The HCT116 cells were cultured in McCoy's 5A medium (M&C Gene Technology Ltd., Beijing, China). The A549, H1975, H1299 and NCM460 cells were cultured in RPMI-1640 (M&C Gene Technology Ltd.). The BEAS-2B, HCoEpic and SW620 cells were cultured in DMEM (M&C Gene Technology Ltd.). All culture media were supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), penicillin and streptomycin. All the cells were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and incubated in a humidified atmosphere of 5% CO₂ at 37°C.

Small interfering RNA (siRNA) transfections were performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. siRNA synthesis was performed by Shanghai GenePharma Co., Ltd. (Shanghai, China) and the siRNA sequences for human SRSF7 were as follows: SRSF7-1, 5′-AGGAGAGUUAGAAAGGGCU-3′; and SRSF7-2, 5′-GCAUCUCCUCGACGAUCAA-3′. The sequence of the control siRNA was 5′-UUCUCCGAACGUGUCACGUTT-3′.

The methods were performed as described previously (29). For western blot analysis, cells were lysed with radioimmunoprecipitation assay cell lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and quantified using a bicinchoninic acid assay Kit (Beijing Solarbio Science & Technology Co., Ltd.). Equal amounts of protein (30 µg) were separated via SDS-PAGE (12% gel) and then transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membrane was blocked for 1 h with 5% skimmed milk at room temperature and then incubated with primary antibodies overnight at 4°C. The primary antibody against SRSF7 (AP12306a, 1:500) was purchased from Abgent, Inc. (San Diego, CA, USA). The primary antibodies against β-tubulin (10068–1-AP, 1:1,000) and β-actin (60008–1-Ig. 1:3,000) was purchased from Proteintech Group, Inc. (Rosemont, PA, USA). Following three washes in Tris-buffered saline with Tween-20, the membrane was incubated with secondary goat anti-rabbit (SA00001-2) or goat anti-mouse (SA00001-1) antibody (Proteintech Group, Inc) for 1 h at 37°C with a dilution of 1:3,000. Finally, the proteins were visualized using EasySee Western Blot Kit (Beijing TransGen Biotech Co., Ltd., Beijing, China), imaged and quantified using ChemiDoc MP Imaging System (Image Lab Software, version 4.1; Bio-Rad Laboratories Co., Ltd., Hercules, CA, USA).

For RT-PCR, total RNA was extracted using TRIzol reagent (Life Technologies; Thermo Fisher Scientific, Inc.), and reverse transcription was performed using a Reverse Transcription system (Promega Corporation, Fitchburg, WI, USA). Total RNA (4 µg) was reverse transcribed using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Beijing TransGen Biotech Co., Ltd.) according to the manufacturer's protocol. The thermocycling protocol was listed as follows: Initial denaturation at 95°C for 5 min, followed by 30 repeats of the threestep cycling program consisting of 30 sec at 95°C (denaturation), 30 sec at 60°C (primer annealing) and 30 sec at 72°C (elongation), followed by a final extension step for 5 min at 72°C. The primers used were as follows: SRSF7, forward 3′-GCGGTACGGAGGAGAAAC-5′ and reverse 3′-TCGGGAGCCACAAATCAC-5′; Fas, forward 3′-GAACATGGAATCATCAAGGAATGCAC-5′ and reverse 3′-AGTTGGAGATTCATGAGAACCTTGG-5′. The primers used to detect the alternative splicing of Fas were as follows: FAS-L, forward 3′-TGCAAAGAGGAAGGATCCAG-5′; FAS-S, forward 3′-CCAAGTGCAAAGAGGAAGTGA-5′; and FAS-L and FAS-S reverse 3′-GGAGATTCATGAGAACCTTGG-5′ (26). The housekeeping gene GAPDH was used as the internal control.

The HCT116 and A549 cells were transfected with control siRNA or SRSF7 siRNA for 60–72 h. Following transfection, 1×10⁶ cells were harvested, washed twice in PBS and double-stained with Annexin V-FITC and PI using an Annexin V-FITC/PI Cell Apoptosis Detection kit (Beijing TransGen Biotech Co.) according to the manufacturer's protocol. Each sample was then quantitatively analyzed with an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA) at 488 nm emission and 570 nm excitation. Cell apoptosis was also examined using an Apo-ONE Homogeneous Caspase-3/7 assay (Promega Corporation) according to the manufacturer's protocol. Caspase substrate and buffer from the kit were added for cell lysis and incubated at room temperature for 18 h. Fluorescence was measured with an F-7000 spectrofluorometer (Hitachi, Ltd., Tokyo, Japan) at 521 nm emission and 499 nm excitation wavelengths.

The BEAS-2B cells were infected with a lentivirus LV5-negative control vector or a LV5-SRSF7 vector, which were purchased from Shanghai GenePharma Co., Ltd. The A549 cells were infected with lentivirus LV3-negative control, LV3-SRSF7 short hairpin shRNA-1 (3′-GATCAAGATCCAGGTCTATTT-5′) or LV3-SRSF7 shRNA-2 (3′-GAACTGTATGGATTGCGAGAA-5′), which were purchased from Shanghai GenePharma Co., Ltd. At 48 h post-infection, the cells were cultured in the aforementioned medium containing puromycin (0.5 µg/ml), and the medium was replenished every 2 days. After ~1 week, stable cell lines were obtained and verified by western blot analysis, as described above.

Immunohistochemistry was performed by Cybrdi, Inc. (Xi'an, China) on a multi-organ (colon, pancreas, lung, breast and prostate) tumor and normal tissue array (cat. no. MC1801, 26 spots for each tumor type), human colon carcinoma (grades I–IV) tissue array (cat. no. CO1801, 90 spots for cancer tissues and paracancerous normal tissues, respectively), and human lung carcinoma (grades I–III) tissue array (cat. no. LC10012a, 45 spots for cancer tissues and paracancerous normal tissues, respectively). The absent or abnormal tissues were not finally counted. Anti-SRSF7 (Abgent, Inc.) antibodies were used at a dilution of 1:50 (for array MC1801), 1:150 (for array CO1801) and 1:100 (for array LC10012a) at 4°C for overnight. Semi-quantitative analysis of the stained sections (H-score) was performed with a light microscope (Model, CX31; Olympus, Tokyo, Japan) by an independent pathologist.

All statistical analyses were performed by two-tailed Student's t-test or a one-way analysis of variance followed by Bonferroni's post-hoc test. Data are presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference. Computer-based calculations were conducted using SPSS 19.0 software (IBM Corp., Armonk, NY, USA).

To evaluate the role of SRSF7 in human cancer, its expression was analyzed on cancer arrays with different tissue origins by immunohistochemical staining. When compared with corresponding normal tissues, a high frequency of increased expression of SRSF7 was found in colon adenocarcinoma (21/24) and lung carcinoma (22/24). This result was confirmed with the specific human colon and lung cancer specimens. As shown in Fig. 1A and B, the immunostaining intensity of SRSF7, compared with paracancerous normal tissues, was more marked in the colon cancer samples (49/71) and lung cancer samples (28/39). These results suggested that SRSF7 offers potential as a marker for colon and lung cancer development, and may be involved in the development of these types of cancer.

The expression of SRSF7 was analyzed in normal and cancerous human cell lines of the colon and lung. Western blot analysis revealed the upregulation of SRSF7 in the colon cancer cell lines (HCT116 and SW620) and lung cancer cell lines (A549 and H1299), compared with the normal cells (Fig. 2A and B). Among the colon and lung cancer cell lines, HCT116 and A549 cells were used in subsequent assays. To examine the function of SRSF7 in colon and lung cancer cell lines, downregulation experiments were performed to reduce the expression of SRSF7. The effect of SRSF7-inhibition on cell proliferation was then evaluated. The knockdown of SRSF7 was achieved in HCT116 and A549 cells using two different siRNAs, and its downregulation was confirmed by western blot analysis (Fig. 2C and D). The results of MTS assays demonstrated that the inhibition of SRSF7 markedly decreased the viabilities of the HCT116 and A549 cells, compared with those of the cells transfected with the control siRNA (Fig. 3A and B). These results suggested that SRSF7 is required for the proliferation of colon and lung cancer cells.

To investigate the effect of the downregulation of SRSF7 on apoptosis, the rates of total apoptosis in the HCT116 and A549 cells were detected and quantified by flow cytometric analysis. The results showed that SRSF7 knockdown significantly increased the percentage of apoptotic cells (P<0.01) in the HCT116 and A549 cells, compared with the cells transfected with control siRNA (Fig. 4A-D). The induction of apoptosis in these cells was also confirmed by an increase in caspase 3/7 activity (Fig. 4E and F). These data indicated that SRSF7 is critical for the survival of colon and lung cancer cells.

To identify the alternative splicing events regulated by SRSF7, which may contribute to its effects on proliferation and apoptosis, the effects of the upregulation or downregulation of SRSF7 on the altered splicing events in cancer were examined. Non-malignant BEAS-2B lung epithelial cells were used to establish a stable SRSF7-overexpression cell line and A549 cells were used to establish a cell line with stable SRSF7 knockdown. In these stable cell lines, it was found that the splicing of Fas receptor was altered upon upregulation or downregulation of SRSF7. It has been reported that the alternative splicing of Fas exon 6 generates either a membrane-bound receptor that promotes apoptosis, or a soluble isoform that inhibits apoptosis (21). The results of the present study revealed that the upregulation of SRSF7 in BEAS-2B cells increased the skipping of Fas exon 6, whereas SRSF7 knockdown in A549 cells increased exon 6 inclusion (Fig. 5A and B). These results suggested that SRSF7 regulated the alternative splicing of Fas and promoted production of the more soluble, pro-survival variant.