Impact of RNAi-Targeted Silencing of Survivin and Hypoxia-Inducible Factor-1α on the Biological Behaviors of Gastric Cancer SGC-7901 Cells In Vitro

Hypoxia-inducible factor-1 (HIF-1) plays crucial roles in the primary transcriptional responses to hypoxia stress by modulating hypoxia gene expression. Survivin is regarded as an effector downstream of HIF-1 that promotes cell survival under hypoxia conditions. HIF-1α and survivin are proposed as potential targets in the treatment of gastric cancer. This study was designed to investigate the anticancer effects of RNA interference targeting survivin and HIF-1α on the gastric cancer cell line, SGC-7901 under hypoxia. In this study, cultured cells were divided randomly into four groups: a blank control group, a scrambled siRNA (SCR) control group, a siRNA-Survivin group (ss group) and a siRNA-Survivin+siRNA-HIF-1α group (sis+siH group). The siRNA targeting survivin and/or HIF-1α genes were transfected into SGC-7901 cells. The expression of survivin and HIF-1α was identied by RT-PCR and Western blotting assay. Apoptosis was determined by ow cytometry. The invasion and migration abilities of the cells were evaluated by transwell and wound healing assays, respectively. Cell growth curves were determined by MTT assay at 48 h indicated that the viability of the SGC-7901 cells was inhibited in a time-dependent manner with a highest inhibitory rate of 82.1 ± 0.85 % at 48 h. The siRNA targeting the survivin gene decreased the expression of survivin and the combined transfection of siRNA targeting survivin and HIF-1α down-regulated the expression of both survivin and HIF-1α (P < 0.05). Compared with the blank control group, the combined siRNA groups exhibited obvious features with decreased invasion and migration abilities (P < 0.05). The apoptosis rate increased and the cell proliferation decreased following gene silencing (P < 0.05). The down-regulation of survivin and HIF-1α may induce an anticancer effect in SGC-7901 cell lines by enhancing cell apoptosis and decreasing proliferation, migration and invasion ability.


Introduction
Gastric cancer is the second most common cancer worldwide [1]. Patients with gastric cancer at even early stage usually have distant metastasis and poor prognosis despite the combined therapy with surgery, chemotherapy and radiotherapy [2]. Hypoxia is a shared feature and form of physiological stress on various cancers. Hypoxia stress can facilitate adaptive changes in tumor cells, which eventually leads to increasing tumor growth, invasion and metastasis [3,4]. Accumulating evidence suggests that hypoxia, resulting from highly active tumor cell proliferation, is related to poor prognosis and development of resistance to chemotherapy and radiotherapy [5,6]. Hypoxia-inducible factor-1 (HIF-1) is an essential transcriptional regulator, also known as hydrocarbon receptor nuclear translocator, which plays crucial roles in the primary transcriptional responses to hypoxia stress by modulating hypoxia gene expression [6]. Structurally, HIF1 consists of HIF-1α and HIF-1β subunits [7]. These heterodimeric transcription molecules activate a series of phosphoglycerate kinase and vascular endothelial growth factor (VEGF) genes by response to hypoxia [8]. Survivin, a member of the inhibitor of apoptosis (IAP) family, acts as a key regulator of angiogenesis in addition to to tumor progression [9].
RNA interference (RNAi) is a mechanism of transcriptional regulation for speci c gene expression silencing in the majority of eukaryotic cells [10,11]. The process is mediated by small RNA (siRNA) molecules 21-23 nucleotides in length [12,13]. There is emerging evidence that RNAi plays an important regulatory role in the articulated molecular mechanism triggered by hypoxia stress. Our previous studies demonstrated that RNAi targeting survivin in BGC-823 cell lines may attenuate the anti-proliferative and apoptosis-inducing effects under hypoxia [14]. However, overexpression of HIF-1α was associated with the poor prognosis in gastric cancer [15]. Currently, it is well known that HIF-1 transactivates the survivin gene by directly targeting the survivin promoter. Both HIF-1α and survivin are activated, along with high over expression of VEGF in tumor cells under hypoxia, which results in endothelial cell proliferation, migration and neovascularizaiton. HIF-1α and survivin are proposed as potential targets in the treatment of gastric cancer. Accordingly, we sought to investigate how RNAi targeting HIF-1α mediates the growth of gastric cancer cell line SGC-7901 combined with survivin.

Materials And Methods
Cell culture and hypoxia treatment The human gastric cancer cell line SGC-7901 was obtained from the China Center for Type Culture and was maintained in RPMI-1640 medium (Hyclone, Beijing, China) containing 10% fetal bovine serum (Sijiqing Bio. Co., Ltd., Hangzhou, China) and 1% penicillin/streptomycin, in a 5% CO 2 humidi ed atmosphere at 37 °C incubation. The medium was changed at alternate days and cells were harvested at 70-80% of con uency for experimental purposes. After seeding for 24 h, cells were incubated in normal or hypoxic conditions for a further 48 h (for hypoxic treatment, cells were maintained in a hypoxia incubator of 1% oxygen concentration infused with 5% CO 2 and nitrogen gas mixture).
The cultured cells were randomly divided into four groups: a blank control group, a siRNA-Survivin group (ss group), a siRNA-Survivin+siRNA-HIF-1α group (sis+siH group) and a SCR control group. For siRNA transfection, SGC-7901 cells were prepared on 6-well plates and incubated overnight to achieve 80-90% con uency. The cells were transfected with 100 nmol/L siRNAs using Hifectin (Applied Gen Co., Ltd., Beijing, China) according to the protocol provided by the manufacturer. Transfection e ciency rates cells displaying green uorescence total count cells×100 . Subsequently, the exponentially growing SGC-7901 cells were maintained under hypoxic conditions for further experiments. Three independent repeats were conducted for all experiments.
The PCR program was as follows: denaturation at 95˚C for 5 min; 30 cycles of 94˚C for 30 s, annealing at 50˚C for 30 s and 72˚C for 1 min, with a nal step at 72˚C for 5 min. PCR products were separated using 2% agarose gel, and the bands were scanned and the relative mRNA expression levels were determined by comparing with the expression of GAPDH.

Western blotting analysis
After treatments, total protein was extracted from cells using radio -immunoprecipitation assay (RIPA) buffer (Beyotime Biotech Co., Ltd., Wuhan, China) and the level of protein was determined using the bicinchoninic acid assay method. Equal amounts of protein lysates (50 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene uoride membranes (EMD Millipore Co., Hayward, CA, USA). The membranes were blocked with 5% skimmed milk for 1 h and then incubated at 4˚C overnight with monoclonal rabbit anti-mouse antibodies against HIF-1α, GAPDH (Boster Co., Ltd., Wuhan, China) and survivin (Biosynthesis Co., Ltd., Beijing, China). On the following day, the membranes were washed with TBST and incubated for 1 h at room temperature with the secondary peroxidase-labeled goat anti-rabbit antibody (Boster Co., Ltd, Wuhan, China) diluted to 1:2000 in skimmed milk/TBST. The protein bands were visualized by enhanced chemi-luminescence and the band intensity was measured using Quantity One v4.6.2 software (Bio-Rad, California, USA). Quantitative analysis of the relative levels of target proteins was determined using the NIH ImageJ software.
Flow cytometry For analysis of apoptosis, an annexin V-FITC apoptosis detection kit was used (Invitrogen, USA). After treatment for 48 h, SGC-7901 cells were harvested, washed twice with PBS and resuspended in 500 μL of binding buffer. Cell suspensions were then incubated with 5 μL of annexin V-FITC and 5 μL of propidium iodide (PI) for 10 min at room temperature away from light. The apoptotic rates were calculated based on the number of these transfected gastric cancer cells in the apoptotic state and evaluated immediately by ow cytometry (BD Biosciences, Franklin Lakes, NJ) . The mean uorescence intensity of annexin-V-FITC/PI was determined by ow cytometry. Then the apoptotic rates were calculated at the mean uorescence intensity. At last, the results were quanti ed using WinMDI 2.9 analysis software.
Wound healing assay Scratch (wound-healing) assays were performed to determine cell migration ability. SGC-7901 cells were seeded in plates at a density of 3×10 5 cells/well and incubated overnight before being treated as described above. After 24 hours, cells were maintained in normoxic or hypoxic conditions and scratched using a sterile 200-μL tip to create a wound. Cells were then washed three times with PBS to remove debris, and were cultured in a serum-free medium for further 24 hours. The width of the wound area was monitored and measured at more than three positions per scratch by using microscopy to compare the migration ratios among the groups.

Invasion assay
Matrigel invasion assays were employed to assess the invasion of SGC-7901 as previously described.

Statistical analysis
Data were expressed as mean values with standard error of the mean (± SEM). Statistical analysis was performed using Student's t test with SPSS11.0 for Windows. All experiments were performed in triplicate. Differences were considered statistically signi cant at P < 0.05.

Rates of siRNA transfection
We observed the expression of transfection e ciency of siRNA under uorescence microscopy for 6 h in SGC-7901 cells. The results showed a higher transfection e ciency with more than 80% of cells displaying green uorescence due to the uorescent-labeled siRNA in both ss group and sis + siH group compared with a SCR control group and blank control group (Fig. 1).

Effect of HIF-1α or/and survivin RNAi on SGC-7901 cell viability under hypoxia
Cell growth curves determined by MTT assay at 48 h showed that cell viability was inhibited in a timedependent manner with the highest inhibitory rate at 82.1 ± 0.85% after 48 h in the siH + sis group compared with the control group and the SCR control group (P < 0.05, Fig. 2).

Individual inhibitory effects of siRNAs targeting HIF-1α and survivin under hypoxia
We further evaluated the silencing effects of siRNA-HIF-1α and siRNA-survivin in SGC-7901 cell. After transfection, cells were maintained under hypoxia. After 48 h, the expression levels of HIF-1α and survivin were determined by RT-PCR and Western blotting analysis. The ndings showed that the expressions of HIF-1α and survivin were inhibited in the siH and sis groups at the mRNA and protein levels. The expression levels of HIF-1α and survivin mRNA and protein were signi cantly reduced simultaneously compared to control cells (P < 0.05, Fig. 3 and Fig. 4).

Effect of HIF-1α or/and survivin RNAi on SGC-7901 cell apoptosis under hypoxia
Apoptosis was assessed for 48 h after transfection by staining cells with Annexin V/PI and analyzed using an FCM. The strongest apoptotic signals were identi ed in the siH + sis groups and the percentages of apoptotic cells were 11.5 ± 2.5. The results indicated that the apoptosis rates of SGC-7901 cells in the siH + sis groups were higher than those in the control group (P < 0.05, Fig. 5).

Effect of HIF-1α or/and survivin RNAi on migration ability of SGC-7901 cells under hypoxia
Woundhealing assays were performed and the width of the wounded area measured at more than three positions per scratch by microscopy at 12 h and 24 h. A signi cant difference was found in the migration ratios amongst the groups. The migration of SGC-7901 cells of the siH + sis group (198.6 ± 14.80 mm) decreased by 45.48% (P < 0.05) compared with control group (464.0 ± 15.04 mm), whilst the SCR group showed no signi cant difference (497.4 ± 25.57 mm, P > 0.05, Fig. 6).

Effect of HIF-1α or/and survivin RNAi on invasive ability of SGC-7901 cells under hypoxia
In the meanwhile, the invasion assay revealed that the invasive number of SGC-7901 cells on the control and SCR groups were 91.33 ± 4.055 and 85.33 ± 4.256 respectively, whereas the invasion cells were 24.41 ± 2.41 in the siH + sis groups (P < 0.05, Fig. 7) .

Discussion
RNAi technique has been widely used in the functional analysis of mammalian genes using synthetic 19-23-nucleotides double-stranded RNAs as siRNA [15,16,17]. RNAi as a target-speci c gene suppression technology has provided a promising way for gene therapy in various diseases, particularly in cancer [18,19,20]. The e ciency of RNAi on the same target usually shows great difference. One of the critical preconditions for RNAi therapy is the appropriate siRNA that can e ciently knock down the expression of target genes [21,22]. While the activated oncogene mRNA was be effectively inhibited by RNAi technology, the tumor cell growth, proliferation, invasion, vascularization and extravasation were also be inhibited [23,24,25]. Tamura [27].
In this study, we employed two pairs of siRNAs targeting survivin and HIF-1a mRNA separately which were then transfected into SGC-7901 cells. On the basis of relative expression of mRNA and protein levels, our ndings con rmed that the two siRNAs were highly e cient in suppressing survivin and HIF-1a expression in SGC-7901 cells. The con rmation of e cient siRNA targeting human survivin and HIF-1a in this study warrants further research on survivin and HIF-1a. Our studies have shown that under hypoxic conditions, the expression of HIF-1a was higher in SGC-7901 cells and the results indicated a highe ciency transfection with more than 80% of cells displaying green uorescence. HIF-1a, as a major transcriptional factor activated by hypoxia, triggers a series of oncogenes in tumor development [28,29].
By activation of a large number of downstream target genes, tumor cells adapt to the hypoxia settings and continue to survive, followed by malignant proliferation, metastasis and even resistance to chemotherapy [30,31]. HIF-1a-survivin pathway provides a means whereby gastric cancer cells can survive and grow under hypoxia conditions, suggesting the pathway as a therapeutic target for the gene treatment of gastric cancer [32,33]. This nding was in an agreement with the publication of others [34,35].
We therefore explored the impact of further knockdown on the expression of survivin and HIF-1a in the gastric epithelial cell line SGC-7901. Compared with the SCR and the blank control group, the inhibitory rate of SGC-7901 had signi cantly lower expression levels of survivin and HIF-1α on the sis + siH. Compared with the blank control group, the expression levels of survivin and HIF-1a mRNA were signi cantly decreased along with survivin and HIF-1a proteins in the sis + siH group. The ow cytometry results showed that the apoptotic rate of SGC-7901 cells in the sis + siH group was signi cantly higher than those in the SCR and blank control groups. The results of the transwell migration assay indicated that the numbers of migration cells in the sis + siH group were signi cantly lower than those in the control group.
There are some limitations to our study. We did not detect the Bcl-2/Bax ratio. More experiments are needed to determine the expression of Bcl-2 and Bax and the exact mechanism underlying this pathway.

Conclusion
Our ndings suggest that RNAi targeting survivin and HIF-1a dramatically reduced cell growth and increased apoptosis in gastric cancer cell in vitro. RNAi against survivin and HIF-1α may be a potentially novel approach for the prevention and treatment of gastric canner. However, the precise underlying mechanisms of survivin and HIF-1a RNAi on proliferation and apoptosis in SGC-7901 cells remain to be fully determined and require further investigation.
Declarations Figure 1 Expression of green uorescence in SGC-7901 cells after transfection for 6 h due to the uorescentlabeled siRNA (×200). Note: Data are representative images (magni cation x 200) in individual groups of cells from three separate experiments.

Figure 3
Individual inhibitive effects of siRNAs on HIF-1α and survivin mRNA levels under hypoxia Note: GAPDH was used as internal control and normalized to 100%. The results were similar in three separate experiments. A, D Blank control group; B, E: SCR control group C, F: siRNA-Survivin+siRNA-HIF-1α group (sis+siH group). *P < 0.05 vs. the control.

Figure 4
Inhibitory effects of siRNAs on HIF-1α and survivin proteins under hypoxia Note: GAPDH was used as internal control and normalized to 100%. The results were similar in three separate experiments. A Blank control group; B: SCR control group; C: siRNA-Survivin group (sis group); D: siRNA-Survivin+siRNA-HIF-1α group (sis+siH group) .*P < 0.05 vs. the control.

Figure 5
Effects of siRNA silencing of HIF-1α and survivin on apoptosis in SGC-7901 cells Note: Cell apoptosis was assayed by ow cytometry. PI was used for cell nucleus staining and annexin V-FITC for cytomembrane staining. Ratio of apoptosis cells was presented as the standard error of the mean (± SEM). *P < 0.05 vs. the control.

Figure 6
Effect of HIF-1α or/and survivin RNAi on the migration abilities of SGC-7901 cells under hypoxia Note: *P < 0.05 vs. the control.