Knockdown of long non‐coding RNA LINC00176 suppresses ovarian cancer progression by BCL3‐mediated down‐regulation of ceruloplasmin

Abstract Ovarian cancer is a common malignancy among women with some clinically approved diagnostic coding gene biomarkers. However, long non‐coding RNAs (lncRNAs) have been indicated to play an important role in controlling tumorigenesis of ovarian cancer. Hereby, the aim of the study was to uncover the function of lncRNA LINC00176 in the development and progression of ovarian cancer by regulating ceruloplasmin (CP). Bioinformatics prediction in combination with RT‐qPCR analysis for the expression pattern of LINC00176 revealed that LINC00176 was highly expressed in ovarian cancer tissues as well as in ovarian cancer cell lines, respectively. LINC00176 was predominantly localized in the nucleus. Delivery of si‐LINC00176, oe‐LINC00176, si‐BCL3 and si‐CP plasmids was conducted to explore the effects of LINC00176 on ovarian cancer. Promoted proliferation, migration and invasion along with reduced apoptosis were observed in cells treated with oe‐LINC00176, while si‐BCL3 and si‐CP were able to block the promoting effects. Investigations with regard to the correlation between LINC00176 and promoter region of CP turned out to be positive via B‐cell CLL/lymphoma 3 (BCL3) by means of dual‐luciferase reporter gene assay, ChIP and RIP assays. Furthermore, oncogenic properties of the LINC00176/BCL3/CP axis were also demonstrated by tumour formation in vivo generated upon injecting cells in nude mice. Our results demonstrate that restored LINC00176 initiates tumorigenesis in ovarian cancer by increasing CP expression via recruiting BCL3, the mechanism of which represented a potential and promising therapeutic target for the disease.


| INTRODUC TI ON
Ovarian cancer ranks as the 5th leading cause of cancer-related mortality in the female population and characterized by histological and genetic tumours from epithelial, sex cord-stromal and germ cells. 1 As a global health issue, diagnosis of ovarian cancer usually is confirmed during a late stage and effective screening strategy at early stage has not been developed up to now. 2 Besides, patients with ovarian cancer typically suffer from dismal prognosis while improvement of therapeutic approaches is still limited, 3 which emphasizes on the urgency to discover novel remedies for ovarian cancer. Tumour cell metastasis is always evident in the late stage of ovarian cancer, where epithelial-mesenchymal transition (EMT) has been identified to function as a critical regulator due to its promoting effects on cell dissemination and tissue invasion. 4 Specifically, long non-coding RNAs (lncRNAs) have emerged to be essential to EMT in gynaecologic cancers. 5 LncRNAs, capable of regulating various pathological and physiological processes by functioning as oncogenes or antioncogenes, have been documented to function in different stages of ovarian cancer, thereby indicating their prognostic and therapeutic significance. 6 LncRNAs have been confirmed to play an essential role in EMT in gynaecologic cancers. 5 Specifically, overexpression of lncRNA DNM3OS, MEG3, and MIAT has been implicated in ovarian cancer. 7 According to the bioinformatics results in our study, LINC00176 was revealed to be a differentially expressed lncRNA in ovarian cancer. LINC00176 is highly expressed in hepatocellular carcinoma while depleted expression LINC00176 can impede proliferation and induce necroptosis, highlighting that reduced expression of lncRNAs may be beneficial for cancer therapy. 8 In addition, LINC00176 associates with the survival condition of patients suffering from oesophageal cancer. 9 Based on the results from the LncMAP website, it was evident that LINC00176 could regulate the expression of ceruloplasmin (CP) via transcription factor B-cell CLL/ lymphoma 3 (BCL3). CP, consisting of 6 copper ions, is a 132-kDa alpha2-glycoprotein produced by the liver and secreted into the circulation. 10 CP can function as a tumour progression marker of epithelial ovarian cancer due to its significantly higher expression in the ascitic fluids of patients with intrinsic chemoresistance. 11 BCL3 has been acknowledged as another important proto-oncogene in context of ovarian cancer that a high BCL3 expression has been evident in human ovarian cancer tissue where ovarian cancer cell survival, proliferation and migration are promoted, respectively. 12 BCL3 involves in key oncogenic pathways in association with cell death and apoptosis in solid tumours and can inhibit ovarian cancer proliferation through interplay with microRNA-125b (miR-125b). 13 However, the functioning of BCL3 cooperates with lncRNA with respect to regulation of cancer progression is unknown. Therefore, we proposed a hypothesis asserting that the LINC00176/BCL3/CP axis may play a novel role in the progression and development of ovarian cancer via confirmation by a series of in vitro and in vivo experiments.

| Ethics statement
The study was conducted under the approval of the Institutional Review Board of Chinese Medicine Hospital in Linyi City. All participants provided written informed consent prior to this study. The animal protocol and experiment procedure were conducted under the approval of the Institutional Animal Care and Use Committee of Chinese Medicine Hospital in Linyi City. Animals in our study were used for research purpose only.

| Microarray-based gene expression profiling
The ovarian cancer-related gene expression data set GSE38666 was downloaded from the Gene Expression Omnibus (GEO) database (https ://www.ncbi.nlm.nih.gov/geo/), followed by conducting a procedure of screening of differential genes using R language with |log2FC| > 1.5 and P Value < .05 as threshold. Heatmap depicting differential gene expression was established subsequently.

| Cell treatment
Plasmids of si-LINC00176, oe-LINC00176, si-BCL3, si-CP and their negative controls (NCs) were transfected into HO8910 cells. The aforementioned target plasmids were purchased from Dharmacon, Lafayette, CO, USA. HO8910 cells were seeded in a 6-well plate at a density of 3 × 10 5 cells/well and then transfected using Lipofectamine 2000 (Invitrogen Inc) upon reaching the cell growth density of 80%.
Then, 250 µL Opti-MEM (Gibco Company) was added to dilute 4 µg target plasmids and 10 µL Lipofectamine 2000, respectively. After standing at room temperature for 5 minutes, the aforementioned components were mixed together and allowed to react for 20 minutes. Following a regimen of 6-hours culture in an incubator at 37°C with 5% CO 2 , the medium was renewed for further culture. Finally, the cells were harvested 36-48 hours after culturing.

| RNA isolation and quantitation
Total RNA was extracted using the TRIzol reagent (15596026, Invitrogen). Then, the extracted RNA was reversely transcribed into complementary DNA (cDNA) in strict accordance with the provided instructions of the reverse transcription kit (RR047A, Takara). The SYBR Premix EX Taq kit (RR420A, Takara) was employed for sample loading. RT-qPCR was performed on an ABI 7500 qPCR instrument (ABI Company) subsequently. Three duplicate wells were set for each sample. The aforementioned primers (Table 1) were synthesized by Shanghai Biotech Co., Ltd.. With regard to β-actin as the internal control, the fold changes of gene expression were calculated on the basis of relative quantification (2 −ΔΔCt method).

| Fluorescence in situ hybridization (FISH)
FISH assay was conducted to identify the subcellular localization of LINC00176. The coverslip was placed in a 24-well plate, where cells were seeded at a density of 6 × 10 4 cells/well. When cell confluence reached about 85%, the coverslip was rinsed using phosphate-buffered saline (PBS), fixed utilizing 1 mL 4% paraformaldehyde solution and treated with proteinase K (2 µg/mL), glycine and acetylation reagent, respectively. Then, the cells were pre-hybridized with 250 µL LINC00176 probes (300 ng/mL) (Eurogentec, Seraing, Belgium) overnight. After 3 rinses using PBS with Tween-20 (PBST), the nucleus was stained using PBST-diluted 4′,6-diamidino-2-phenylindole (DAPI) (1:800) in a 24-well plate for 5 minutes. After that, the cells were sealed using fluorescence decay-resistant medium. Five fields were randomly selected for microscopic observation and photography under a fluorescence microscope (Olympus Optical Co., Ltd).

| RNA binding protein immunoprecipitation (RIP) assay
The BCL3-induced enrichment of LINC00176 was determined using a RIP Kit (Millipore). After a pre-cooled PBS rinse, the HO8910 cells were lysed with equal volume of lysis in ice bath for 5 minutes. The supernatant was attained through centrifugation at 21 912 × g and 4°C for 10 minutes. A portion of the cell extract was used as input, while the remaining was probed with the BCL3 antibody (ab27780, Abcam lnc) for coprecipitation. Immunoglobulin G (IgG) antibody (ab2410, Abcam Inc) served as NC. RNA was extracted from the sample and input after protease K detachment, followed by RT-qPCR detection.

| Chromatin immunoprecipitation (ChIP) assay
HO8910 cells were fixed using formaldehyde solution for 10 minutes to initiate DNA-protein cross-link. Chromatin fragments were obtained by application of ultrasonic sound (10 seconds each time at an interval of 10 seconds, 15 times in total). The supernatant was collected through centrifugation at 12 000 × g and 4°C for 10 minutes, subpacked into 2 tubes, and incubated with 2 µg NC antibody to IgG (ab2410, Abcam Inc) and 2 µg target protein-specific antibody to BCL3 (ab27780, Abcam Inc) and p50 (sc-166588, Santa Cruz Biotechnology, Inc) at 4°C overnight. The DNA-protein complex was precipitated using protein agarose/Sepharose, followed by centrifugation at 16 099 × g for 5 minutes. The supernatant was discarded, and the non-specific complex was washed.
De-cross-linking was conducted at 65°C overnight. DNA fragment was purified and extracted using phenol/chloroform. The binding of BCL3 to the CP promoter was determined by RT-qPCR using CPspecific primer.
After extensive washing, the beads were boiled for 5 minutes at 100°C. The protein was separated using sodium dodecyl sulphatepolyacrylamide gel electrophoresis and transferred onto the nitrocellulose membrane (Merck Millipore), followed by application of Western blot analysis.

| Dual-luciferase reporter gene assay
According to the bioinformatics analysis, target genes of LINC00176 were predicted. The promoter region of CP was constructed onto the pGL3-Basic vector (Promega Corp.) to produce pGL3-CP recombinant vector. The correctly sequenced luciferase reporter plasmid was successfully acquired and cotransfected with oe-LINC00176, oe-NC, si-LINC00176 and si-NC into HEK293T cells, which were then collected and lysed 48 hours later. The luciferase activity was measured using a dual-luciferase detection kit (K801-200, Biovision Inc) in a dual-luciferase reporter gene analysis system (Promega Corp.). Renilla luciferase served as an internal reference. The relative activity of luciferase was indicative of the activity degree of target reporter gene, which equalled the ratio of relative light unit (RLU) of firefly luciferase to RLU of Renilla luciferase.

| Transwell invasion assay
The extracellular matrix (ECM) gel was placed at 4°C overnight and diluted to 1 mg/mL the next day with serum-free medium at a ratio of 1:9. Subsequently, 40 µL ECM was added to the polycarbonate membrane on the apical chamber and subjected to incubation at 37°C with 5% CO 2 for 5 hours until the ECM polymerized into gel.
Next, 70 µL DMEM was added into each well, followed by incubation at 37°C for 0.5 hour to prepare a hydrated gel. Subsequently,

| Western blot analysis
Total protein was extracted from the tissues or cells using radioimmunoprecipitation assay (

| Xenograft tumour in nude mice
A total of 36 BALB/c mice of either sex (age: 4 weeks; weight:

| LINC00176 is highly expressed in ovarian cancer
Initially, the ovarian cancer-related microarray data in combination with Gene Expression Profiling Interactive Analysis (GEPIA) (http:// gepia.cancer-pku.cn/index.html) were performed to identify the respective differentially expressed lncRNAs, the results of which demonstrated LINC00176 as a significantly highly expressed lncRNA in ovarian cancer ( Figure 1A  Subcellular localization of LINC00176 in HO8910 cells detected using FISH assay ( Figure 1E) demonstrated that LINC00176 was evenly distributed in the nucleus, indicating that LINC00176 was primarily localized in the nucleus. Taken together, these findings suggested that LINC00176 was expressed at a high level in ovarian cancer.

| Silencing LINC0176 impedes EMT of ovarian cancer cells
After identification of the high expression of LINC00176 in ovarian cancer, the aim was shifted onto assessing the effects of LINC00176   Figure 2I and J). The aforementioned findings demonstrated that silencing LINC00176 to be sufficient to weaken the ovarian cancer cell capabilities of proliferation, migration and invasion as well as to enhance the apoptotic capability, leading to suppressed EMT.

| Overexpressed LINC00176 up-regulates expression of CP via BCL3
Subsequently, an investigation was conducted to explore the molecular mechanism of LINC00176 in ovarian cancer. According to the LncMAP website (http://bio-bigda ta.hrbmu.edu.cn/LncMA P/), LINC00176 regulated CP expression by BCL3, and the GEPIA database revealed that CP was highly expressed in ovarian cancer ( Figure 3A). The results obtained from RIP assay presented in Figure 3B showed that BCL3 was verified to specifically bind to LINC00176 as evidenced by significantly more LINC00176 enrichment caused by BCL3 than IgG (P < .05). Then, RT-qPCR and Western blot analysis were conducted to quantify CP expression in ovarian cancer and adjacent tissues, the results of which showed that CP was abnormally overexpressed in ovarian cancer as the mRNA and protein expression of CP was much higher in ovarian cancer tissues than adjacent tissues (P < .05, Figure 3C   verification purposes and the results demonstrated that site 2 was crucial to the relative luciferase activity of CP as the capability of oe-BCL3 activating CP was significantly weakened with the truncated or mutant site 2 (P < .05), while no significant difference was observed with the truncated or mutant site 1 (P > .05, Figure 3J and K), thereby suggesting site 2 as the binding site between BCL3 and CP promoter.
Moreover, the binding ability of BCL3 to CP DNA on site 2 was detected by ChIP assay, the results of which showed that relative enrichment of CP DNA by BCL3 significantly increased compared to IgG (P > .05, Figure 3L), ascertaining site 2 of CP DNA as the binding site to BCL3. The aforementioned findings elucidated that LINC00176 could promote the transcription expression of CP by recruiting BCL3.
In order to further explore the regulatory mechanism among LINC00176, BCL3 and CP, rescue experiments were designed with transfection of oe-LINC00176 + si-NC and oe-LINC00176 + si-BCL3, followed by RT-qPCR for quantification of CP expression ( Figure 3M). Results showed that transfection of oe-LINC00176 + si-BCL3 significantly diminished CP expression while transfection of oe-LINC00176 + si-NC did not, suggesting that silencing BCL3 impeded the promoting effect of LINC00176 on CP expression. The abovementioned findings offered evidence proving that LINC00176 promoted CP expression in ovarian cancer through recruitment of BCL3.

| LINC00176/BCL3 involves in the regulation of EMT in ovarian cancer
With results determining the functionality of the regulatory mechanism regarding LINC00176, BCL3 and CP, effects induced by such mechanisms in ovarian cancer were then investigated with regard to cell proliferation, apoptosis, migration, invasion and EMT after transduction of si-BCL3, si-CP and oe-NICL00176. It was observed that both si-BCL3 and si-CP exerted inhibitory effects on cell proliferation ( Figure 4A and B), migration ( Figure 4E and F) and invasion ( Figure 4G and H) accompanied by accelerated apoptosis ( Figure 4C and D), increased E-cadherin protein level and decreased protein levels of N-cadherin and vimentin ( Figure 4I and J

| LINC00176 regulates CP expression via promotion of the interaction between BCL3 and NF-κB1 P50
A former study indicated that BCL3 could closely bind to NF-κB P50 or p52 homodimer and trans-activated. 14 In our study, we initially conducted the Co-IP experiment and found that BCL3 and P50 bound and interact with each other. Overexpression of LINC00176 promoted the binding of BCL3 to P50, while silencing of LINC00176 impeded the binding of BCL3 to P50 ( Figure 5A). Further results of ChIP revealed that overexpressed LINC00176 could promote the binding of BCL3 to P50 in the CP promoter, while silenced LINC00176 could inhibit the binding of BCL3 to P50 in the CP promoter ( Figure 5B).
These results suggested that LINC00176 stimulated the interaction F I G U R E 5 LINC00176 regulates CP expression by promoting the interaction between BCL3 and NF-κB1 P50. A, The binding of BCL3 to P50 upon oe-LINC00176 or si-LINC00176 treatment evaluated using Co-IP. B, The binding of BCL3 to P50 in the CP promoter and fold enrichment of CP promoter upon oe-LINC00176 or si-LINC00176 treatment detected using ChIP assay an RT-qPCR, respectively. All data were measurement data and expressed as mean ± standard deviation. The diagram on the left indicates oe-LINC00176 treatment, *P < .05 vs the oe-NC group (HO8910 cells treated with oe-NC). The diagram on the right indicates si-LINC00176 treatment, *P < .05 vs the si-NC group (HO8910 cells treated with si-NC). Each reaction was run in triplicate   An initial conclusion was comprehended as that LINC00176 was highly expressed in ovarian cancer tissues and cells featured with strengthened cell capabilities of proliferation, migration, invasion and EMT along with weakened apoptotic property. Hitherto, limited studies have enclosed the role of LINC00176 in malignancy. A contrary expression pattern of LINC00176 has been detected in oesophageal cancer and patients exhibiting a higher LINC00176 expression are expected to have a longer overall survival. 9 In consistency with our results, LINC00176 expression has been found to be expressed at high levels in hepatocellular carcinoma while depleted LINC00176 can induce necroptosis. 8 To assess the involvement of LINC00176 in the biological behaviours of ovarian cancer cells, we treated HO8910 cells with si-LINC00176. Consequently, depletion of LINC00176 in HO8910 cells significantly induced cell apoptosis and inhibited cell proliferation, migration and invasion. EMT is the initial process characterized by the metastasis of tumour cells. 17 The conversion of epithelial cells to mesenchymal cells is vital for the invasion and metastasis of ovarian cancer while the switch from E-cadherin to Ncadherin plays a crucial role illustrated by decreased E-cadherin and increased N-cadherin. 18 E-cadherin, N-cadherin and vimentin have been identified as phenotype markers for EMT. 19 Vimentin is ubiquitous in normal mesenchymal cells by functioning as a critical regulator of cellular integrity and overexpression of vimentin is indicative of enhanced tumour growth, accelerated invasion and dismal prognosis. 20 We identified EMT to be suppressed by silencing LINC00176 as evidenced by higher E-cadherin level and lower levels of N-cadherin and vimentin. Likewise, overexpressed lncRNA SPRY4 intronic transcript 1 has been demonstrated to up-regulate E-cadherin and down-regulate N-cadherin and vimentin, which would consequently impede metastasis through EMT in ovarian cancer. 21 Furthermore, in other types of cancers, EMT is closely increased by many lncRNAs such as TRE, MALTA1 and HOTAIR. 22 Therefore, it could be speculated that lncRNAs usually participate in the EMT of tumorigenesis.

| D ISCUSS I ON
Finally, it is well acknowledged that lncRNA recruiting a variety of transcription factors is a common pattern that regulates viability and metastasis of tumour cells. 23 Transcription factors have been elucidated to mediate gene expression and are involved in the regulation of proliferative status, cellular differentiation and cell fate. 24 LncRNAs usually recruit these proteins forming RNP complex and specifically binding to the promoter region of target genes. For instance, the regulatory role of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in cell cycle progression is facilitated via the oncogenic transcription factor B-MYB. 25 Currently, no study has reported the relation between BCL3 and lncRNAs. In our study, a novel discovery revealed that LINC00176 could positively regulate the expression of CP through regulation of the transcription factor BCL3.
BCL3 is known as a transcription factor associated with the activity of NF-κB concerning the inflammatory response in pancreatic and biliary tissues. 26 However, future researches are required regarding the regulatory possibility of the NF-κB signalling pathway in ovarian cancer. Critically, the proto-oncogene BCL3 has been detected to express highly in human ovarian cancer tissues where ovarian cancer cell survival, proliferation and migration are promoted, 12 supporting our results with regard to the rescue properties of silencing BCL3.
To conclude, LINC00176 functions as an oncogene, and the reduction in its expression is associated with suppressed tumorigenicity and EMT through inhibition of CP via BCL3 (Figure 7). However, it is uncertain that such effects may be induced by different downstream signal pathways like the NF-κB signalling pathway. Therefore, their exact functions require further confirmation in future studies.
The present study can serve as an insight for identifying these factors in early diagnosis or providing a new approach for the treatment of ovarian cancer.

ACK N OWLED G EM ENTS
We would like to give our sincere appreciation to the reviewers for their helpful comments on this article.

CO N FLI C T O F I NTE R E S T
We declare that we have no conflicts of interest.