NF‐κB p65 promotes ovarian cancer cell proliferation and migration via regulating mortalin

Abstract Previous studies show that mortalin, a HSP70 family member, contributes to the development and progression of ovarian cancer. However, details of the transcriptional regulation of mortalin remain unknown. We aimed to determine whether NF‐κB p65 participates in the regulation of mortalin expression in ovarian cancer cells and to elucidate the underlying mechanism. Chromatin immunoprecipitation and luciferase reporter assay were used to identify mortalin gene sequences, to which NF‐κB p65 binds. Results indicated that NF‐κB p65 binds to the mortalin promoter at a site with the sequence ‘CGGGGTTTCA’. Using lentiviral pLVX‐NF‐κB‐puro and Lentivirus‐delivered NF‐κB short hairpin RNA (shRNA), we created ovarian cancer cell lines in which NF‐κB p65 was stably up‐regulated and down‐regulated. Using these cells, we found that downregulation of NF‐κB p65 inhibits the growth and migration of ovarian cancer cells. Further experimental evidence indicated that downregulation of NF‐κB p65 reduced mortalin, and upregulation of mortalin rescued the proliferation and migration of ovarian cancer cells reduced by NF‐κB p65 knockdown. In conclusion, NF‐κB p65 binds to the mortalin promoter and promotes ovarian cancer cells proliferation and migration via regulating mortalin.

factor that was first discovered in B cells. It specifically binds to the enhancer κB sequence of the immunoglobulin kappa light chain gene and is involved in the body's inflammatory and immune responses. 17 NF-κB is present in various tissues and cells and plays critical roles in cell proliferation and cell death. It also regulates cell differentiation, inflammation and other pathological processes. 18,19 NF-κB has been implicated in various diseases such as allergies, 20 neurodegenerative diseases, 21,22 ophthalmic diseases 23,24 and cancer. [25][26][27][28] There are five members of the NF-κB family, p65 (RelA), RelB, c-Rel, p105/ p50 (NF-κB1) and p100/52 (NF-κB2). 29 NF-κB p65 subunit is considered as the most potent transcriptional activator of the family. 30 This study was prompted by our earlier study that used bioinformatics techniques to predict and determine the potential transcription factors of mortalin. Results showed NF-κB p65 and MZF-1 were the potential transcription factors of mortalin (Supporting Information Table S1). We selected NF-κB p65 for further study and assessed whether it participates in the regulation of mortalin expression in ovarian cancer cells.
A2780CP and A2780S were transfected with NF-κB p65 overexpression and interferon expression lentiviral vectors to generate stable cell lines. Additionally, we introduced the mortalin-overexpression vector into the NF-κB p65 down-regulated A2780CP cells. After transfection, cells were cultured for 72 hours and then screened after approximately 1 week using 2 µg/mL puromycin to obtain the stable cell lines.
293T cells were cultured in DMEM containing 10% FBS, at 37°C and in 5% CO 2 . Logarithmic phase 293T cells were trypsinized, seeded in 10 cm culture dishes and used for transfection once they reached 60%-80% confluence. Three systems of plasmid, including the constructed vectors psPAX2 and PMD2G were used to transfect 293T cells. HilyMax (Dojindo, Kumamoto, Japan) was used to transfect the cells following the manufacturer's instructions. The 48 and 72 hours supernatants were collected and used for further infections.

| Real-time quantitative PCR
Total RNA was extracted from cells using the RZ reagent according to the instructions in the RNAsimple total RNA Kit (TIANGEN, Beijing, China). First-stand cDNA was synthesised using the HiScript 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). NF-κB p65-chip -Rv: 5'-AGGAGTACGAGGCAG-3'. Membranes were then incubated with secondary antibody for 2 hours at room temperature. Proteins were then detected using Immobilon Western chemiluminescent HRP substrate (Bio-Rad, Hercules, CA, USA) and a Gel Doc XR System (Bio-Rad). Results were analysed with Image J software (NIH, Bethesda, MD, USA).

| Cell viability assay
Cell viability was assessed using a Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Cells were seeded in 96-well plates at a density of 5000 cells/well and cultured at 37°C for 24 hours. After incubation with CCK-8 reagent for 2 hours at 37°C, the optical density was measured at 450 nm by using a Multiskan MK3 microplate reader (Thermo Fisher Scientific, MA, USA).

| Colony formation assay
Cells were seeded into 6 cm dishes at 1000 cells per dish. After incubation for 2 weeks, cells were washed with PBS, fixed with methanol for 10 minutes, stained with 10% Giemsa solution for 15 minutes, washed with running water and dried before being photographed.
Visible colonies were counted.

| Cell migration assay
Cell migration was detected by wound healing assay. Cells were seeded into a 6-well plate at the density of 2 × 10 6 cells per well.
When cells reached full confluence, a wound was scratched across the middle of each well using a micropipette tip. Cells were washed twice with PBS and cultured with fresh DMEM medium containing mitomycin C (10 ug/mL; Sigma-Aldrich, MO, USA). Cells were photographed at the beginning (0 hour) using a DM2500 fluorescence microscope (Leica, Germany). And after the cells incubated for 24 hours, the same fields were photographed again (24 hours).
ImageJ software (NIH, USA) was used to evaluate the average extent of wound closure by measuring the width of the wound.

| Chromatin immunoprecipitation assay
The chromatin immunoprecipitation (CHIP) assay was performed according to the instructions provided with the CHIP assay kit (Sigma-Aldrich, MO, USA). Briefly, 10% of the chromatin was saved to act as the input control and remainder diluted in CHIP dilution buffer. The diluted chromatin was incubated with 5 μL anti-NF-κB p65 antibody or normal immunoglobulin G (IgG). Immunoprecipitated DNA was analysed using PCR and RT-qPCR.

| Luciferase reporter assay
NF-κB p65 possible binding sequence of mortalin promoter (TCAGTAGAGACGGGGTTTCACCGTGTTAGC) was cloned into the pGL4.10-luc2 vectors to generate luciferase reporter plasmid (pGL4.10-proMortalin-luc). HEK-293FT cells, A2780CP cells and A2780S cells were seeded into 24-well plates. Once reaching a confluence of 60%, the cells were transfected with different plasmids (100 ng of pGL4.10-basic-luc or pGL4.10-proMortalin-luc and 5 ng of pRL-TK vectors; 100 ng of pGL4.10-proMortalin-luc, 100ng NF-κB p65 overexpressing vectors and 5 ng of pRL-TK vectors). The luciferase activities were detected 36 hours after transfection using the Dual-Luciferase Reporter Assay System (Beyotime, Shanghai, China) according to the manufacturer's protocol. Firefly luciferase activities were normalised to renilla luciferase values, and expressed as relative luciferase units. SPSS 12.0 statistical software (IBM) was used to analyse the experimental data. Independent sample t-tests and single factor analysis of variance (One -way ANOVA) were used for statistical analysis. P < 0.05 was considered statistically significant. The relationship between NF-κB p65 and mortalin was confirmed using a CHIP assay coupled with RT-qPCR. Four primer pairs were designed (Supporting Information Table S2). RT-qPCR results showed that there were no significant differences between anti-NF-κB p65 and the control IgG, if the m2, m3, m4 primers were used (Supporting Information Figure S1). The results of PCR, in which the m1 primers were used, showed the anti-NF-κB p65 and input group shared a similar band separation both in A2780CP and A2780S cells ( Figure 1E).

| NF-κB binds to the mortalin promoter
RT-qPCR showed similar results that the value of anti-NF-κB p65 group was higher than that of IgG group (P < 0.05) ( Figure 1F). The CHIP results suggest that NF-κB p65 could combine with mortalin promoter, and the possible binding site is 'CGGGGTTTCA'.
All these indicated that NF-κB p65 could bind to the sequence 'CGGGGTTTCA' in mortalin promoter.

| NF-κB p65 regulates the expression of mortalin
NF-κB p65 mRNA and protein expression were assessed in stable NF-κB p65 down-regulated and up-regulated cell lines using qRT-PCR and Western blotting. As shown in Figure 2A, the expressions of NF-κB p65 mRNA and protein in NF-κB p65 down-regulated cells (A2780CP NF-κB p65-shRNA, A2780S NF-κB p65-shRNA) were lower than in control cells (A2780CP-Ctrl, A2780S-Ctrl). Conversely, the mRNA and protein expression of NF-κB p65 in NF-κB p65 overexpression cells (A2780CP NF-κB p65-OV, A2780S NF-κB p65-OV) was higher than that in control cells ( Figure 2B). Then we detected the expression of mortalin in NF-κB down-regulated and up-regulated cells. Results showed ( Figure 2C,D) that mRNA and protein expression of mortalin in A2780CP NF-κB p65-shRNA and A2780S NF-κB p65-shRNA cells were lower than in control cells and mortalin mRNA and protein expression in A2780CP NF-κB p65-OV and A2780S NF-κB p65-OV cells were higher than in control cells. Immunofluorescence results ( Figure 2E) showed that the fluorescent signal was fainter F I G U R E 3 NF-κB p65 promotes proliferation and migration of ovarian cancer cells. (A) Cell viability was measured using a CCK-8 assay, which showed that proliferation decreased in NF-κB p65 down-regulated cells compared to control group. In contrast, NF-κB p65 up-regulated cells exhibited significantly higher growth rates compared to vector controls. (B) Colony formation assays showed that colony size decreased in NF-κB p65 down-regulated cells and increased in their NF-κB p65 up-regulated counterparts. (C) Wound healing assays showed that NF-κB p65 overexpression promotes ovarian cancer cells migration. *P < 0.05 in the cytoplasm and nucleus of NF-κB p65-shRNA cells than in the control cells. At the same time, immunofluorescence results ( Figure 2F) also showed that mortalin cytoplasm expression in A2780CP NF-κB p65-shRNA and A2780S NF-κB p65-shRNA cells was lower than in the control cells. Together, these results indicate that NF-κB can regulate the expression of mortalin in nucleus.

| NF-κB p65 promotes proliferation and migration of ovarian cancer cells
The CCK-8 and colony formation assays were used to investigate the effect of NF-κB p65 on the proliferation of ovarian cancer cells.
The viabilities of A2780CP NF-κB p65-shRNA and A2780S NF-κB p65-shRNA cells were significantly lower compared with the control groups. In contrast, A2780CP NF-κB p65-OV and A2780S NF-κB p65-OV cells grew faster than A2780CP-NC and A2780S-NC control cells ( Figure 3A). As Figure 3B showed that stable NF-κB p65 down-regulated cells formed much smaller colonies compared to the control groups. And stable NF-κB p65 up-regulated cells formed much larger colonies than control cells. These results indicated that NF-κB p65 affects the proliferation of ovarian cancer cells.
Wound healing assay was used to assess cell migration. Results ( Figure 3C) showed that NF-κB p65 down-regulated cells migrated more slowly than control cells. In contrast, NF-κB p65 up-regulated cells closed the wound more rapidly compared to controls.
These results indicated that NF-κB affects ovarian cancer cell migration.
Wound healing assay results ( Figure 4E) showed that the migration of A2780CP-NF-κB p65-shRNA-mortalin-OV cells was higher than the A2780CP-NF-κB p65-shRNA cells. Taken together, mortalin overexpression can partially rescue the proliferation and migration of ovarian cancer cells reduced by NF-κB.

| D ISCUSS I ON
Mortalin, a member of the HSP70 family, is widely presented in cells and plays important roles in oxidative stress, regulation of mitochondrial membrane potential, intracellular transport and immune response. It regulates cell proliferation through interacting with a variety of molecules such as P53, GRP94 and VDAC, and participates in a number of molecular pathways. [32][33][34] Studies show that mortalin expression is elevated in some tumour cells and tissues. 35 Mortalin overexpression promotes tumorigenesis by inhibiting tumour cells apoptosis and promoting proliferation.
In tumour cells, mortalin overexpression can inhibit P53 activity and activate telomerase and hnRNP-K to promote tumour progression. 36 In addition, it can inhibit apoptosis by suppressing conformational changes in P53 and BAX. 37 As an integral part of the ATPase complex, mortalin also plays an important role in maintaining mitochondrial homeostasis, which is critical for the translocation of most mitochondrial membrane and matrix proteins. 38,39 The energy generated by mitochondria is essential for the process of tumour development. 40 Our previous studies have found that mortalin can promote cell proliferation, invasion and metastasis by activating Raf/Mek/Erk1/2 cascade signalling pathway and by regulating cell cycle progression in ovarian cancer cells. 11 However, the mechanism of mortalin expression in the tumour cells remained unclear. We predicted some transcription factors and their binding sites in the mortalin promoter using bioinformatics tools and identified NF-κB, MZF-1, LBX-1 and others as candidates. Li et al also showed that NF-κB may bind to mortalin in human skin keratinocytes (HaCaT) and regulate mortalin expression. 41 We therefore focused on NF-κB in this study.
Previous studies have shown that overexpression of mortalin is correlated to the malignancy of ovarian cancer, and that downregulation of mortalin can significantly enhance the sensitivity of cisplatin resistance in ovarian cancer cells and reduce their proliferation and invasion. 42 Thus, the cisplatin-resistant human ovarian cancer cell line A2780CP and the cisplatin-sensitive human ovarian cancer cell line A2780S were selected to study the interaction between NF-κB p65 and mortalin. We found that NF-κB p65 expression in A2780CP cells was significantly higher than that in A2780S cells, which was mirrored by mortalin expression. When cells were treated with the NF-κB p65 inhibitor PDTC, they exhibited different sensitivities to the drug. Low concentrations of PDTC could significantly inhibit NF-κB and mortalin expression in A2780S cells, whereas higher concentrations were needed in A2780CP cells, suggesting that NF-κB may be associated with ovarian cancer cell drug resistance. to mortalin mRNA and decrease its expression in K562 cells. 52 Our experiments have shown that overexpression of mortalin can partly reverse the proliferation and migration ability of ovarian cancer cells induced by NF-κB p65 downregulation. Other factors may also regulate mortalin in ovarian cancer cells, and these mechanisms need to be studied further.
In summary, the present study suggests that NF-κB can bind to the promoter of the molecular chaperone mortalin and promote ovarian cancer cell proliferation and migration via regulating mortalin.

ACK N OWLED G EM ENTS
This work was supported by National Natural Science Foundation of China (81773203 and 81670177). We wish to thank Prof. Yunlong Yang (Fudan University) for revising of our manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.