Mechanism Research Of LncRNA WDFY3-AS2 Promotes Cisplatin Resistance In Ovarian Cancer

Background: Ovarian cancer is the third most common gynecologic malignancy with the highest incidence and mortality rate.Although most patients are sensitive to initial treatment,70-80% of patients experience recurrence within the subsequent 1-2years and eventually develop platinum-resistant recurrent ovarian cancer with a 5-year survival rate of less than 40%.Existing targeted and chemotherapeutic regimens have prolonged the survival of ovarian cancer patients to a certain extent,but there are still problems such as drug resistance and adverse effects,so exploring new therapeutic modalities is the key to current research.Tumor stem cells(CSCs)mediate the development of drug resistance in ovarian cancer,and long noncoding RNA(lnc RNA)plays an important role in regulating the stemness-mediated drug resistance.In this study,based on transcriptome sequencing(RNA-seq),we revealed that lnc RNA WDFY3-AS2 was differently transcribed in platinum-resistant and platinum-sensitive ovarian cancers,and explored its mechanism of mediating platinum-resistance and tumor stemness in ovarian cancers in vivo and in vitro.And verified the regulatory effect of cinobufotalin on lnc RNA WDFY3-AS2.Which will provide a new direction for the clinical treatment of platinum-resistant recurrent ovarian cancer.Materials and methods: In the first part of the study,total RNA transcripts of platinum-resistant and platinum-sensitive ovarian cancers in the GEO database were retrieved and included in the dataset to obtain lnc RNA expression profiles.The differentially expressed lnc RNAs were screened and extracted using a range condition of P < 0.05,|log2 Fold Change| ≥ 1.Venn Diagram was drawn to determine the overlapping genes among the three.Single-gene ROC curves were plotted using tumor gene expression profile data from the TCGA database.The levels of lnc RNA WDFY3-AS2 in A2780/A2780-DDP cells and platinum-sensitive/platinum-resistant ovarian cancer tissues were verified by q RT-PCR.In the second part of the study,the viability of A2780,A2780-DDP cells was evaluated by CCK-8 assay.q RT-PCR was used to detect the levels of lnc RNA WDFY3-AS2,mi R-139-5p and SDC4 in A2780-DDP cells.Flow cytometry was performed to detect the level of apoptosis and the number of CD44+,CD166+ positive cells.transwell assay was performed to detect the effect of lnc RNA WDFY3-AS2 on migration and invasion of A2780-DDP cells.Tumor spheroid assay was performed to enrich ovarian CSCs in A2780-DDP cells.protein blotting assay(WB)was performed to detect the expression of SDC4 and CSCs markers SOX2,OCT4,and Nanog.the RNA pull down assay,WB,and dual-luciferase reporter gene assay confirmed the regulatory relationship between RNAs.In addition,nude mouse xenograft tumors were utilized to assess the effect of lnc RNA WDFY3-AS2 on cisplatin resistance in ovarian cancer in vivo.In the third part of the study,the effects of cinobufotalin on the viability and cisplatin resistance of A2780-DDP cells were evaluated by CCK-8 assay.q RT-PCR was used to detect the levels of lnc RNA WDFY3-AS2,mi R-139-5p,and SDC4 in A2780-DDP cell lines.transwell assay was used to detect the effects of lnc RNA WDFY3-AS2 on the migration and invasion of A2780-DDP cells.Tumor ball formation assay was performed to detect the enrichment of ovarian CSCs in A2780-DDP cells.Flow cytometry was performed to detect the number of CD44+,CD166+ positive cells in the tumor spheres.WB was performed to detect the expression of SDC4 and CSCs markers SOX2,OCT4,Nanog.Results: In the first part,by searching the included GSE51373,GSE176218,GSE198042 datasets,the analysis results showed that there were multiple differentially expressed lnc RNAs in platinum-resistant ovarian cancers versus platinum-sensitive ovarian cancers,among which WDFY3-AS2,MNX1-AS1,LINC01224 were differentially expressed.Only Lnc RNA WDFY3-AS2 showed consistent up-regulation in all expression profiles,with a statistically significant difference of P value < 0.05.Therefore,lnc RNA WDFY3-AS2 was selected in this study for subsequent further study.The q RT-PCR assay was performed to show the up-regulation of lnc RNA WDFY3-AS2 levels in platinum-resistant ovarian cancer cells and tissues,which was consistent with the results of the biosignature analysis.In the second part,lnc RNA WDFY3-AS2 was highly expressed in ovarian cancer A2780-DDP cells.silencing WDFY3-AS2 significantly attenuated the proliferation,migration and invasion ability of A2780-DDP cells,and promoted apoptosis,down-regulated the phenotype of CSCs,and inhibited the sphere-forming ability and stem-cell properties of A2780-DDP cells.Based on bioinformatics analysis,it was hypothesized that lnc RNA WDFY3-AS2 regulates SDC4 expression by competitively inhibiting hsa-mi R-139-5p in ovarian cancer cells.Upon silencing of WDFY3-AS2 or up-regulation of mi R-139-5p,SDC4 expression was decreased,which inhibited the proliferation,migration,invasion,and formation of tumor spheres in ovarian cancer cells,increased cisplatin sensitivity,and promoted apoptosis of tumor cells.Subcutaneous xenograft tumors were constructed in nude mice for in vivo validation,and transfection of si-WDFY3-AS2 inhibited tumor growth in vivo.mi R-139-5p inhibitor or overexpression of SDC4 restored the inhibitory effect of silencing lnc RNA WDFY3-AS2 on tumor growth.In the third part,cinobufotalin enhanced A2780-DDP sensitivity to cisplatin,inhibited invasive migration ability,down-regulated stem cell phenotype,and limited spheroidogenic ability.Cinobufotalin down-regulated the level of lnc RNA WDFY3-AS2 in A2780-DDP,modulated the expression of mi R-139-5p/SDC4 axis,and enhanced the regulation of cisplatin resistance and tumor stemness by si-WDFY3-AS2.Conclusion: In this study,we revealed the differences in lnc RNA transcripts in platinum-resistant and platinum-sensitive ovarian cancers based on RNA-seq.lnc RNA WDFY3-AS2 expression was significantly up-regulated in platinum-resistant ovarian cancers,which was in agreement with the results of ex vivo and in vivo characterization.Further studies explored that lnc RNA WDFY3-AS2 induces cisplatin resistance and promotes ovarian CSCs characterization in vitro and in vivo by regulating the expression of mi R-139-5p / SDC4 axis in platinum-resistant ovarian cancers.Cinobufotalin has the effect of reversing A2780-DDP cisplatin resistance to inhibit tumor stemness,and it inhibits the proliferation,invasion and metastatic ability of A2780-DDP by regulating the expression of lnc RNA WDFY3-AS2 affecting the mi R-139-5p / SDC4 axis.Lnc RNA WDFY3-AS2 is a potential target for reversing platinum resistance and cinobufotalin can modulate lnc RNA WDFY3-AS2 for the treatment of platinum-resistant recurrent ovarian cancer,which is of great clinical value.