Linc00312/miR-196a-5p Axis Reverses Cisplatin Resistance By Targeting ZMYND11 In Ovarian Cancer

Objective:Ovarian cancer is the leading cause of women’s cancer-associated death Many drugs have been used to treat ovarian cancer for improving patients’prognosis Platinum-based chemotherapy is the first-line treatment for locally advanced or metastatic patients.However,chemotherapy resistance has been observed in many patients,leading to ineffective treatment.How to reverse the chemo-resistance of ovarian cancer has become a hot spot at home and abroadLncRNAs,a class of RNA longer than 200 nucleotides that cannot encode proteins,are mRNA-like transcripts that have been increasingly identified as the key regulators in the tumorigenesis and progression of various human cancers.At present,many sequencing technologies have shown that LncRNAs have differential expressions between drug-resistant and drug-sensitive cells in various types of cancer.It may affect chemo-resi stance of cancer through cell signaling pathways,DNA damage repair,DNA methylation and so on.Therefore,it is of great clinical significance to study the roles and potential molecular mechanisms of LncRNAs in development,progression,radiosensitivity and chemosensitivity of many tumorsIn the meantime,it was found that LncRNAs can act as competing endogenous RNAs,and directly bind to miRNAs and their downstream target genes through microRNA response elements,regulating the expression levels and biological functions of each other.Linc00312 is transcribed from the genomic region of chromosome 3p25.3,and has been found to be significantly dysregulated in a wide range of diseases and play a key role as a tumor suppressor gene.Furthermore,Linc00312 plays a vital role in cell proliferation,cell apoptosis,differentiation,and metastasis by interacting with particular microRNAs or target genes.However,there is no evidence to prove whether Linc00312 is connected to drug resistance,especially in ovarian cancerThis study is the first to describe the functions of Linc00312 in resistance to cisplatin in ovarian cancer.Quantitative real-time PCR and in situ hybridization were used to test for expression of Linc00312 in chemotherapy resistant and sensitive tissue samples and cells in ovarian cancer.After constructed siRNAs and overexpression plasmids,the cell counting kit-8 assay,flow cytometry and Western Blot were used to explore the molecular mechanism of Linc00312 in reversing the chemo-resistance of ovarian cancer.MiRNA and target gene which Linc00312 binds to directly were predicted by the bioinformatics software.Then,the role of miR-196a-5p and ZMYND11 in reversing the chemoresistance of ovarian cancer was verified.Luciferase reporter assay,qRT-PCR and Western Blot were used to test the relationship between the three.This study would clarify that Linc00312 can regulate the expression of ZMYND11 by inhibiting miR-196a-5p directly,thereby reversing the chemo-resi stance of ovarian cancer and provide a new idea for exploring the treatment of ovarian cancer.Methods:Firstly,qRT-PCR and in situ hybridization were used to detect the expression of Linc00312 in serous ovarian cancer tissues.The correlation between Linc00312 and clinicopathological features of ovarian cancer was analyzed by follow-up of patients.qRT-PCR was used to test the differential expression of Linc00312 in ovarian cancer chemo-sensitive cell line SKOV3 and the chemo-resistant cell line SKOV3/DDP.Linc00312 siRNA and overexpression plasmid were transfected into SKOV3 and SKOV3/DDP respectively.CCK-8 assay was used to detect cell viability.The 50%inhibitory concentration value for cisplatin treatment was estimated based on the cell viability curve.Flow cytometry was used to measure cell apoptosis.qRT-PCR and Western Blot were performed to confirm changes in mRNA and protein levels of drug resistance-related proteins(MDR1 and MRP1)and apoptosis-related proteins(Bcl-2,Bax,Caspase-3 and Caspase-9).Secondly,search for one microRNA which Linc00312 may interact derictly by bioinformatics software.qRT-PCR was used to detect the differential expression of miR-196a-5p in chemo-resi stance and chemo-sensitive tissues and cells of ovarian cancer.Pearson correlation co-efficient was used to determine the correlation between Linc00312 and miR-196a-5p.After transfection of miR-196a-5p mimics and inhibitor,CCK-8,Annexin V-FITC apoptosis assay and Western Blot were used to detect the mechanism of miR-196a-5p to chemotherapy resistance of ovarian cancer.The regulatory relationship between Linc00312 and miR-196a-5p was detected by qRT-PCR.PMIR-Linc00312 was constructed and the direct regulation relationship between Linc00312 and miR-196a-5p was detected by luciferase reporter gene assay.Thirdly,immunohistochemistry and Western Blot were used to detect the expression of ZMYND11 in chemo-resistant and chemo-sensitive tissues of ovarian cancer,and the correlation between ZMYND11 and clinicopathological features of ovarian cancer was statistically analyzed.The differential expression of ZMYNDll in SKOV3 and SKOV3/DDP cells was detected by qRT-PCR and Western Blot.After transfection of ZMYND11 siRNA and overexpression plasmid,the effect and molecular mechanism of ZMYND11 to chemotherapy resistance in ovarian cancer was explored by CCK-8 assay,Annexin V-FITC cell apoptosis assay and Western Blot.The Linc00312 siRNA,overexpression plasmid and miR-196a-5p mimics,inhibitor were transfected into SKOV3 and SKOV3/DDP cells.The mRNA and protein levels of ZMYNDll were detected by qRT-PCR and Western Blot,verifing the regulation of ZMYND11 by Linc00312 and miR-196a-5p.The binding site of miR-196a-5p and ZMYND11 3’UTR was predicted by TargetScan and verified by luciferase reporter gene assayResults:Initially,the expression of Linc00312 in chemo-resistant tissues of serous epithelial ovarian cancer was significantly lower than that of chemo-sensitive tissues and mainly localized in cytoplasm.The low expression of Linc00312 was related to the FIGO stage of ovarian cancer,but not to the ages of patients,tumor differentiation and lymph node metastasis.The expression of Linc00312 decreased significantly in SKOV3/DDP cells compared to that in SKOV3 cells.Down-regulation of Linc00312 could decrease inhibition and apoptosis rates in SKOV3 cells,and the IC50 of cisplatin was 6.308±0.299 μg/ml,which was higher than 4.297 ± 0.148 μg/ml in control SKOV3 cells Moreover,the mRNA and protein levels of MDR1,MRP land Bcl-2 increased,while the expression of Bax,Caspase-3 and Caspase-9 were lower than that in SKOV3-NC cells.When Linc00312 overexpression plasmid was transfected into SKOV3/DDP cells,the reverse trends were evidentNext,in chemo-resistant tissues,the expression of miR-196a-5p was significantly higher than that in chemo-sensitive tissues.Compared with SKOV3 cells,the expression of miR-196a-5p increased in SKOV3/DDP cells.In SKOV3 cells,the cell inhibition and apoptosis rates of miR-196a-5p mimics group were significantly decreased compared with control group,and the IC50 for cisplatin treatment was higher than NC group.In SKOV3/DDP cells,compared with NC group,miR-196a-5p inhibitor group had significantly higher cell inhibition and apoptosis rates,and the IC50 was lower.There was a target binding site between Linc00312 and miR-196a-5p,which could negatively regulate each other’s expression,and they could co-inhibit the expression of MDR1 and MRP1 proteins,thereby reversing the resistance of ovarian cancer to cisplatin.Finally,compared with drug-sensitive tissues and cells of ovarian cancer,ZMYND11 had a lower expression in drug-resistant tissues and cells,but its lower expression was not associated with age,FIGO stage,tumor differentiation and lymph node metastasis of ovarian cancer patients.The down-expression of ZMYND11 decreased inhibition rate and apoptosis rate of SKOV3 cells,and increased IC50 value and the expression of MDR1 and MRP1.However,after up-expression of ZMYND11 in SKOV3/DDP cells,the above trends were just the opposite.After knockdown Linc00312 expression protein expression level of ZMYND11 was significantly reduced,while the protein expression level grew with Linc00312.MiR-196a-5p and ZMYND11 also had a target binding site.Linc00312 and miR-196a-5p could regulate the mRNA and protein levels of ZMYND11 together,thereby affecting the chemo-sensitivity of ovarian cancer to cisplatin.Conclusion:1.The expression of Linc00312 in chemo-resistant tissues and cell line SKOV3/DDP were significantly lower than that of chemo-sensitive tissues and cell line SKOV3,which was related to the FIGO stage of ovarian cancer.Linc00312 could enhance the sensitivity of ovarian cancer cells to cisplatin by promoting cell apoptosis via the Bcl-2/Caspase-3 signaling pathway.2.The expression of miR-196a-5p was higher in chemo-resistant tissues and cells of ovarian cancer.Linc00312 could reverse the chemotherapy resistance of ovarian cancer to cisplatin by inhibiting miR-196a-5p.3.Compared with the chemo-resistant tissues and cells,ZMYND11 was significantly lower than that in chemo-sensitive ones.Its lower expression had no correlation with age,FIGO stage,tumor differentiation and lymph node metastasis of ovarian cancer patients.Linc00312 could regulate the target gene ZMYND11 through miR-196a-5p,reversing the chemo-resistance of ovarian cancer to cisplatin.