| Objective: For exploring mechanism of hsa-mir-429 in the treatment of ovarian cancer,we analyzed the basic information of hsa-mir-429 by bioinformatics method.Then we predicted its target gene,and annotated its target gene.Methods: We predicted the target genes of hsa-mir-429 by four prediction software including mi RDB,Targetscan,Target Miner,RNACentral and Satrbase v3.0 database platform.The target genes of mir-429 were analyzed by the chip database GSE56967 retrieved from the GEO database,and the predicted target genes were annotated with GO function.Results: Twenty-five target genes of hsa-mir-429 were predicted by four prediction software.At the same time,seven predicted target genes(ZEB1,ATP2A2,NAB1,VLDLR,CHD1,SEC23 A,FRMD4B)were predicted in Satrbase v3.0 database platform.A data chip search of ovarian cancer related to mir-429 in the GEO database showed that overexpression of mir-429 would mediate the down-regulation of ZEB1.The predicted target gene of hsa-mir-429 showed enrichment in biological processes such as embryonic organ development,transcriptional regulation,MAPK family signaling pathway regulation,signal regulation,signal transduction,and lipid metabolism(P < 0.05).Combined with the clinical data related to the differential expression profile of ovarian cancer and mi RNA in previous studies,we found that mi R-429 was regulated down by 35% in the drug-resistant group of ovarian cancer,and the differential expression of mi R-429 and its target genes ZEB2 and KANK2 were also correlated with OS and PFS in patients.Conclusion: Hsa-mir-429 may regulate a variety of target genes.The kind of target genes may play a key role,that including ZEB1,ATP2A2,NAB1,VLDLR,CHD1,SEC23 A and FRMD4 B.Meanwhile,they may be the key regulatory mechanism that transcriptional regulation,signal regulation and signal conduction of MAPK family signaling pathway in ovarian cancer.Subsequently,these target genes will be tested and clinically verified simultaneously.Objective: Previous studies indicated that the target gene of mir-429 may be ZEB1,so we verified the expression level of ZEB1 in drug-resist ant ovarian cancer cell lines and their xenograft tumors of nude mice.Methods: Lentivirus infected target cells.QRT-PCR was performed to detec t the expression levels of a series of cells which are SKOV3,SKOV3/D DP2,SKOV3/DDP2-mi R-429 mimics,SKOV3/DDP2-mi R-NC,SKOV3/DDP2-mi R-429 inhibitor.A series of cells were transplanted into mice which are S KOV3,SKOV3/DDP2,SKOV3/DDP2-mi R-429 mimics,SKOV3/DDP2-mi R-N C,SKOV3/DDP2-mi R-429 inhibitor,and the growth of transplanted tumor was monitored.The expression of mir-429 in the transplanted tumor tissue s was detected by q RT-PCR.Immunohistochemistry was used to observe t he expression of ZEB1 in xenograft tumors of nude mice.HE(hematoxyl in-eosin)staining was used to observe the cell morphology of transplanted tumor in nude mice.Results: The growth rate of transplanted tumor of SKOV3/DDP2-mi R-429 mimics was slower than the control group.Mi R-429 was highly expressed in transplanted tumor of SKOV3/DDP2-mi R-429 mi mics.Immunohistochemical detection showed that the expression of ZEB1 in the five transplanted tumors decreased with the increase of the number of cisplatin injections,and the expression level of ZEB1 in xenograft tu mors SKOV3/DDP2-mi R-429 mimics was lower than that in the control gro up.Conclusion: In the study of xenograft tumor in nude mice,mir-429 c an target ZEB1,and the up-regulation of mir-429 inhibited the expression of ZEB1.Objective: It was explored that the changes in autophagy process of cisplatin resistant in ovarian tumor cells SKOV3/DDP2 after mi R-429 was overexpressed and the effect on the autophagy pathway of PI3K-AKT1,and then it was verifed in nude mice.Methods: CCK8 was used to detect the IC50 of SKOV3/DDP2 and its parental cells SKOV3,and resistance index was calculated.The IC50 values of cells were detected by CCK8 t hat are SKOV3/DDP2,SKOV3/DDP2-mi R-429 mimics,SKOV3/DDP2-mi R-NC,SKOV3/DDP2-mi R-429 inhibitor.We observed the distribution of autophagy protein LC3A/B with different concentrations of cisplatin for 24 hours b y immunofluorescence.The autophagosomes were observed with a certain cisplatin concentration for 24 hours by transmission electron microscopy.Western Bolt was used to detect the expression of intracellular autophagy proteins(LC3A/B-II)/(LC3A/B-I)and p62 at different cisplatin concentratio ns for 24 h.Western Bolt showed that overexpression of mi R-429 affected the expression of PI3K-AKT1 pathway in ovarian cancer cells.Western Bolt was used to detect the expression of autophagy proteins(LC3A/B-II)/(LC3A/B-I)and p62 in the transplanted tumor.HE(hematoxylin-eosin)staini ng was used to observe the transplanted tumor tissue in nude mice.The e xpression of LC3 B in transplanted tumor tissue of nude mice was observe d by immunohistochemistry.Results: After cisplatin induction,SKOV3/DD P2-mi R-429 mimics was showed no autophagosome formation.After cisplati n treatment at a certain concentration,Western Bolt showed that the ratio of(LC3A/ b-II)/(LC3A/ b-I)decreased and p62 increased at mimics cells.Overexpression of mi R-429 increased the expression of AKT1,p-AKT1,m TOR and p-m TOR in the PI3K-AKT1 autophagy pathway;meanwhile,the expression of Atg7 and AMPK in SKOV3/DDP2-mi R-429 mimics was up-regulated.The expression of LC3A/B was lower than the control grou p in SKOV3/DDP2-mi R-429 mimics tumor detected by Western Bolt in nud e mice.Conclusion: Overexpression of mir-429 can weaken cisplatin-induc ed autophagy in SKOV3/DDP2 cells and increase the sensitivity of cisplati n-resistant,and up-regulated mi R-429 can effectively inhibit the growth of drug-resistant ovarian tumor and weaken cisplatin-induced autophagy.It ca n not only study the signaling pathway mechanism in the development of ovarian cancer resistance and its clinical application further,but also serv e as an effective tool to guide the chemotherapy response and the treatme nt of multi-drug resistance. |
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