LncRNA WDFY3-AS2 promotes cisplatin resistance and the cancer stem cell in ovarian cancer by regulating hsa-miR-139-5p/SDC4 axis

Background Ovarian cancer (OC) is a high-mortality gynecological cancer that is typically treated with cisplatin, although such treatment often results in chemoresistance. Ovarian cancer resistance is usually related to cell stemness. Herein, we explored the function of lncRNA WDFY3-AS2 in OC cell resistance to cisplatin (DDP). Methods Cisplatin resistant OC A2780 cell lines (A2780-DDP) were established by long-term exposure to cisplatin. CCK-8 assay were performed to evaluate the viability of A2780, and A2780-DDP cells. Quantitative RT-PCR was used to examine the expression of lncRNA WDFY3-AS2, miR-139-5p, and SDC4 in A2780-DDP cell lines. After treatment with cisplatin, cell apoptosis and CD44+CD166+-positive cells were measured by flow cytometry. The transwell assays were employed to measure the effect of WDFY3-AS2 on cell migration, and invasion. In addition, tumorsphere formation assay was used to enrich OC cancer stem cells (CSCs) from A2780-DDP cells. The expression of CSC markers (SOX2, OCT4, and Nanog) was detected by western blotting. The regulatory mechanism was confirmed by RNA pull down, and luciferase reporter assays. Furthermore, xenograft tumor in nude mice was used to assess the impact of WDFY3-AS2 on cisplatin resistance in OC in vivo. Results WDFY3-AS2 was highly expressed in OC A2780-DDP cells, and silencing WDFY3-AS2 significantly inhibited proliferation, migration and invasion but increased apoptosis in OC A2780-DDP cells. Additionally, WDFY3-AS2 significantly promoted the A2780-DDP cells tumorspheres. WDFY3-AS2 was predicted to impact OC by sponging miR-139-5p and regulating SDC4. The xenografts inoculated with A2780-DDP cells additionally confirmed that tumor growth in vivo was reduced by si-WDFY3-AS2 transfection. MiR-139-5p inhibitor or SDC4 overexpression could restore the suppressive influence of silenced WDFY3-AS2 on tumor growth. Conclusions Together, WDFY3-AS2 may lead to change of cisplatin resistance by the expression of miR-139-5p/SDC4 in the OC A2870-DDP cells both in vitro and in vivo. Our finding may provide a drug target for the drug resistance of OC. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-021-01993-x.

clinically treating OC [5]. The prognosis of OC is very poor on account of late diagnosis and limited effective treatment; it is mainly because of chemotherapy resistance post-surgery [6]. The mechanisms governing OC therapeutic resistance thus warrant further study.
Cancer stem cells (CSCs) are important drivers of therapeutic resistance in many tumor types [7], and represent a population of tumor cells that exhibit stem-like properties including the ability to differentiate and undergo selfrenewal, thus allowing them to support sustained tumor growth and heterogeneity [8,9]. Owing to their properties, CSCs are thought to be important drivers of tumor metastasis and other malignant processes [10,11], and developing approaches to eliminating these cells is therefore vital to decrease the odds of OC therapeutic resistance or relapse [12].
The molecular networks are found to provide targeted approaches to treating OC development [13]. Non-coding RNAs including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) have been characterized in recent years as key regulators of gene expression and may be viable targets of therapeutic intervention in OC [14]. LncRNAs are > 200 nt long and do not encode proteins [15], yet can post-transcriptionally regulate diverse biological processes in many forms of cancer [16][17][18]. Increasing evidences have shown lncRNAs to control invasion, metastasis, differentiation, apoptosis, cell cycle progression, and tumor development [19][20][21][22]. An increasing number of lncRNAs, including lncRNA MEG3, lncRNA UCA1, and lncRNA LINC00511, are related to OC onset and progression [23][24][25]. The previous report revealed that WDFY3-AS2 suppressed ovarian cancer progression by sponging miR-18a [26], WDFY3-AS2 may suppress the proliferation and invasion in oesophageal squamous cell carcinoma by regulating miR-2355-5p/SOCS2 axis [27], and WDFY3-AS2 also was reported to promote lung adenocarcinoma progression via targeting miR-491-5p/ZNF703 axis [28]. These findings highlighted the role of WDFY3-AS2 in tumour development and progression, however, too little is known regarding the role of WDFY3-AS2 in cisplatin resistance of OC. Also, the understanding of their mechanisms in the tumor stem cell is inadequate. These topics thus warrant further detailed study.
In light of these prior reports, this study sought to explore the modulatory effects of lncRNA WDFY3-AS2 in OC drug resistance. Since lncRNAs can usually be used as miRNA molecular sponges to regulate miRNA target gene expression, in this experiment, the ceRNA mechanism was used as the main mechanism for mediating lncRNA WDFY3-AS2 to regulate drug resistance in OC. In our study, we also demonstrate that lncRNA WDFY3-AS2 function as a sponge of hsa-miR-139-5p which in turn regulates the expression of SDC4 on tumorigenic function of CSCs and will provide crucial information for its application for the treatment of OC.

Transwell assays for migration and invasion
Transwell assays were performed by using transwell chambers (Corning, USA). After 48 h transfection, OC A2780-DDP treated with 20 μΜ cisplatin, cells migration and invasion were detected as described in the past [26].

Flow cytometry for cell apoptosis
An Annexin V-FITC kit (Biosea Biotechnology Co., Beijing, China) was used as previously described [31] to measure cell apoptosis.

Tumorsphere formation assay
The transfected cells with 20 μM cisplatin 48 h treatment were cultured in serum-free DMEM/F12 containing 20 ng/mL EGF, 20 ng/mL bFGF, and B27. Following plating into 24-well ultra-low attachment plates (5000 cells/ well), growth media was changed every 2 days and light microscopy (Nikon, Japan) was used to monitor tumorspheres.

Cell surface marker analysis (CD44, CD166)
The CD44 and CD166 antibody were obtained from Beijing biosynthesis biotechnology CO., LTD. The transfected cells after treatment with 20 μM cisplatin were cultured in serum-free medium (SFM). After a 7-day period, the number of CD44 + and CD166 + positive cells was assessed via flow cytometry assay. Detection of CD44 + and CD166 + positive cells was carried out according to provided directions.

In vivo tumorigenesis assay
All BALB/C female nude mice (6 weeks old) were purchased from the Changzhou card Vince laboratory animal Co., Ltd (SCXK(Su) 2011-0003, Changzhou, China). Centre (ARASC), Health Campus, Universiti Sains Malaysia (USM). Mice were housed in specific pathogenfree facility with a 12 h light cycle and food and water ad libitum in Anhui Medical University. Animals studies were consistent with the NIH Guide for the Care and Use of Laboratory Animals and received approval from the Animal Care and Use Committee of the First Affiliated Hospital of Anhui Medical University. The A2780-DDP cells (3 × 10 6 , 200 μL) transfected with si-WDFY3-AS2, si-WDFY3-AS2 + inhibitor-NC, or si-WDFY3-AS2 + miR-139 inhibitor, si-WDFY3-AS2 + pcDNA-NC, si-WDFY3-AS2 + SDC4, and control were subcutaneously implanted in the right flank of nude mice (9 mice per group). Subsequently, cisplatin, at a dose of 2.5 mg/ kg/2 days, was administrated into the abdominal cavity of the nude mice. Every 7 days, the growth of these tumors was assessed, with volume being quantified as follows: volume = (length × width 2 )/2 (mm 3 ). Mice were anesthetized by exposure to 1-3% isoflurane 4 weeks following tumor implantation, at which time these tumors were collected, weighed, and imaged.

Statistical analysis
GraphPad Prism 7.0 (GraphPad, CA, USA) and SPSS 22.0 (SPSS Inc. IL, USA) were used for analyzing data, which are given as means ± SD (standard deviation) and were compared via one-way ANOVAs with Tukey's post hoc tests. All experiments were carried out in triplicate, and P < 0.05 was the significance threshold.

The levels of lncRNA WDFY3-AS2 in OC tissues and cells with different levels of cisplatin sensitivity
qRT-PCR revealed that WDFY3-AS2 expression patterns in OC cell line were as follows: the expression of WDFY3-AS2 in A2780-DDP cells was significantly higher than that in A2780 cells (Fig. 1A, P < 0.001), suggesting higher WDFY3-AS2 levels in cisplatin-resistant OC cells compared to cisplatin-sensitive OC cells. Furthermore, to determine whether lncRNA WDFY3-AS2 was involved in cisplatin resistance in OC, firstly, CCK-8 assay showed that in cisplatin-resistant cells (A2780-DDP) and A2780 cells, after cisplatin treatment with an increasing concentration (0, 5, 10, 15, 20, 25, 30 μM), the viability of cisplatin-sensitive cells was reduced with the increase of cisplatin concentration. Cisplatin-resistant cells had no significant changes in cell viability under the treatment of 0-20 μM cisplatin, whereas the viability of cisplatin-resistant cells was decreased under the treatment of 20-30 μM (P < 0.001, Fig. 1B). A 20 μM cisplatin dose was thus used in subsequent analyses of A2780 cells.
To additionally assess the function of WDFY3-AS2 in cisplatin-resistant OC cell migration and invasion, Transwell assays were conducted. A2780-DDP cells after cisplatin treatment (20 μM). The data showed that WDFY3-AS2 significantly enhanced both invasion and migration (P < 0.001); in contrast, si-WDFY3-AS2 overexpression markedly suppressed cell invasion and migration (P < 0.001, Fig. 2D, E). Taken together, these data indicate that WDFY3-AS2 could induce resistance to cisplatin in OC A2780-DDP cells.

The impact of WDFY3-AS2 on CSCs of OC
To examine whether WDFY3-AS2 was able to suppress OC CSCs, we treated the transfected sphere-forming cells with 20 μM concentrations of cisplatin. Seven days post-treatment, WDFY3-AS2 overexpression promoted the formation of A2780-DDP tumorspheres; in contrast, si-WDFY3-AS2 significantly inhibited the formation tumorspheres (Fig. 3A). In addition, we examined the expression of stem cell marker proteins SOX2, OCT4 and Nanog by Western blot analysis. After 7 days of SFM culture, A2780-DDP sphere-forming cells with the WDFY3-AS2 transfection expressed markedly higher levels of CSC markers including the SOX2, OCT4 and Nanog (all P < 0.001); while si-WDFY3-AS2 transfection could decrease the protein levels of SOX2, OCT4 and Nanog (P < 0.01, P < 0.001, Fig. 3B). Furthermore, we also detected the CD44 + , CD166 + -positive population in A2780-DDP sphere-forming cells. As shown in Fig. 3C, WDFY3-AS2 overexpression significantly induced the number of CD44 + , CD166 + -positive cells in A2780-DDP tumorspheres (P < 0.001). These above data noted that WDFY3-AS2 seemed to be more efficient in the promotion of A2780-DDP cells tumorspheres.

Discussion
OC is a gynecological tumor, and most advanced OC patients undergo combination surgical and platinumbased chemotherapeutic treatment using first-line agents such as cisplatin or carboplatin [32][33][34][35]. However, drug resistance often leads to therapy failure. In our study, we sought to explore the functions of lncRNA WDFY3-AS2 on cisplatin resistance in OC. We found that overexpression of the WDFY3-AS2 increased cell viability, migration, and invasion, inhibited apoptosis, as well as OC CSC traits induction, as shown by induced tumorsphere formation, CD133-positive cell numbers, and the expression levels of CSC markers both in vitro and in vivo. Moreover, we demonstrated that lncRNA WDFY3-AS2 sponged miR-139-5p to downregulate its expression, and WDFY3-AS2 increased SDC4 expression by sponging miR-139-5p. These findings suggested that WDFY3-AS2 influenced cisplatin resistance and OC-CSCs via regulation of miR-139-5p/SDC4 axis.
The dysregulation of lncRNAs is a common feature of OC wherein they can influence malignant cancer cell phenotypes [36]. WDFY3-AS2 expression level was reportedly low in both OC cells and tissues [26]. Also WDFY3-AS2 has been identified as a potential prognostic biomarker of diffuse glioma given that its overexpression in this cancer type is linked to longer patient OS [37]. The upregulation of WDFY3-AS2 has also been detected in lung adenocarcinoma (LUAD), with the knockdown of this lncRNA being sufficient to impair the proliferative, migratory, and invasive activity of LUAD cells while promoting their apoptotic death [28]. The role of WDFY3-AS2 in OC chemoresistance has not been assessed previously. Herein, we found that WDFY3-AS2 expression was increased in cisplatin resistant A2780-DDP OC cells compared to A2780 cells. si-WDFY3-AS2 inhibited the proliferative, migratory, invasive, and tumorsphere formation of OC A2780-DDP cells, while driving their apoptotic death. These results suggested that WDFY3-AS2 is a key regulator of the chemoresistance of OC. However, our observation was in contrast to prior findings that lncRNA WDFY3-AS2 suppresses tumor progression in ovarian cancer [26]. We speculate that this may be due to the specificity of ovarian tissue chemotherapy. The mechanisms of their associations require investigation in future studies.
In general, cytoplasmic lncRNAs can control gene expression via a ceRNA mechanism [38], and WDFY3-AS2 is expressed at high levels in the cytoplasm [26].
Through several experiments we found that WDFY3-AS2 sponged miR-139-5p and thereby downregulated it, while WDFY3-AS2 could upregulate SDC4 by sponging miR-139-5p. Many cancers exhibit the downregulation of miR-139-5p, which can in turn suppress the malignant properties of many cancer cells [39,40]. Such miR-139-5p downregulation has been linked to multi-drug chemoresistance in breast, colorectal cancer [41,42] and OC [43]. In this present study, we also found that significant miR-139-5p downregulation was evident in cisplatin-resistant OC A2780-DDP cells relative to their parental A2780 cells. The miR-139-5p inhibitor reversed the cisplatin resistance of OC induced by si-WDFY3-AS2 in vitro and in vivo. These data thus indicated that overexpressing WDFY3-AS2 regulated the cisplatin resistance in OC via targeting miR-139-5p.
CSCs are key mediators of oncogenesis in humans [44]. Although CSCs represent only about 2-5% population of cells in the tumor, they are reported to be chemoresistant and result in tumor relapse and recurrence [45,46]. CSCs are characterized by their ability to for 3D spheres in serum-free media, and such spheroid-forming cell populations are considered to by CSC-enriched [47]. SDM tumorpshere formation can therefore be measured to readily assess the biology of CSCs [48]. CSCs are also distinguished by specific cell markers. SOX2, OCT4 and Nanog are considered pluripotent genes and stem cell markers to regulate CSC activity [49,50]. CD44 and CD166 were reported as markers for adult stem cells [51,52]. Herein, we employed a tumorsphere formation assay to enrich for A2780-DDP CSCs, revealing that these cells grown in SFM were able to form these spheroid structures and expressed higher levels of known CSC marker proteins (SOX2, OCT4, and Nanog) in addition to expressing higher levels of CD44/CD166. And we also (See figure on next page.) Fig. 7 LncRNA WDFY3-AS2 induced the traits of CSCs by regulating miR-139-5p/SDC4. A Representative A2780-DDP sphere-forming cell images were acquired (100 μm). The transfected A2780-DDP cells were treated for 7 days with cisplatin (20 μM). B Western blotting was performed to assess CSC markers protein expression including the SOX2, OCT4 and Nanog in A2780-DDP sphere-forming cells. C CD44 + , CD166 + -positive cells in A2780-DDP sphere-forming cells detected via flow cytometry. D The weight of transplanted tumors in nude mice injected with si-WDFY3-AS2, miR-139-5p inhibitor or SDC4. E Assessment of tumors in nude mice. F The volume curve of xenograft tumors. ***P < 0.001 vs. control, ### P < 0.001 vs. si-WDFY3-AS2 + inhibitor NC, &&& P < 0.001 vs. si-WDFY3-AS2 + pcDNA-NC found that the properties of CSCs in these cells were regulated by WDFY3-AS2 via miR-139-5p/SDC4 axis.

Conclusions
In summary, these results indicated that WDFY3-AS2 may facilitate cisplatin resistance via the expression of miR-139-5p/SDC4 in the OC A2870-DDP cells in vitro and in vivo. Additionally, si-WDFY3-AS2 could lead to the decrease of the cell proliferation, migration, invasion and CSC-like state, and apoptosis induction in OC A2870-DDP cells. Our data thus highlight a novel target that may be modulated to overcome chemoresistance in OC.