LncRNA LINC00665 Promotes Ovarian Cancer Cell Proliferation and Inhibits Apoptosis via Targeting miR-181a-5p/FHDC

Previous reports indicate that long intergenic non-coding RNA LINC00665 naturally occurred vital effects in various cancers. Herein, the role of LINC00665 in ovarian cancer progress was explored. We found that LINC00665 was upregulated in ovarian cancer cell lines. Besides, a series of assays including flow cytometry, wound-healing, transwell, cell counting Kit-8 (CCK-8), and EdU assay confirmed that the knockdown of LINC00665 could reduce the viability, proliferation, and migration of SKOV-3 and OVCAR-3 cells. Accumulating evidence indicates that many lncRNAs can function as endogenous miRNA sponges by competitively binding common miRNAs. In this study, the bioinformatics analysis suggests that LNC00665 specifically binds to miR-181a-5p. LINC00665 downregulated the miR-181a-5p in SKOV-3 and OVCAR-3 cells. The knockdown of miR-181a-5p evidently reverses the inhibitory effect of sh-LINC00662. Besides, FH2 domain containing 1 (FHDC1) has been proved to deed as an effective target of miR-181a-5p. The results reveal the knockdown of LINC00665 facilitates ovarian cancer via development by sponging miR-181a-5p and up-regulating FHDC1 expression. These may contribute to ovarian cancer therapy.


Introduction
Ovarian cancer (OC) is one of the highest lethal tumors worldwide [1]. In the USA, there were about 22,000 new cases diagnosed and 14,000 deaths related to ovarian cancer in 2018 [2]. In China, there were about 25,000 ovarian cancer death cases in 2015 [3]. Although surgery, chemotherapy, and radiotherapy have made significant progress in the treatment of OC, advanced OC patients with distant metastasis or recurrence remain incurable, and the median overall survival (OS) time of patients with metastatic disease has not been improved in the past decades [4]. It is urgently needed to develop an effective strategy to improve the early detection of OC. However, despite increasing reports on the molecular mechanisms of OC development, it is little known about the exact molecular mechanisms of ovarian cancer pathogenesis.
Long non-coding RNAs (lncRNAs) are the long intergenic non-coding RNA which naturally occurs without coding proteins [5]. During the past few decades, a great number of reports have disclosed that lncRNAs can act as scaffolds or sponges to modulate gene expression [6] so as to play some vital roles in cell proliferation, differentiation, or apoptosis [7,8]. Clear evidence provided by numerous studies that the aberrant lncRNAs play oncogenic or tumor-suppressive roles in ovarian cancer [9,10]. The dysregulation of lncRNAs was expected to become a potential prognostic and diagnostic biomarker, and a therapeutic target for ovarian cancer treatment. LINC00665 was first identified in oral premalignant lesions [11]. Then LINC00665 was observed in several other tumors, including hepatocellular carcinoma (HCC) [12] and lung adenocarcinoma [13].
Although LINC00665 was reported to promote several kinds of human tumor progression, its involvement in ovarian cancer remained unknown. In this study, the important role of LINC00665 was elucidated as an oncogene to promote ovarian cancer progression. First, we showed the function of LINC00665 in ovarian cancer cells. Then, a series of experiments were carried out to demonstrate that LINC00665 upregulated FHDC1 expression by sponging miR-181a-5p. All results indicated that LINC00665 might take part in the inactivation of the miR-181a-5p-FHDC1 pathway. These may contribute to the novel ovarian cancer therapy.

Cell culture
Human ovarian epithelial cell line (ISOE80) and human ovarian cancer cell lines (SKOV-3, A2780, OVCAR-3 and HO8910) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). After resuscitation, ISOE80 was cultured in a DMEM medium, while other cells were cultured in a RPMI-1640 medium containing 10% fetal bovine serum (FBS, Beyotime, Beijing, China) in a humidified environment. When cells attained the confluence between 70 and 80%, transfection experiments could be conducted.

Cell Transfection
The LINC00665-targeting shRNA sequences were designed and inserted into pENTR/U6 plasmids to construct LINC00665 shRNA. The NC mimic and miR-181a-5p mimic were purchased from Ribobio Corporation (Guangzhou, China). Cell transfection was performed by cell electroporation system operator H1 (Suzhou Etta Biotech Co. Ltd., Suzhou, China.) according to the manual. Cell transfection efficiency was measured by qRT-PCR at 48 h post-transfection.

CCK8 Assay
After cell transfection, SKOV-3 and OVCAR-3 were seeded into 96-well plates at 5 × 10 3 per well. After incubated for 0 h, 24 h, 48 h, and 72 h, 10-μL CCK8 solution were added and incubated for 2 h in the dark. Then absorbance at 450 nm was determined using a Vari-oskanTM LUX microplate reader (Thermo Fisher Scientific).

EdU Assay
EdU assay was conducted as described [14]. Briefly, after the electroporation treatment, 5 × 10 3 SKOV-3 and OVCAR-3 cells were seeded into each well of the 96-well plates and cultured for another 72 h. Then, the cells were incubated with 10-μM EdU for 4 h and stained DAPI. Finally, the EdU-positive cells were analyzed by fluorescence microscope (Leica, Hilden, Germany) at 200 × , and densitometric analyzed the percentage using ImageJ (Bethesda, MD, USA).

Cell Cycle and Cell Apoptosis
After cell electroporation, SKOV-3 and OVCAR-3 cells were seeded into 6-well plates at 5 × 10 5 per well and cultured cells for the next 72 h. Then, the tumor cells were gained for cell cycle and apoptosis analysis.
For cell cycle analysis, the cells were incubated in ice-cold ethanol for 2 h and then processed with RNase A (0.2 mg/mL, Sigma-Aldrich). Next, the cells were incubated with propidium iodide (2 µL) at room temperature for 40 min. Finally, the cell cycle detection was analyzed.
For analysis, the tumor cells with indicated treatments were washed once with PBS. Then, the cells were resuspended in 100-µL binding buffer and incubated with 10 µg/mL Annexin V-fluorescein isothiocyanate and 10 µg/mL PI (both Sigma-Aldrich) at room temperature for 30 min. Apoptosis analysis was conducted using a FACScan flow cytometer (Becton Dickinson).

Wound Healing Assay
To study the migration of SKOV-3 and OVCAR-3 cells transfected with different reagents, the wound-healing assay was conducted as described [15]. Briefly, 5 × 10 5 transfected SKOV-3 or OVCAR-3 cells were seeded into each well in a 6-well plate. When the cells were cultured to 90% confluence, a wound was made by a 100-μl size pipette tip. After another 48 h, the wound recovery area was evaluated under a light microscope.

Transwell Assay
To detect the capacity of migration, the Transwell assay was conducted as described [16]. Briefly, 3 × 10 4 transfected SKOV-3 or OVCAR-3 cells were seeded into the upper chamber of Matrigel-precoated transwell and cultured in RPMI-1640 medium. The lower chamber contained a 10% FBS growth medium. After 48 h of culture, the transwell cells present in the lower chamber were fixed and stained with crystal violet. The pictures were taken under a light microscope, and individual cell colonies were counted.

Luciferase Reporter Assay
The Dual-Luciferase system was generated by inserting the cDNA fragments containing the putative miR-181a-5p binding site from LINC00665 or FHDC1 3'-UTR into pmirGLO Dual-Luciferase miRNA target expression vectors (Promega, Madison, WI, USA). PmirGLO/LINC00665 or pmirGLO/ FHDC1 3'-UTR constructs along with miR-181a-5p mimics were co-transfected into SKOV-3 and OVCAR-3 cells. Then the cells were seeded into a 24-well plate to culture for about 48 h. Finally, the cells were lysed to measure the Dual-Luciferase Reporters' luciferase activity according to the manufacturer's protocol.

Immunofluorescence Staining
After cell transfection, SKOV-3 and OVCAR-3 cells were seeded in 96-well plates at 5 × 10 3 per well and cultured for the next 72 h. Then, the cells were fixed in 4% paraformaldehyde and permeabilized with xylene. Blocked with 5% BSA in PBS, the cells were immunolabeled with primary antibody, FHDC (1:100; Proteintech), and then incubated with FITC-conjugated secondary antibody after being washed by xxx. The nuclei were counterstained using DAPI (Invitrogen), and the cells were observed under a fluorescence microscope (Olympus, Japan).

Western Blot
The cultured cells were gained in a lysis buffer and incubated on ice for 30 min. The supernatant was collected by centrifugation with 15,000 g for 5 min at 4 °C. Then the proteins (40 μg) were separated on 12% SDS-PAGE and transferred into PVDF membranes (Millipore) using a MiniGenie blotting system (Bio-Rad). Next, the membranes were blocked for 1 h with TBST containing 1% skimmed milk powder and then incubated overnight at 4 °C with rabbit antibodies against human FHDC1 (1:1000; cat. ab49822; Abcam), Cleaved Caspase-9 (1:1000; cat. no. 20750S; Cell Signaling Technology), or GAPDH (1:1000; cat. no. 5174S; Cell Signaling Technology). Next, the membranes were washed with TBST and incubated with goat-anti-rabbit secondary antibody (1:10,000; cat. no. 14708S; Cell Signaling Technology). Finally, the protein bands on the membranes were visualized with an enhanced chemiluminescence (ECL) system. Densitometric analysis of the bands was conducted using the ImageJ.

Statistical Analysis
All experiments were conducted in triplicates. GraphPad Prism (Version 6.01 for Windows) statistical software was used to conduct statistical analysis. Student t tests were employed to identify the significant differences between groups. One-way ANOVA and Tukey test were used to identify differences among three or more groups. Statistical significance difference was set at p < 0.05.

LINC00665 Is Upregulated in Ovarian Cancer Cell Lines
First of all, to confirm whether LINC00665 took part in ovarian cancer, we employed RT-qPCR to compare its expression level in different human ovarian cancer cell lines (SKOV-3, A2780, OVCAR-3, and HO8910) and human ovarian epithelial cell line (ISOE80). The results demonstrated that the LINC00665 level was significantly upregulated in SKOV-3, A2780, OVCAR-3, and HO8910 cell lines compared with the control ISOE80 cell line (Fig. 1A). As the expression level of LINC00665 in SKOV-3 and OVCAR-3 was highest among the human ovarian cancer cell lines, we chose SKOV-3 and OVCAR-3 as cell models to explore the biological function of LINC00665 in vitro.

Knockdown of LINC00665 Inhibits Ovarian Cancer Cell Proliferation and Promotes the Apoptosis
To further explore the role of LINC00665 in the progression of ovarian cancer, we transfected sh-RNA targeting LINC00665 (sh-LINC00665) into SKOV-3 and OVCAR-3 cells. RT-qPCR showed that sh-LINC00665 significantly downregulated the expression of LINC00665 in SKOV-3 and OVCAR-3 cells (Fig. 1B, p < 0.01). CCK-8 assay showed that sh-LINC00665 remarkably reduced the viability of SKOV-3 and OVCAR-3 cells as compared with a negative control (sh-NC) (Fig. 1C, p < 0.01), while EdU assay showed that sh-LINC00665 remarkably reduced the EdU-positive cells compared with a negative control (sh-NC) in both SKOV-3 and OVCAR-3 cells (Fig. 1D, p < 0.01). Next, the data from the flow cytometry assay indicated that sh-LINC00665 prominently increased the percentage of cells at the G0/G1 (p < 0.01) and decreased the percentage of S and G2/M phase in SKOV-3 and OVCAR-3 cells (Fig. 1E, p < 0.05).
Meanwhile, the result of western blot showed that sh-LINC00665 remarkably reduced the expression of Cyclin D1 and increased the expression of p21 in SKOV-3 and OVCAR-3 cells (Fig. 1F, p < 0.01). Besides, the flow cytometry analysis showed that knocking down LINC00665 observably facilitated ovarian cancer cell apoptosis (Fig. 1G, p < 0.01). Additionally, the western blot analysis showed that sh-LINC00665 upregulated the protein levels of Bax, Cleaved Caspase-3, and Cleaved Caspase-9 and down-regulated the protein level of Bcl-2 in SKOV-3 and OVCAR-3 cell (Fig. 1H, p < 0.01).

Knockdown of LINC00665 Inhibits Ovarian Cancer Cell Migration and Invasion
Subsequently, the effect of LINC00665 on cell invasion and migration was examined using wound healing assay and transwell assay. The wound-healing assay showed that the reduced level of LINC00665 remarkably repressed the migratory ability of (H) Western blot analysis was used to assess the expression of cell apoptosis-related proteins. Three independent experiments were conducted. The graph showed the mean ± SD calculated for at least three experiments. *P < 0.05, **P < 0.01 SKOV-3 and OVCAR-3 cells compared with the control (Fig. 2A, p < 0.01). The transwell assay showed that sh-LINC00665 markedly inhibited the invasion of SKOV-3 and OVCAR-3 cells compared with sh-NC (Fig. 2B, p < 0.01). Additionally, the western blot was explored to measure the cell migration-related proteins. The result showed that sh-LINC00665 remarkably downregulated the expression levels of MMP-2 and MMP-9 in SKOV-3 and OVCAR-3 cells compared with sh-NC (Fig. 2C, p < 0.01). Taken together, these findings demonstrated that LINC00665 promoted ovarian cancer cells proliferation, migration, and invasion and inhibited cell apoptosis in vitro.

Fig. 2 LINC00665 knockdown inhibits migration and invasion of SKOV-3 and OVCAR-3 cells. Wound healing assay (A) and transwell assay (B) were conducted to assess the migration and invasion of SKOV-3 and OVCAR-3 cells transfected with sh-LINC00665 or sh-NC. (C)
Western blot analysis was used to assess the expression of MMP-2 and MMP-9 proteins. Three independent experiments were conducted. The graphs showed the mean ± SD of at least three experiments. * P < 0.05, **P < 0.01 of the LINC00665-WT reporter vector (Fig. 3C). The downregulation of the luciferase activity of the LINC00665 reporter vector was abrogated when the predicted binding site was mutated (Fig. 3C). Moreover, our results showed that LINC00665 knockdown upregulated miR-181a-5p expression in SKOV-3 and OVCAR-3 cells (Fig. 3D). In addition, the result of RT-qPCR demonstrated that the expression level of miR-181a-5p was significantly lower in SKOV-3, A2780, OVCAR-3, and HO8910 cell lines compared with the control ISOE80 cell line (Fig. 3E). Overall, these results demonstrated that LINC00665 directly interacted with miR-181a-5p and influenced its expression in ovarian cancer cells. The binding region between miR-181a5p and LINC00665 was predicted, and LINC00665-Mut sequences were constructed. (B) RT-qPCR was taken to assess the expression of miR-181a-5p in OVCAR-3 and SKOV-3 cells transfected with miR-181a-5p mimic or NC mimic. (C) The luciferase reporter constructs containing the wild type (LINC00665-WT) or mutant LINC00665 (LINC00665-Mut) sequences were co-transfected into OVCAR-3 and SKOV-3 cells with miR-181a-5p mimic or NC mimic, followed by detecting of the relative luciferase activity. RT-qPCR was taken to assess the expression of miR-181a-5p in OVCAR-3 and SKOV-3 cells transfected with sh-LINC00665 or sh-NC. (D) RT-qPCR analysis was used to determine the expression of miR-181a-5p in ovarian cancer cells and ovarian epithelial cells. Three independent experiments were conducted. The graph showed the mean ± SD calculated for at least three experiments. *p < 0.05, **p < 0.01

MiR-181a-5p Regulates FHDC1 Expression in SKOV-3 and OVCAR-3 Cells
Next, we explored the target genes of miR-181a-5p. Bioinformatics analysis via Targetscan (http:// www. targe tscan. org) showed that one of the target genes of miR-181a-5p was FHDC1 (Fig. 4A). We conducted a dual-luciferase reporter assay to verify this interaction. The result indicated that the overexpression of miR-181a-5p significantly downregulated the FHDC1-WT reporter vector's luciferase activity (Fig. 4B, p < 0.01). Meanwhile, the overexpression miR-181a-5p with a mutation within the predicted miR-181a-5p binding site did not influence luciferase activity of the FHDC1 reporter vector (Fig. 4B). We determined both the RNA and protein expression level of FHDC1 in the SKOV-3 and OVCAR-3 cells overexpressed miR-181a-5p with RT-qPCR, western blot, and immunofluorescence. The results demonstrated that FHDC1 was significantly downregulated in the SKOV-3 and OVCAR-3 cells overexpressed miR-181a-5p compared with the negative control (Fig. 4C, 4D and 4E). Similarly, the expression level of FHDC1 level was significantly upregulated in SKOV-3, A2780, OVCAR-3, and HO8910 cell lines compared with the control ISOE80 cell line (Fig. 4F). These data confirmed that miR-181a-5p could regulate FHDC1 expression in ovarian cancer. Fig. 4 FHDC1 is a target gene of miR-181a-5p. (A) Targetscan was adopted to predict the target genes regulated by miR-181a-5p. A binding region between miR-181a-5p and FHDC1 was predicted, and a FHDC1-Mut sequence was designed. (B) The luciferase reporter constructs containing the wild type (FHDC1-WT) or mutant FHDC1 (FHDC1-Mut) sequences were co-transfected into OVCAR-3 and SKOV-3 cells with miR-181a-5p mimic or the corresponding negative control (NC mimic), followed by the detection of the relative luciferase activity. RT-qPCR analysis (C), western blot (D), and immunofluorescence (E) were used to detect the expression level of FHDC1 in OVCAR-3 and SKOV-3 cells transfected with miR-181a-5p mimic or NC mimic. (F) RT-qPCR analysis was used to detect the expression level of FHDC1 in ovarian cancer cells and ovarian epithelial cells. Three independent experiments were conducted. The graph showed the mean ± SD calculated for at least three experiments. *P < 0.05, **P < 0.01

MiR-181a-5p/FHDC1 Mediated the Effect of LINC00665 Knockdown on SKOV-3 and OVCAR-3 Cells
To confirm the interaction of miR-181a-5p, FHDC1, and LINC00665 in OC, we cotransfected SKOV-3 cells with sh-LINC00665 or sh-LINC00665 with miR-181a-5p inhibitor or their combinations with sh-FHDC1. RT-qPCR results showed that after transfection of miR-181a-5p inhibitor, the expression of miR-181a-5p in cells was significantly downregulated. However, in sh-LINC00665-transfected cells, FHDC1 was significantly downregulated (Fig. 5A, p < 0.01). CCK8 assay revealed that the cells transfected sh-LINC00665 and NC inhibitor showed lowest cell viability, while the Fig. 5 Compensation experiment reveals the relationship among FHDC1, miR-181a-5p and LINC00665 in SKOV-3 cells. Sh-LINC00665 or sh-NC along with miR-181a-5p inhibitor and sh-FHDC1 were co-transfected into SKOV-3 cells. After transfection, the cells were seeded into appropriate cell culture plates for further assays. (A) RT-qPCR was used to assess the expression of miR-181a-5p in SKOV-3 cells with miR-181a-5p inhibitor or the corresponding negative control (NC inhibitor). (B) RT-qPCR was used to assess the expression of FHDC1 in SKOV-3 cells with sh-LINC00665 or sh-NC. Cell viability of SKOV-3 cell lines was measured employing CCK-8 assay (C) and EdU assay (D). Wound healing assay (E), and transwell assay was (F), which were conducted to assess the migration and invasion of SKOV-3 cells. Three independent experiments were conducted. The graph showed the mean ± SD calculated for at least three experiments. *P < 0.05, **P < 0.01 viability of cells transfected sh-LINC00665 along with miR-181a-5p inhibitor and sh-FHDC1 markedly increased, compared with the cells transfected sh-LINC00665 and miR-181a-5p (Fig. 5B, p < 0.05). Besides, as shown in Fig. 5C and Fig. 5D, the cells transfected sh-LINC00665 and NC inhibitor showed the highest level of cell apoptosis. Downregulation of miR-181a-5p remarkably inhibited the cell apoptosis induced by sh-LINC00665. Meanwhile, sh-FHDC1 markedly reversed the inhibition effect of the miR-181a-5p inhibitor on SKOV-3 cells apoptosis (p < 0.05). Furthermore, the wound healing assay delineated that the downregulation of miR-181a-5p remarkably improved the migration of the SKOV-3 cells with LINC00665 knockdown (Fig. 5E), compared with the cells co-transfected with sh-LINC00665 and NC inhibitor, while sh-FHDC1 markedly reversed the improvement from miR-181a-5p inhibitor (Fig. 5F, p < 0.05). Similarly, the transwell assay delineated that the downregulation of miR-181a-5p markedly aggravated the invasion of SKOV-3 cells with LINC00665 knockdown. And sh-FHDC1 remarkably inhibited such a restoration effect (Fig. 5F, p < 0.05).

Discussion
Ovarian cancer is one of the most common and aggressive malignancies among women around the world. Revealing the molecular mechanisms of ovarian cancer pathogenesis and finding the potential diagnostic biomarkers and therapeutic targets may be some of the best ways to reduce the harm of ovarian cancer to women. In this research, we demonstrated that LINC00665 was upregulated in ovarian cancer cell lines. Knockdown of LINC00665 dramatically reduced the cell visibility, arrested the cell cycle at the G0/G1 phase, and inhibited the migration and invasion of SKOV-3 and OVCAR-3 cells in vitro. This preliminary research indicated that LINC00665 might be a promising candidate for ovarian cancer therapy.
Increasing evidence indicated that dysregulation of lncRNAs played an important role in tumorigenesis and metastasis of human cancer [17]. In ovarian cancer, several lncR-NAs have been identified as indicators for patients' prognosis. For example, the expression of lncRNA ROR has been studied significantly higher in the plasma of ovarian cancer patients than that in healthy controls. And the enhanced level of plasma lncRNA ROR could be used as a potential biomarker for the diagnosis of ovarian cancer [18]. By contrast, the expression of lncRNA CASC15 was lower in ovarian cancer tissues. And the Kaplan-Meier survival analysis showed that patients with low CASC15 expression levels had poorer overall survival and progression-free survival than those with high CASC15 expression [19]. In this research, we demonstrated that LINC00665 expression level was significantly higher in ovarian cancer cell lines (SKOV-3, A2780, OVCAR-3 and HO8910) compared with the human ovarian epithelial cell line (ISOE80).
LINC00665 was first reported in oral premalignant lesions as potential molecular pathogenesis and biomarker of high-risk oral premalignant lesions [20]. Next, LINC00665 was observed upregulated in several other tumors, including hepatocellular carcinoma [12] and lung adenocarcinoma [13]. These were consistent with our results. As the analysis of the cancer genome atlas and the gene expression omnibus showed, overexpression of LINC00665 in patients with HCC was significantly associated with gender, tumor grade, stage, and tumor cell type. And overexpression of LINC00665 in patients with HCC was significantly associated with overall survival (OS) (HR = 1.47795%; CI: 1.046-2.086) [12].
Besides, the bioinformatics analysis identified ten identified core genes of LINC00665 that regulated pathways involved in the cell cycle. This indicated that LINC00665 facilitated the development and progression of HCC [12]. In gastric cancer, LINC00665 might serve as a ceRNA for miR-149-3p to regulate the expression of RNF2. And LINC00665 suppression significantly reduced GC cells viability and invasion ability in vitro [21]. In prostate cancer, LINC00665 was the sponge for miR-1224-5p. LINC00665 knockdown markedly attenuated growth and metastasis of PC cells and impaired tumor propagation in vivo [22]. This study demonstrated that LINC00665 knockdown could inhibit the proliferation, migration, and invasion of ovarian cancer cells (SKOV-3 and OVCAR-3).
The evidence above suggested that LINC00665 could play a role in miRNA sponges to downregulate the activity of target miRNAs in many cancers [21,22]. Here, bioinformatics analysis identified miR-181a-5p as the target miRNAs of LINC00665, as they had complementary binding sites. The binding sites of miR-181a-5p were also found in the 3'-UTR of FHDC1, and they were validated with the luciferase assay. A previous study showed that miR-181a-5p was downregulated in aggressive human breast and colon cancers and indicated that miR-181a-5p could inhibit tumor cell growth and invasion by attenuation elevated MMP-14 expression [20]. Most importantly, one hundred sixty-four matched tumor biopsies study showed that miR-181a-5p was associated with overall and progression-free survival (p < 0.05), residual tumor volume, and Pt-free interval inv ovarian cancer [23]. As previous studies reported, endogenous FHDC1 regulated Golgi ribbon formation and had an apparent preferential association with the Golgi-derived microtubule network. Knockdown of FHDC1 expression resulted in defective Golgi assembly and suggested a role for FHDC1 in the maintenance of the Golgi-derived microtubule network [24]. Besides, FHDC1 could affect the cell cycle and cell division [25]. We found that LINC00665 could downregulate the expression of miR-181a-5p via direct interaction and upregulate the expression of FHDC1 in SKOV-3 and OVCAR-3 cells. Also, we found that miR-181a-5p decreased the expression of FHDC1 and reversed the effect of LINC00665 knockdown in SKOV-3 cells. Meanwhile, knockdown FHDC1 could reverse the effect of miR-181a-5p inhibitor in SKOV-3 cells. All of these data indicated LINC00665 might take part in the inactivation of the miR-181a-5p-FHDC1 pathway.
In summary, the present study proved that LINC00665 modulated miR-181a-5p/FHDC1 pathway to facilitate ovarian cancer progression. The data suggest that LINC00665-miR-181a-5p-FHDC1 signal axis may provide a new therapeutic target for ovarian cancer therapy.
Author Contribution S.W conducted most of the experiments. J.W. interpreted and analyzed the data. Y.W wrote the first draft of the article. J.L. finalized the manuscript. J.W. conceived the study and revised the manuscript. All authors read and approved the final manuscript.
Data Availability All data generated or analyzed during this study are included in this published article.

Declarations
Ethics Approval and Consent to Participate Not applicable.

Competing interests
The authors declare no competing interests.