The tumor suppressor gene RBM5 inhibits lung adenocarcinoma cell growth and induces apoptosis

This study was approved by the Institutional Review Board of the Second Affiliated Hospital of Jilin University, Changchun, China. Thirty pairs of tissues and the information of disease history were collected from the patients, who had been diagnosed with primary lung adenocarcinoma. All participants underwent surgery and provided written informed consent. Samples were snap-frozen in liquid nitrogen at the time of study and stored at −80°C until RNA or protein was extracted following the routine protocol. All cases were reevaluated by pathologists for confirming tumor histology and tumor content.

A549 cell line was purchased from the American Tissue Type Collection (Manassas, VA). Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum as previously described [21].

A549 cells were transiently transfected with pcDNA3.1 or pcDNA3.1-RBM5 plasmids using the Lipofectamine 2000 reagent (Roche, Switzerland) for the indicated times according to the manufacturer’s instruction. Briefly, 1 × 10⁵ cells were seeded into six-well plates containing an antibiotic-free medium and incubated overnight. For each well, 2 μg DNA (pcDNA3.1 or pcDNA3.1-RBM5) was mixed with 95 μl RPMI-1640 with 10% fetal bovine serum. The mixture was then combined with a solution of 5 μl Lipofectamine (Roche, Switzerland) in 95 μl RPMI-1640 with 10% fetal bovine serum. After a 20-min incubation period at room temperature, the mixture was applied to the cells in an appropriate volume of OPTI-MEM I so as to achieve a final volume of 2 ml. Then the cells were cultured for an additional 24 h/48 h at 37°C before analysis.

Cell proliferation assay was evaluated using MTT according to the manufacturer’s instruction. Briefly, A549 cells transfected with pcDNA3.1 or pcDNA3.1-RBM5 plasmids were seeded in a 96-well plate at a density of 1 × 10⁴ cells per well (0 hour). At 24 h and 48 h, 20 μl of 5 mg/ml MTT in PBS was added, and the cells were subsequently incubated for 4 h at 37°C in 5% CO₂. Cells were then washed twice with PBS, and the precipitate was solubilized in 150 μl of 100% dimethylsulfoxide (Sigma, USA) by shaking for 5 min. Absorbance was measured using a microplate reader (Bio-Rad, Richmond, CA) at a wavelength of 570 nm. All experiments were carried out in triplicate.

Total RNAs were isolated using Trizol (Invitrogen, USA) according to the manufacturer’s instruction. Reverse transcription was performed with 3 μg of total RNA at a final volume of 10 μl, containing 10 mM dNTP, 0.5 μg oligo dT, 20 U RNasin and 200 U M-MLV reverse transcriptase (Promega Corp., USA). The primer sequences were RBM5: 5′-GCACGACTATAGGCATGACAT-3′ and 5′-AGTCAAACTTGTCTGCTCCA-3′, GAPDH: 5′-GAAGGTGAAGGTCGGAGTC3′ and 5′-GAAGATGGTGATGGGATTTC-3′. PCR was performed at 95°C for 3 min and 25–30 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min and 72°C for 10 min. All densitometry scanning was carried out using ImageMaster VDS Software (Pharmacia Biotech, USA).

Total cellular proteins from both lung tissues and A549 cells were extracted according to the previous study [22]. Protein samples (50 ug) were then separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA). The primary antibodies were rabbit anti-human RBM5, Bcl-2, PARP, cleaved-PARP, caspase-3, cleaved caspase-3 and β-actin antibodies from Abcam (MA, USA). The primary antibodies of rabbit anti-human caspase-9 and cleaved caspase-9 antibodies were from Cell Signaling Technology (USA). The second antibody was a goat anti-rabbit IgG-HRP from Santa Cruz Biotechnology (CA, USA). Western blot was carried out as previously described [22]. The protein bands were visualized by SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA), and the membranes were subjected to X-ray autoradiography. Band intensities were determined with Quantity One software (Bio-Rad, Hercules, CA, USA). Furthermore, we confirmed the reproducibility of the experiments at least three times.

A549 cells were transfected with pcDNA3.1 or pcDNA3.1-RBM5 plasmids using the Lipofectamine 2000 reagent (Roche, Switzerland) for 48 h, followed by harvesting, counting (1 × 10⁶ cells) and resuspending in 100 μl of phosphate-buffered saline (PBS). Afterward, 5 μl of AnnexinV (1 μg/ml) (Beckman Coulter, Fullerton, CA) was added and incubated at RT for 15 min, then 10 μl of propidium iodide (PI 1 μg/ml) was added and incubated for additional 5 min at room temperature in the dark. Finally, the cells were subjected to flow cytometry (FCM) to measure the apoptosis rate with an Epics-XL-MCL flow cytometer (Beckman Coulter, USA).

The models of A549 xenografts were established using BALB/c nude mice (6 weeks old) that were acquired from the Institute of Zoology, Chinese Academy of Sciences (Beijing, China). Use of animals was in accordance with Animal Care guidelines, and the protocol was approved by Jilin University Animal Care Committee. Cultured cells were washed and resuspended in phosphate-buffered saline (PBS). The suspension (5 × 10⁶ cells in 150 μl/ per mouse) was inoculated subcutaneously into the right flanks of nude mice. The sizes of the tumors were measured starting from day 7 after cell injection until day 21 using calipers. These tumor-bearing mice were then divided randomly into two groups (six mice per group): (1) PcDNA3.1 as the control group; (2) pcDNA3.1-RBM5 as the RBM5 group. Plasmids were carried by bioengineered attenuated strains of Salmonella enterica serovar typhimurium using electrotransfection as previously described [22, 23]. Briefly, plasmids were electrotransfected into Salmonella typhi Ty21a competent cells before use. Mice in each of these groups were inoculated with bacteria with control vector (pcDNA3.1) or pcDNA3.1-RBM5 plasmids [10⁸ colony-forming units (CFU) per 50 μl] via tail vein injection two times (on day 28 and 35). Tumors were measured using calipers every 4 days for 42 days in total, and the data were plotted using the Kaplan-Meier method to analyze the tumor growth curves. In addition, the wet weight and sizes of tumors were measured when mice were killed. To ensure the tumor-preferable distribution of the bacteria, an additional pilot study was performed before the above-described animal experiments. Tissue samples of the primary tumor, liver, lung, spleen, heart and kidney from mice were used for bacterial distribution analysis on day 3 and 7 after injection of attenuated Salmonella carrying plasmid. Equal amounts of tissues were collected, minced, and homogenized. Afterward, the homogenized tissues were plated in triplicate onto Luria-Bertani agar containing ampicillin (100 mg/ml) for 24 h, followed by counting of bacterial colonies and CFU evaluation.

The χ² and Fisher’s exact tests were used to analyze the association of mutations with clinical characteristics. All p values were calculated based on a two-tailed hypothesis. p <0.05 was considered statistically significant.

To assess the expression of RBM5 mRNA and protein in human lung adenocarcinoma, we performed RT-PCR and Western blot analysis on 30 pairs of primary lung tumor versus adjacent non-tumor tissues. Our results showed that the expression of both RBM5 mRNA and protein was decreased in lung tumor compared to that in the non-tumor counterpart (Figure 1C, χ 2 = 2.814, P < 0.05; Figure 1D, χ 2 = 2.963, P < 0.05). When the bands were quantitatively compared, expression of RBM5 mRNA was found to be lower in tumor compared to the non-tumor counterpart in the majority of the paired samples (except in 9 of the 30 patients, Figure 1A, C). Similarly, when the bands were quantitatively compared, the expression of RBM5 protein was found to be lower in tumor compared to the non-tumor counterpart in the majority of the paired samples (except in 7 of the 30 patients, Figure 1B, D). Our result confirms the previous finding that RBM5 expression was decreased in human lung adenocarcinoma tissues compared to that in the corresponding non-tumor tissues.

To further determine the relationship between RBM5 gene and tumor growth, we performed the proliferation assay in A549 cells to explore the effect of RBM5 overexpression on proliferation of tumor cells. We transiently transfected A549 cells with pcDNA3.1-RBM5, then the expression level of RBM5 in A549 cells was determined by RT-PCR and Western blot, presented in Figure 2. MTT assays were performed at 24 h and 48 h, respectively, after the transfection. Results showed that there was a significant inhibition of cell proliferation in RBM5-overexpressing A549 cells compared to that in controls (Figure 3 A, B). Along with the extension of the time, the inhibition rate was increased.

We next explored the mechanisms underlying growth inhibition by RBM5. To determine the contribution of cell death induced by RBM5, we employed FCM to detect apoptotic cells, which are characterized by phosphatidyl serine (PS) translocation on the outer cell membrane. Cells were double-stained with AnnexinV and PI after 48 h transfection. Representative raw FCM data are as follows: Cells transfected with RBM5 showed a higher proportion of early and late apoptosis (13.41% and 18.28%) as compared to the control cells (4.01% and 7.94%), respectively (Figure 4). The early and the late apoptotic cells were distributed in the Q2 and Q3 regions, respectively. The necrotic cells were located in the Q4 region. The result suggested that overexpression of RBM5 significantly increased apoptosis in lung cancer cells.

To further study the possible molecular mechanisms underlying RBM5-induced cell apoptosis, we next examined the expression of some apoptosis-related genes including Bcl-2, caspase-3, caspase-9 and PARP. We observed that the expression of Bcl-2 protein was decreased significantly when RBM5 was overexpressed, while the expression of cleaved caspase-3, caspase-9 and PARP proteins was significantly increased in the RBM5 overexpressing cells as compared to that in the control cells. Accordingly, the expression of caspase-9 protein was slightly decreased in RBM5 overexpressing cells as compared to that in the control cells, while the expression of caspase-3 and PARP had no significant difference in either group (Figure 5). This result again suggested that RBM5, by decreasing Bcl-2 expression, could induce caspase-3, caspase-9, PAPP cleavage and promoted apoptosis.

To further test our hypothesis, the model of A549 xenograft was established as described in Materials and methods. At the 28th day after implantation, the tumor-bearing mice were inoculated with bacteria carrying the pcDNA3.1 vector tumor with 10⁸ CFU in 50 μl PBS per mouse. To ensure that attenuated Salmonella typhi Ty21a carrying plasmids preferentially localized in the tissue, we monitored the kinetics of bacterium distribution in the xenograft tumor and different organs of the tumor-bearing area at certain post-injection times (Figure 6A). On day 3 (72 h) after injection, bacteria could be found predominantly in the tumors compared to other organs (Figure 6 A, B; p < 0.05), which indicated the tumor-targeting character of attenuated Salmonella typhi Ty21a. We found that the concentration of bacteria gradually decreased in the tumors, however, which was found decrease or vanish significantly in other organs on day 7 after injection (Figure 6 A, C). We concluded that the bacteria could be accumulated in the tumors in the course of treatment since we injected the mice every 7 days from day 28 to day 42. Quantitative analysis of the bacteria by CFUs as described in Materials and methods (Figure 6B and C) confirmed the predominant distribution of bacteria in the tumor tissues.

The potential therapeutic effect on lung adenocarcinoma xenograft growth by RBM5 was examined in tumor-bearing mice treated with bacteria carrying RBM5 plasmid (Figure 7A). Dynamic tumor growth was monitored from day 7 to day 42 after injection. We showed that, while the sizes of the tumor xenografts between RBM5 and control groups were similar before day 28, the growth of tumor xenografts in the mice treated with RBM5 decreased after the 28^th day (Figure 7B). In addition, the weight of the tumor xenografts from mice treated with RBM5 became significantly lighter than that in the control group at the 42^nd day when the mice were killed (Figure 7C). This result suggested that accumulative and stable expression of RBM5 in A549 xenograft BALB/c nude mice significantly retarded the tumor growth rate in vivo. Moreover, we set up a novel animal model using BALB/c nude mice treated with attenuated Salmonella as a vector carrying plasmids to determine RBM5 function in vivo.

To further explore whether it possesses the same molecular mechanisms underlying RBM5 functions on both in vitro and in vivo, we examined the expression of Bcl-2 and caspase-3 proteins in A549 xenograft. Similarly, we found that the expression of Bcl-2 was significantly decreased in A549 xenograft BALB/c nude mice treated with RBM5 as compared to the control mice, while the expression of cleaved caspase-3 was significantly increased in RBM5-treated mice (Figure 8). This result, consistent with the cell line study, suggested again that RBM5 inhibited Bcl-2 expression, triggered cleavage of caspase-3, promoted apoptosis and suppressed the tumor growth in vivo.