Si-RNA mediated knockdown of CELF1 gene suppressed the proliferation of human lung cancer cells

To evaluate the levels of CELF1 expression in lung cancer tissues and normal tissues, real time PCR was performed in 10 lung cancer tissues and 10 normal tissues. Results showed that the relative expression levels of CELF1 were higher in lung cancer tissues compared with the normal tissues (Figure 1A and Table 1). Moreover, the expression levels of CELF1 in lung cancer tumors varied depending on tumor grade. CELF1 expression levels were higher in low-grade cancers compared with high-grade cancers (Figure 1B). A higher expression level of CELF1 was observed in patients with the increase of T stage, indicating that when tumors begin to grow larger, gradually some other mechanism(s) may play a more important role to promote cancer cell growth, so the CELF1 expression level had some decrease. Furthermore, comparison of the survival rate of patients with N1 and N2 lymph node metastasis and without lymph node metastasis showed that the survival rates were significantly higher in the absence of lymph node metastasis (Figure 1C). In comparing the survival rate with CELF1 expression levels, no significant difference in survival rate was observed between patients with higher CELF1 expression and lower expression (Figure 1D). Together these data indicate that CELF1 expression is not related to postoperative survival in lung cancer patients.

Next, CELF1 mRNA and protein levels in A549 and H1299 lung cancer cells were evaluated by real time PCR and western blot, respectively. As shown in Figure 2, both gene and protein expression of CELF1 were detected in A549 and H1299 cells. The CELF1expressing lung cancer cells were then infected with lentivirus containing CELF1 shRNA or non-silencing control shRNA, and successful infection was confirmed by green fluorescence of infected cells (Figure 3A and B). Fluorescence analysis showed that the lentiviral infection rate was higher in H1299 cells than in A549 cells. Infection of cells with lentivirus containing CELF1 shRNA significantly reduced CELF1 gene and protein expression levels in both A549 and H1299 cells (Figure 3C-F). In contrast, the non-silencing siRNA infection had no effect on CELF1 levels, confirming that CELF1 expression levels were reduced specifically from CELF1 siRNA.

The effect of CELF1 knockdown on lung cancer cell survival was analyzed using a MTT assay performed over a five-day time course. In both cell lines, significant differences in cell survival were not observed until three days after infection. From the 4^th day of infection onwards, significant differences in cell survival were observed in both cell lines. The survival rate of CELF1 knockdown cells was markedly (P < 0.0001) reduced at the 5^th day of infection compared with the non-infected and control siRNA infected A549 and H1299 cells (Figure 4). The reduction in cell survival due to CELF1 knockdown was higher in A549 cells than in H1299 cells.

Lung cancer cells tend to form large cell colonies while in culture; therefore, we next evaluated the effect of CELF1 silencing on the colony forming ability of lung cancer cells. As shown in Figure 5A and B, the size of colonies in CELF1-silenced A549 and H1299 cells was markedly reduced compared with the control groups. Similarly, the number of colonies was also significantly (P < 0.001) reduced with the CELF1 gene knockdown. Together these results indicate that CELF1 plays an important role in the growth progression of lung cancer cells.

Fifty-three pulmonary cancer samples of fresh frozen tissue were acquired from the Department of Thoracic Surgery, Beijing cancer hospital, under approval from the Ethical Committee. Written consent statements were obtained from all patients before operation. None of the patients received any neoadjuvant therapy prior to surgery. The tissues were collected immediately after surgical resection at the Beijing Cancer Hospital and stored at the Tissue Bank of Peking University Oncology School. Clinicopathological characteristics of the tumors were defined according to the TNM staging system criteria of UICC. Clinicopathological factors are shown in Table 1.

AgeI, EcoRI, and SYBRGreen Master Mix Kits were purchased from TaKaRa (Dalian, China). pHelper1.0, pHelper 2.0, and pGCSIL-GFP plasmids were purchased from Genechem Co. Ltd (Shanghai, China). The RNeasy Midi Kit was from Qiagen (Valencia, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI 1640) and fetal bovine serum (FBS) were obtained from Hyclone (Logan, UT, USA). Lipofectamine2000, TRIzol and SuperScriptII reverse transcriptase were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals were obtained from Sigma (St. Louis, MO, USA). The following antibodies were obtained from Santa Cruz: anti-CELF1 (1:1000 dilution), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase, 1:3000 dilution) and anti-mouse HRP (1:5000 dilution).

Human lung cancer cells (A549 and H1299) and human embryonic kidney (HEK) 293 T cell lines were obtained from the cell bank of Shanghai Institute of Cell Biology. A549 and 293 T cells were maintained in DMEM supplemented with 10% heat-inactivated FBS and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO₂. H1299 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO₂.

The sequences of CELF1 siRNA and non-silencing control siRNA were 5′-CTAGCCGGGATTGAAGAATGCCGGATATTCAAGAGATATCCGGCATTCTTCAATCTTTTTAAT-3′ and 5′-CTAGCCCGGTTCTCCGAACGTGTCACGTATCTCGAGATACGTGACACGTTCGGAGAATTTTTTTAAT-3′, respectively. The nucleotide sequences were inserted into the plasmid through the pFH-L vector (Shanghai Hollybio, China) and the generated lentiviral-based shRNA-expressing vectors were confirmed by DNA sequencing. Lentiviruses were generated by transfection of 293 T cells at 80% confluence with generated plasmids. The cells were starved for 2 h before transfection, and pFH-L-shCELF1 or -shCTRL and the packaging vector carriers pVSVG-I and PCMVΔR8.92 were transfected into cells using Lipofectamine 2000. The supernatant was collected 48 h after transfection and lentiviral particles were harvested by ultracentrifugation (4000 g) at 4°C for 10 min. The collected virus particles were filtered through a 45 μM filter and the filtrate was centrifuged (4000 g at 4°C) for 15 min to collect the viral concentrate.

A549 and H1299 cancer cells were then infected with CELF1 shRNA- or control shRNA-expressing lentiviruses at a MOI of 30 for A549 cells or MOI of 15 for H1299 cells. The cells were seeded (5 × 10⁴ cells per well) in six-well plates, and after 24 h of incubation, the culture medium was replaced with Opti-MEM medium containing the appropriate amount of the virus. The cells were then incubated with the virus for another 48 h. Successful transfection was confirmed by observation through a fluorescence microscope (Leica, Germany) for expression of green fluorescence protein.

RNA was obtained using the Total RNA Isolation Reagent (ABgene™) according to the manufacturer’s instructions (Abgene, Surrey, UK). Total RNA was converted to cDNA using DuraScript™, a commercial reverse transcription kit from Sigma Aldrich. Real-time quantitative PCR analysis for the RNA extracted from lung cancer tissues and cultured lung cancer cells was performed using the SYBR Green Master Mix Kit (Applied Biosystems, Foster City, CA). In brief, each PCR reaction mixture contained 10 μl of 2 × SYBR Green Master Mix, 0.4 μl of sense and antisense primers (2.5 μM) and 10 ng of cDNA. Reactions were run for 40 cycles, including denaturation at 95°C for 10 min and annealing at 60°C for 1 min in a total volume of 20 μl using an ABI 7500 Real Time PCR platform. The primer sequences for PCR amplification of the CELF1 gene were 5′-ACCTGTTCATCTACCACCTG-3′ and 5′-GGCTTGCTGTCATTCTTCG-3′. Primer sequences for the internal control GAPDH were 5′-GACCCCTTCATTGACCTCAAC-3′ and 5′-CTTCTCCATGGTGGTGAAGA-3′. Relative gene expression levels were calculated using 2-ΔΔCT analysis.

For the cell viability analysis, A549 and H1299 cells were first seeded (2 × 10³ cells/well) into a 96-well plate and infected with CELF1 silencing or non-silencing siRNA-containing lentivirus for 72 h. Following infection, 20 μl of MTT solution (5 mg/ml) was added to each well and cells were incubated at 37°C for 4 h. The medium and MTT from the wells was removed and 200 μl of DMSO was added to each well. The optical density was measured using a microplate reader at 490 nm. Experiments were performed in triplicate.

Lung cancer cells seeded in six-well plates (2 × 10² cells/well) were infected with CELF1 silencing and non-silencing siRNA-containing lentivirus for 72 h. The cells were continuously incubated, and medium was replaced with new medium every three days until 8 days of culture. The cells were then washed with PBS and fixed with 4% paraformaldehyde. The fixed cells were stained with freshly prepared diluted Giemsa stain for 20 min. The cells were washed with double distilled water and colonies were counted using a fluorescence microscope.