-2,The two puptative miR-199a-3p binding sites were mutated,Luciferase activities were assessed using Dual-Luciferase? Reporter Assay System.and miR-199a-3p targeted ITGB8 and the binding sites were determined by dual luciferase reporter gene verification.5.By liposome transfection of miR-199a-3p mimic and inhibitor,the changes of ITGB8 expression in SKOV3/DDP and SKOV3 cells were detected,which further proved the target role regulation of miR-199a-3p to ITGB8.6.ITGB8 over expression vector was constructed using recombinant plasmid Technology:through the NCBI website to find the sequence of ITGB8 gene and find the ORF of ITGB8,according to the pc DNA3.1 vector map to select suitable restriction sites,primers were designed by means of PCR gene cloning,then digested into pc DNA3.1 to construct the recombinant plasmid of pc DNA3.1-ITGB8.7.After pc DNA3.1-ITGB8 was constructed,SKOV3 cells were transfected with miR-199a-3p inhibitor,ITGB8 over expression vector or both;at the same time,SKOV3/DDP cells transfected with miR-199a-3p mimic,ITGB8 over expression vector or both.8.Methods including drug resistance assay(MTT),Wound healing assay and transwell cell invasion assay was used to assess the difference functions between SKOV3 and SKOV3/DDP transfected differently.9.Cell cycle and apoptosis were detected by flow cytometry to assess the difference functions between SKOV3 and SKOV3/DDP transfected differently.10.Orthotopic ovarian xenograft model was established.Mice were anesthetized with a ketamine/xylazine cocktail,and the left ovary enshrouded in its oviductal fimbriae was exposed by a dorsal incision.SKOV3 and SKOV3/DDP cells were injected into the ovarian bursa.10 weeks after xenograft,the number,size and weight of local tumors as well as peritoneal metastatic nodules were recorded.11.q RT-PCR to assess the different expression level of miR-199a-3p in SKOV3/DDP tumors and in SKOV3 tumors,12.q RT-PCR and western blot to assess the different level of ITGB8 m RNA and protein levels in SKOV3/DDP tumors and SKOV3 tumors.Results1.The clinical rule of miR-199a-3p in EOC and the relationship with cisplatin chemo-resistance(1)Compared with normal ovarian tissues,the expression of miR-199a-3p in benign is almost same,and slightly decreased in borderline ovarian tumor,but obviously decreased in EOC.(2)Compared with the sensitive group of epithelial ovarian cancer,the drug resistance group had lower level of miR-199a-3p,and the results were statistically significant;(3)according to the surgical pathological staging of ovarian cancer,the expression of miR-199a-3p gradually decreased with stage increased;expression of miR-199a-3p in different tissues grade also have obvious difference,the degree of differentiation is worse,the expression level of miR-199a-3p is lower;there are obvious differences in the expression of miR-199a-3p between lymph node metastasis and no lymph node metastasis.Compared with no lymph node metastasis,the expression of miR-199a-3p obviously decreased,indicating that there were relationship between miR-199a-3p difference expression level and EOC staging,differentiation degree and lymph node metastasis,the difference was statistically significant,but there was no obvious difference expression of miR-199a-3p in different histological types;2.The expression of miR-199a-3p in ovarian cancer cell and its influence to biological behavior of ovarian cancer cell.(1)There were significantly stronger ability of SKOV3/DDP than SKOV3 in the drug resistance,migration and invasion ability and the difference was statistically significant;(2)The expression level of miR-199a-3p in SKOV3/DDP was significantly lower than that of SKOV3;(3)miR-199a-3pmimic and inhibitor transfected SKOV3/DDP and SKOV3 cells,drug resistance index,migration and invasion and apoptosis were detected in SKOV3/DDP and SKOV3 cell after transfection.After miR-199a-3p mimic transfected SKOV3/DDP resistance to cisplatin decreased,sensitivity increased,cell invasion decreased and metastasis apoptosis increased;the functions of miR-199a-3pinhibitor tranfected SKOV3 were on the other hand;3.miR-199a-3p regulates ITGB8 as a target gene(1)Searching possible target genes of miR-199a-3p,detection of the m RNA gene expression level and protein expression level of possible target genes in SKOV3 and SKOV3/DDP by q RT-PCR and WB methods,ITGB8 may be the target gene because of its significant expression and changes;(2)Through biological information analysis,we founded that ITGB8 associated strongly with miR-199a-3p,some researches had found that integrin is related to chemoresistance and development in cancer;(3)The decreased expression of miR-199a-3p in ovarian cancer cell can increase expression of ITGB8,the increased expression of miR-199a-3p on the other coincide.(4)Two miR-199a-3p seed sequences were identified by Target Scan 7.1(www.target scan.org)within ITGB8 3'-UTR at position 203-209 and 729-735.Tagging ITGB8 3'-UTR fragment containing both miR-199a-3p seed sequences to the 3'-end of renilla luciferase coding sequence caused about 65% reduction of luciferase activity when whereas the same DNA fragment with mutated miR-199a-3p seed sequences had no effect on luciferase activity.Cotransfecting miR-199a-3p inhibitor strongly increased luciferase activity while miR-199a-3p mimics inhibited luciferase activity of psi CHECK-ITGB8.However,psi CHECK-ITGB8 with mutant miR-199a-3p seed sequences was insensitive to either miR-199a-3p mimics or inhibitor;4.miR-199a-3p limited biological behavior of cancer cells through target ITGB8.(1)Constructed ITGB8 over expression vector;(2)Cells transfected with miR-199a-3p inhibitor,ITGB8 over expression vector or both had significantly higher ITGB8 protein levels and drug resistance towards cisplatin.On the other hand,miR-199a-3p mimic markedly inhibited ITGB8 protein level and cisplatin resistance of SKOV3/DDP cells.The inhibition of ITGB8 level and drug resistance by miR-199a-3p mimic was abrogated by ITGB8 expression vector;(3)The migration and invasion abilities of SKOV3 cells was substantially increased by miR-199a-3p inhibitor,ITGB8 expression vector,or both.On the contrary,miR-199a-3p mimics inhibited the motility and invasiveness of SKOV3/DDP cells,which was reversed by ITGB8;(4)Reducing miR-199a-3p level in SKOV3 cells by its inhibitor or over expressing ITGB8 significantly reduced apoptosis caused by cisplatin.Cocurrent transfection miR-199a-3p inhibitor and ITGB8 expression vector resulted in further reduction of apoptosis.Introducing miR-199a-3p mimic into SKOV3/DDP cells significantly increased cisplatin apoptosis caused by cisplatin,which was largely blocked by ectopic ITGB8.(5)Nude mice with SKOV3/DDP cells injected into the ovarian bursa had drastically larger number of metastatic nodules on omentum,mesentery,and peritoneal surface of abdominal flanks than mice implanted with SKOV3 cells.Similarly,SKOV3/DDP implanted mice had more and bigger primary tumors.The expression level of miR-199a-3p was significantly lower in SKOV3/DDP tumors than that in SKOV3 tumors whereas ITGB8 m RNA and protein levels were markedly increased in SKOV3/DDP tumors compared to SKOV3 tumors.Conclusions1.In clinical tissue level,the expression of miR-199a-3p in EOC tissue decreased according to the degree of malignancy and staging increased;the expression of miR-199a-3p was so different in platinum sensitive tissues from platinum resistant tissues,miR-199a-3p may be associated with the development and platinum resistance of EOC;2.miR-199a-3p can increase cells chemotherapy sensitivity to platinum based on downing regulate expression of target gene ITGB8;3.miR-199a-3p can increase cells invasion and metastasis of ovarian cancer at the same time decrease apoptosis based on downing regulate expression of target gene ITGB8;4.Reduced miR-199a-3p level in ovarian cancer cells was associated with strong tumor growth and metastasis in vivo.5.miR-199a-3p/ITGB8 signaling pathway may play a role in the development and the platinum resistance of EOC,while decreasing the expression of miR-199a-3p improved cells invasion and metastasis to improve the development of ovarian cancer through the upregulation of the expression of the target gene of ITGB8,at the same time reduced the apoptosis of ovarian cancer cells which led to cisplatin chemoresistance by the after-target mechanism,miR-199a-3p is expected to become a new target of ovarian cancer biological therapy.