Identification of differentially expressed long non-coding RNAs in human ovarian cancer cells with different metastatic potentials

Cell lines and cell culture

The human ovarian cancer cell lines SKOV3 and SKOV3.ip1 were obtained from the American Type Culture Collection and M.D. Anderson Cancer Center, respectively. The SKOV3.ip1 cell line was derived from ascites from a nude mouse injected i.p. with SKOV3 cells and demonstrated a higher metastatic potential compared with the parental cell line⁷. Both cell lines were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and cultured in a humidified incubator (5% CO₂) at 37 °C.

Validation of invasive capability of ovarian cancer cells

In vitro invasion assay was performed using 24-well transwell units with polycarbonate filters (pore size: 8 μm) coated on the upper side with reconstituted basement membrane matrix (BD Biosciences, USA). The cells were harvested, and 4×10⁴ cells in 100 μL of serum-free medium were placed in the upper part of the transwell unit and were allowed to invade the membrane for 72 h at 37 °C. Successfully penetrating cells were fixed, stained, and quantified at optical density of 570 nm after extraction. The results are reported as the averages of three individual experiments containing three replicates per condition.

RNA preparation

Total RNA was extracted from the cell samples using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The RNA integrity was analyzed using the Agilent 2100 Bioanalyzer (Agilent Technologies).

Microarray

Microarray analysis was performed by a commercial company (Oebiotech, PRC), using SurePrint G3 Human Gene Expression 8×60K v2 (Agilent Technologies) that is designed to contain approximately 12,000 lncRNAs. Briefly, samples were used to synthesize cDNA, and labeled cRNA was then synthesized and hybridized to the microarray. After hybridization and washing, processed slides were scanned with the Agilent Microarray Scanner (Agilent Technologies), and the acquired array images were analyzed using Agilent Feature Extraction Software (Agilent Technologies), which performs background subtractions. Quantile normalization and subsequent data processing were performed using the GeneSpringGX v. 11.0 software package (Agilent Technologies). A threshold of fold change >2 was used to screen upregulated or downregulated lncRNAs.

Real-time quantitative PCR validation

To validate the microarray data, we selected nine differentially expressed lncRNAs (MALAT1, H19, XIST, UCA1, CCAT1, LOC645249, LOC100128881, LOC728228, and LOC100292680). MALAT1, H19, XIST, UCA1, and CCAT1 were selected because they had been associated with cancer. The four remaining lncRNAs were selected randomly from all aberrantly expressed lncRNAs.

Quantification was performed with a two-step reaction process, i.e., through reverse transcription (RT) and PCR. Each RT reaction consisted of 0.5 μg RNA, 2 μL of PrimerScript Buffer, 0.5 μL of oligo dT, 0.5 μL of random 6 mers, and 0.5 μL of PrimerScript RT Enzyme Mix I (TaKaRa, Japan), in a total volume of 10 μL. Reactions were performed in a GeneAmp® PCR System 9700 (Applied Biosystems, USA) for 15 min at 37 °C, followed by heat inactivation of RT for 5 s at 85 °C. The 10 μL RT reaction mix was then diluted ×10 in nuclease-free water and held at –20 °C.

Real-time PCR was performed using LightCycler® 480 II Real-time PCR Instrument (Roche, Swiss) with 10 μL PCR reaction mixture that included 1 μL of cDNA, 5 μL of 2× LightCycler® 480 SYBR Green I Master (Roche, Swiss), 0.2 μL of forward primer, 0.2 μL of reverse primer, and 3.6 μL of nuclease-free water. Reactions were incubated in a 384-well optical plate (Roche, Swiss) at 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 30 s. At the end of the PCR cycles, melting curve analysis was performed to validate the specific generation of the expected PCR product. Three independent experiments were performed with each sample run in triplicate. The primers were synthesized by Generay Biotech (Generay, PRC) and are listed in Table 1. The expression levels of lncRNAs were normalized with respect to GAPDH and were calculated using the 2^–ΔΔCt method.

Statistical analysis

Student’s t-test was performed to evaluate any significant difference. A value of P<0.05 was considered statistically significant.

Increased invasive capability of SKOV3.ip1 cells

The abilities of the two cell lines to penetrate a uniform layer of extracellular matrix-resembling material were tested using Transwell invasive assay to compare their invasive capabilities. Cells were seeded on top of a porous membrane. Fetal bovine serum was used as a chemoattractant in the lower compartment. The results showed a marked increase in the invasive ability of the SKOV3.ip1 cell line compared with that of the SKOV3 cell line (Figure 1).

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1

SKOV3.ip1 cells are more invasive than SKOV3 cells (*P<0.01).

Differentially expressed metastasis-related lncRNAs

A total of 4,956 lncRNAs were detected in the microarray. Data analysis showed that, compared with the parental SKOV3 cells, more than a 2-fold deregulation was found in 1,161 lncRNAs in SKOV3.ip1 cells, of which 583 were lncRNAs were upregulated and 578 downregulated. The expression levels of nine lncRNAs were validated using RT-qPCR, and seven confirmed the deregulation found by microarray analysis (Table 2).