LncRNA KCNQ1OT1 promotes the metastasis of ovarian cancer by increasing the methylation of EIF2B5 promoter

Collection of clinical tissues

Thirty-two pairs of human tissue samples, including tumor tissues and adjacent healthy tissues, were acquired during surgery at the Third Xiangya Hospital of Central South University, then immediately preserved at − 80 °C. All tissues were confirmed by pathological diagnosis. Before tissue collection, we had got permission from the Ethics Committees of the Third Xiangya Hospital of Central South University and informed consent from all these 32 OC patients. None of OC patients had received preoperative therapy. Clinicopathological characteristics of all OC patients were listed in Table 1.

Table 1 Clinical characteristics of OC patients with high and KCNQ1OT1 risk scoresCell culture and transfection

Human ovarian surface epithelial cells (IOSE-80#, LMAIBio, Shanghai, China), OC A2780 cells (CL-0013#, Procell, Wuhan, China), Anglne (CL-0024#, Procell), SKOV3 (HTB-77#, ATCC, Manassas, VA, USA), SW626 (HTB-78#, ATCC), COV362 (07071910#, ECACC, European Collection of Authenticated Cell Cultures), CAOV3 (HTB-753#, ATCC) and OVCAR-3 (HTB-161#, ATCC) cells were cultured in a humidified incubator with 5% CO2 at 37 °C. Cells were maintained in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) mixed with 10% FBS (Gibco, Gran Island, NY, USA) and 1% penicillin–streptomycin (Sigma-Aldrich). Lentiviral-based shRNA targeting KCNQ1OT1 (sh-KCNQ1OT1; GenePharma, Shanghai, China) or EIF2B5 (sh-EIF2B5; GenePharma) was stably transfected into SKOV3 and SW626 cells to silence KCNQ1OT1 or EIF2B5 expression, with sh-NC (GenePharma) as negative control.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA of clinical tissues or cells was extracted using TRIzol Reagent (Invitrogen, Vienna, Austria), then RNA quality was determined using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Then cDNA was synthesized with 1 μg RNA template and M-MLV Reverse Transcriptase (Invitrogen). RT-qPCR was performed using SYBR Green PCR Kit (Qiagen, Frankfurt, Germany) to evaluate the expression of KCNQ1OT1, EIF2B5, DNMT1, DNMT3A and DNMT3B, with GAPDH as internal control. Relative expression was analyzed using 2−ΔΔCt method (Livak and Schmittgen 2001). All RT-qPCR primers were synthesized by Sangon Biotech (Shanghai, China), and primer sequence was shown in Table 2.

Table 2 The primer sequence for RT-qPCR assayWestern blotting

Clinical tissues or cells were lysed in RIPA buffer (Beyotime, Nantong, China) to extract protein samples, and a BCA Kit (Beyotime) was used to determine protein concentration. Then, 40 μg protein samples were loaded onto 12% fresh SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany). Membranes were blocked with 5% non-fat milk and incubated with rabbit primary antibody anti-EIF2B5 (ab181033; Abcam, Shanghai, China), anti-E-cadherin (ab40772; Abcam), anti-N-cadherin (ab76011; Abcam), anti-DNMT1 (ab188453; Abcam), anti-DNMT3A (ab2850; Abcam), anti-DNMT3B (ab2851; Abcam) or Loading Control anti-GAPDH (ab199554; Abcam), then probed with secondary antibody (ab205718; Abcam). Signals were detected using ECL detection kit (Millipore), and analyzed by Image J software (NIH, Bethesda, MD, USA).

Cell viability assessment

After transfection, 3 × 103 SKOV3 and SW626 cells seeded in 96-well plates were incubated with 20 μL MTT (Beyotime) for additional 4 h. After removing medium, DMSO was added to dissolve the formazan. 10 min later, the absorbance at 570 nm was recorded using a microplate reader (Bio-Rad, Hercules, CA, USA).

Colony formation assay

600 transfected SKOV3 and SW626 cells were placed in 6-well plates and continually cultured for 2 weeks at 37 °C. Visible colonies were fixed and stained with 4% paraformaldehyde (Sigma-Aldrich) and crystal violet (Beyotime), respectively. Then, colonies were counted utilizing Image J software.

Cell migration and invasion assay

OC cells are prone to migrate and invade into the peritoneum and underlying organs (Zhang and Zou 2015). Here, cell migration was examined by wound healing assay. After transfection, SKOV3 and SW626 cells were maintained in 6-well plates containing FBS-free RPMI-1640 medium until confluence reached 80%. A sterile pipette tip (10 μL; Corning Inc., Corning, NY, USA) was applied to create a scratch through the monolayer. Then, PBS was used to wash away detached cells, and cell migration was monitored at same position using a microscope (Nikon, Tokyo, Japan) at 0 h and 24 h after scratch-making.

Transwell chambers (Corning Inc.) pre-enveloped with Matrigel (BD Bioscience, San Jose, CA, USA) were utilized to detect cell invasion. Transfected SKOV3 and SW626 cells suspended in FBS-free medium were transferred to upper chambers. Additionally, complete medium was added to lower ones. 24 h later, invaded cells were fixed with 4% paraformaldehyde, stained with crystal violet and counted under a microscope.

Nuclear-cytoplasmic fractionation

The current assay combined with RT-qPCR assay were applied to investigate the subcellular location of lncRNA KCNQ1OT1 in OC cells. Firstly, cytoplasmic and nuclear RNA derived from SKOV3 and SW626 cells were separated using a Purification Kit (Norgen Biotek, Thorold, Canada) as instructed by the supplier. Furthermore, GAPDH and U6 snRNA served as internal control for cytoplasmic and nuclear fractions, respectively.

Fluorescence in situ hybridization (FISH) analysis

FISH assay was executed to determine the cellular localization of in lncRNA KCNQ1OT1 in OC cells. The oligonucleotide probe for KCNQ1OT1 was supplied by Biosense (Guangzhou, China). After mixture with probe mixture (10 μL, Biosense) in the dark overnight, cell nuclei of SKOV3 and SW626 was dyed with 4,6-diamidino-2-phenylindole (DAPI; Beyotime) and visualized under a fluorescence microscope (Olympus).

MS-PCR assay

This assay was executed to detect the methylation status of the EIF2B5 promoter referring to a former research (Real et al. 2018). The genomic DNA of SKOV3 and SW626 cells was isolated using a commercial kit (TIANGEN, Beijing, China), and a DNA Methylation-Gold™ kit (D5005, Zymo Research, Irvine, CA, USA) was exploited to assess the methylation level of EIF2B5 promoter. Methylated (M) and unmethylated (U) primers were: EIF2B5-M-forward, 5′-GTGATAATGTTGAGGTTAAGGAAC-3′ and EIF2B5-M-reverse, 5′-AAACAACGTAATATTTAAACCCACG-3′; EIF2B5-U-forward, 5′-TGATAATGTTGAGGTTAAGGAAT-3′ and EIF2B5-U-reverse, 5′-AACAACATAATATTTAAACCCACAACC-3′. PCR reaction products were then detected by agarose gel electrophoresis, then images were collected and analyzed.

RIP assay

The combing potency of KCNQ1OT1 to DNMT1, DNMT3A and DNMT3B was estimated by RPIseq database (http://pridb.gdcb.iastate.edu/RPISeq/). Then RIP assay was carried out to demonstrate the binding potential of KCNQ1OT1 to DNMT1, DNMT3A and DNMT3B proteins. SKOV3 and SW626 cells were lysed in lysis buffer (Beyotime), then generated cell lysate was incubated with the mixture of magnetic beads re-suspended in RIP Wash Buffer (Merck Millipore) and rabbit anti-DNMT1, anti-DNMT3A, anti-DNMT3B or NC anti-IgG (ab133470; Abcam) at 4 °C overnight. After treatment with Proteinase K (Invitrogen), RNA levels were evaluated via RT-qPCR assay.

ChIP assay

Until cell confluence reached 80%, SKOV3 and SW626 cells were fixed with 1% formaldehyde for 10 min, allowing intracellular DNA and protein to crosslink, followed by ultrasonic treatment. After centrifugation (13,000 rpm, at 4 °C), generated supernatant was incubated with anti-DNMT1, anti-DNMT3A and anti-DNMT3B, or incubated with negative control anti-IgG at 4 °C overnight. Then endogenous DNA–protein complex was precipitated as previously described (Chen et al. 2019). And enrichment of EIF2B5 promoter fragment binding to DNMT1, DNMT3A, and DNMT3B was analyzed by RT-qPCR assay with specific primers for promoter region of EIF2B5 gene: forward, 5′-CTTTCCATGTTTCGCCATCT-3′ and reverse, 5′-AAAGTCCACTGGCACCAAAC-3′.

Statistical analysis

All assays in this work were performed ≥ 3 times. Data analysis was implemented by SPSS 21.0 software and exhibited as mean ± SD. Student’s t-test or ANOVA was applied to compare differences in groups. The correction between levels of KCNQ1OT1 and EIF2B5 in OC tissues was evaluated by Spearman’s correlation coefficient. P < 0.05 meant statistically significant.

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