{'fraction_non_alphanumeric': 0.03235147730871906, 'fraction_numerical': 0.031989502737432694, 'mean_word_length': 5.009244154431756, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Lentivirus-mediated RASSF1A expression suppresses aggressive phenotypes of gastric cancer cells in vitro and in vivo

Loss of Ras association domain family protein 1 isoform A (RASSF1A) expression is associated with the development of a variety of human cancers and the expression of carcinoembryonic antigen (CEA) frequently occurs in gastric cancer. This study investigated the effects of RASSF1A expression restoration using a hypoxia-inducible CEA promoter-driven vector on xenograft tumor growth in nude mice and on the in-vitro regulation of gastric cancer cell viability, cell cycle distribution, apoptosis, colony formation and invasion capacity. The data showed that the level of CEA mRNA and protein was much higher in gastric cancer SGC7901 cells than in a second gastric cancer cell line, MKN28, or in the MCF-10A normal epithelial breast cell line. RASSF1A expression was restored in SGC7901 cells compared with the negative control virus-infected SGC7910 cells. RASSF1A expression restoration significantly inhibited gastric cancer cell viability, colony formation and invasion capacity, but induced cell cycle arrest and apoptosis in vitro, especially under hypoxic culture conditions. At the gene level, restoration of RASSF1A expression under hypoxic culture conditions significantly suppressed matrix metalloproteinase-2 expression and prevented cyclinD1 expression. A nude mouse xenograft assay showed that the restoration of RASSF1A expression reduced gastric cancer xenograft formation and growth. In conclusion, the restoration of RASSF1A expression using a hypoxia-inducible and CEA promoter-driven vector suppressed aggressive phenotypes of gastric cancer cells in vitro and in vivo. These results suggest that LV-5HRE-CEAp-RASSF1A gene therapy may be a promising novel approach to treat advanced gastric cancer.


INTRODUCTION
Gastric cancer is one of the most commonly diagnosed cancers and is the most frequent cause of cancer death in the world. 1,2 Although there have recently been significant advancements in gastric cancer treatment, including new surgical, chemotherapeutic and radiotherapeutic methods, many patients are still dying after being diagnosed with advanced gastric cancer and developing metastasized disease. 3 Thus, gastric cancer remains a major clinical challenge. Tumor-targeted gene therapy may be a promising new approach for the effective control of gastric cancer. Gene therapy is defined as the use of vectors to bring nucleic acids into cells to alter gene expression and prevent, halt or reverse a pathological process. The number of ongoing or completed gene therapy clinical trials approved worldwide since 1996 has reached 1274 and 63.8% of these were for human cancer. 4 Although significant progress has been made in the development and use of tumor-selective delivery systems in the past 20 years, there are still many challenges that must be overcome to develop therapeutic genes that are specifically expressed in cancer cells, but not in normal cells. To achieve this goal, strategies have been developed that include the use of specific gene promoters, enhancers and 5′-untranslated region that respond to tumor-specific transcription factors, to drive or knock down gene expression to treat human cancers.
We focused on the Ras association domain family protein 1 isoform A (RASSF1A), because lost RASSF1A expression is associated with the development of a variety of human cancers, including gastric cancer or other gastrointestinal malignancies, and this suggests the possibility that RASSF1A may have a tumor suppressor function in these human cells. [5][6][7] Restoration of RASSF1A expression, therefore, could potentially control gastric cancer progression. Moreover, carcinoembryonic antigen (CEA) is a glycosylated protein that is also highly expressed in a number of cancers, including gastric cancer. 8 The CEA promoter has been used in many cancer studies and can specifically target CEApositive cancer cells. 9,10 Hypoxia-inducible factors (HIFs) are another potential target. Hypoxia frequently occurs in solid tumor lesions. 11 Accumulating data shows that 50%-60% of locally advanced solid tumors develop hypoxia. 12 Hypoxia promotes tumor progression, invasion of adjacent tissues and metastasis, and hypoxic tumors are resistant to radiotherapy and chemotherapy. 13 HIF-1, a well-studied HIF, is a heterodimer of the HIF-1α and HIF-1β subunits. One subunit, HIF-1α, is an O 2regulated subunit that induces HIF-1 activity and expression in response to a reduction of O 2 availability, whereas the other, HIF-1β, is a constitutive subunit. 14 HIF-1α binds to the hypoxiaresponsive element (HRE), a conserved transcription promoting sequence in the regulatory regions of target genes. 15 Previous studies have shown that HIF-1α contributes to the malignant phenotypes of gastric cancer. [16][17][18] In this study, to restore RASSF1A expression, we constructed an expression vector carrying RASSF1A under the control of hypoxiainducible and CEA promoters, to specifically restore RASSF1A expression in gastric cancer cells. We then assessed the effects of RASSF1A expression on xenograft tumor growth in nude mice and the regulation of gastric cancer cell viability, cell cycle distribution, apoptosis, colony formation and invasion capacity in vitro. The results of this study could help the future development of an inducible tumor-specific lentiviral vector that combines RASSF1A with CEAp and 5HRE (LV-5HRE-CEAp-RASSF1A) for gastric cancer gene therapy.

RESULTS
Expression of endogenous CEA and RASSF1A mRNA and protein in gastric cancer cells We first detected expression of endogenous CEA mRNA using quantitative reverse-transcription PCR in several gastric cancer cell lines as well as in a normal breast cell line. The results showed that the level of CEA mRNA in gastric cancer SGC7901 cells was much higher than that in both the MKN28 gastric cancer and normal breast MCF-10A cell lines (Figure 1a). The same was true for endogenous CEA protein (Figure 1b). The mean density of the CEA protein, as quantified using the immunohistochemical staining results, was 0.111 ± 0.024, 0.076 ± 0.016 and 0.002 ± 0.000 in SGC7901, MKN28 and MCF-10A cells, respectively (P o 0.001 between SGC7901 or MKN28 and MCF-10A cells; Po 0.01 between SGC7901 and MKN28 cells). These data clearly indicate that the level of endogenous CEA protein was highest in SGC7901 cells, moderate in MKN28 cells and lowest in MCF-10A cells. Furthermore, endogenous RASSF1A protein was not expressed in SGC7901 or MKN28 cells, but was expressed in MCF-10A cells ( Figure 1c). Thus, due to the levels of CEA and RASSF1A expression, we selected SGC7901 cells (CEA mRNA+, CEA protein+, RASSF1A protein−) for the experimental group and MKN28 cells (CEA mRNA − , CEA protein+, RASSF1A protein − ) for the negative control.
Restoration of RASSF1A expression in gastric cancer cells using a CEAp or 5HRE-driven expression vector We stably restored RASSF1A expression in gastric cancer cells using CEAp or 5HRE-driven expression vectors. RASSF1A expression in SGC7901/5HC-RASSF1A and MKN28/5HC-RASSF1A cells was confirmed by quantitative reverse-transcription PCR and western blotting (Figures 2a and b). SGC7901/5HC-RASSF1A cells showed very high levels of RASSF1A mRNA under hypoxic conditions (300 μm l − 1 cobalt chloride (CoCl 2 )), but only slightly elevated levels of RASSF1A mRNA under normoxic conditions. Similarly, RASSF1A protein levels were significantly increased under hypoxic conditions. However, RASSF1A expression was low in SGC7901, SGC7901/NC, MKN28/5HC-RASSF1A and MKN28 cells under both normoxic and hypoxic conditions. These data indicated that both RASSF1A mRNA and protein were expressed in SGC7901/5HC-RASSF1A cells, especially under hypoxic conditions. The hypoxic inducibility, CEA-targeting and RASSF1A expression-driving ability of the 5HRE-CEAp element was confirmed using a dual luciferase assay ( Figure 2c). SGC7901, MKN28 and MCF-10A cells were transfected with pGL4.20-5HRE-CEAp-Luc or control vectors and cultured for 24 h under normoxic or hypoxic conditions. The luciferase activity fold change was measured and the data showed a significant increase in luciferase activity in SGC7901 cells under normoxic conditions (P o0.05; 59.63 ± 24.24 in SGC7901 vs 27.82 ± 7.19 in MKN28 and 1.25 ± 0.57 in MCF-10A). However, luciferase activity in SGC7901 and MKN28 cells was further increased in the presence of CoCl 2 (300 μm l − 1 ) when compared with luciferase activity under aerobic conditions (SGC7901, P o0.001; 519.75 ± 97.14 vs 59.63 ± 24.24; MKN28 Po 0.001; 87.80 ± 7.78 vs 27.82 ± 7.19).
Effects of RASSF1A expression on regulation of gastric cancer cell viability, apoptosis, cell cycle distribution, colony formation and invasion capacity in vitro The viability of cells expressing RASSF1A was assessed using a CCK-8 assay. The data showed that RASSF1A expression significantly reduced SGC7901/5HC-RASSF1A cell viability when compared with either SGC7901 or SGC7901/NC cell viability under both normoxic and hypoxic conditions. Under hypoxic conditions, RASSF1A expression further decreased tumor cell viability ( Figures  3a and b). However, this result was not replicated in MKN28/5HC-RASSF1A cells (Figure 3b), because MKN28 cells do not express CEA and, thus, the vector did not restore RASSF1A expression in these cells (Figure 2b).
Moreover, a tumor cell apoptosis assay showed that the rate of apoptosis was increased in SGC7901/5HC-RASSF1A cells under hypoxic conditions (Figure 4a). Cell cycle analysis revealed that SGC7901/5HC-RASSF1A cells accumulated at the G1 phase of the cell cycle under hypoxic, but not normoxic, conditions (P o0.05; Figure 4b). Colony formation assays also showed a significant reduction in the numbers of colonies that were formed on the plate by SGC7901/5HC-RASSF1A cells under hypoxic conditions (P o 0.05; Figures 5a and b). In addition, the Transwell assay showed that the number of tumor cells passing through the    is also true that there was no RASSF1A expression in normal mouse hepatocytes in the liver tissues (Figure 7d).

DISCUSSION
Gene therapy is a promising option for the treatment human cancers; however, two keys to success, a tumor-selective delivery system and the persistent expression of anti-cancer gene products, remain elusive. In the current study, we developed a lentivirus vector for hypoxia-inducible, CEA promoter-driven and tissue-specific expression of RASSF1A, LV-5HRE-CEAp-RASSF1A.
Assessing this vector showed that the following: and specific promoter-driven RASSF1A expression (CEA is usually overexpressed in these cancers) for future control of gastric cancer. Thus, our current data strongly supports the potential usefulness of LV-5HRE-CEAp-RASSF1A as an effective anti-tumor treatment option for advanced gastric cancer. A tumor-selective delivery system is the key to successful tumor gene therapy. Over time, many gene therapy strategies have been developed and these strategies have been accompanied and shaped by an increasing knowledge of tumor pathogenesis. Hypoxia, caused by rapid tumor growth, has a key role in cancer progression and is the focus of many cancer treatment strategies. 19 For example, Shibata et al. 20 constructed an NTR/ CB1954 vector under the control of five copies HRE and a minimal cytomegalovirus promoter (CMV) promoter (5HRE/CMVmp) and confirmed specific anti-tumor activity under hypoxic conditions. They showed that among various HRE constructs and promoter elements, 5HRE/CMVmp had optimal activation at a low oxygen tension, which is true in the gastrointestinal tract. Harvey et al. 21 developed an adenovirus vector for hypoxia-targeted gene therapy using the herpes simplex virus thymidine kinase and bacterial nitroreductase prodrug-activating genes. They showed that 5HRE derived from vascular endothelial growth factor and linked to the CMV minimal promoter could induce optimum luciferase reporter gene expression.
In the current study, we constructed a lentiviral vector under the control of a 5HRE element and a CEA promoter to enhance RASSF1A expression in tumor lesions. This 5HRE regulatory element showed hypoxia-inducible activity and its design conformed to the HRE described by Shibata et al. 20 In addition to a hypoxia-driven strategy for tumor targeting, the inclusion of the CEA promoter serves as a secondary tumor targeting strategy. The CEA promoter has been used in many studies, is highly expressed in a number of tumors and is a known tumor marker of digestive cancer. Nyati et al. 22 used the CEA promoter and enhancer to selectively drive prodrug yeast cytosine deaminase expression in CEA-positive cancer cells and demonstrated that their vector had the ability to suppress tumor growth. Liu et al. 23 constructed a CMV enhancer and CEA promoter regulatory element, and demonstrated the ability to specifically target CEApositive gastric cancer. Although the CEA promoter was used as part of a strategy of specific cancer gene delivery, the CEA promoter alone could only weakly drive target transgene expression, leading to insufficient anti-cancer gene expression for cancer gene therapy. 24 To overcome this challenge, various enhancers were added in front of the CEA promoter. [22][23][24] We used 5HRE as the CEA promoter enhancer.
Although it has only been studied for 14 years, RASSF1A is reported to be a putative tumor suppressor gene. Dammann et al. 25 showed that RASSF1A is localized at chromosome 3p21.3 and that it was inactivated by methylation of the CpG-island promoter. They also showed that RASSF1A expression was lost in lung tumors and re-expression of RASSF1A had anti-oncogenic effects in lung carcinoma cells. Since then, accumulating data has demonstrated that through multi-mechanisms, RASSF1A is a tumor suppressor gene in the vast majority of human cancers. 26 Our data, reported here, also demonstrated that RASSF1A has effective anti-tumor activity. Shivakumar et al. 27 showed that RASSF1A expression blocked cell cycle progression at the G1/S phase by inhibiting cyclinD1 accumulation. Agathanggelou et al. 28 found that transfection of RASSF1A cDNA into A549 cells induced cell cycle arrest at the G1 phase and downregulated cyclinD1 expression. Our current study further confirmed these results and showed that the effects were significantly increased under hypoxic culture conditions. However, the cell cycle impact of RASSF1A under hypoxic conditions may be due to other unknown mechanisms. Apoptosis promotion is an important tumor suppressor function of RASSF1A and many studies have investigated the pro-apoptotic mechanisms of RASSF1A. [29][30][31][32] Our current study demonstrated the effect of RASSF1A on the induction of SGC7901 apoptosis under hypoxic conditions. Furthermore, other studies have demonstrated that RASSF1A can suppress the migration of tumor cells. 33 Our current study revealed that the mechanism by which RASSF1A inhibits the invasive capacity of SGC7901 cells under hypoxic conditions might be related to the inhibition of MMP-2 expression. Although hypoxia can induce tumor progression, our current data indicate that LV-5HRE-CEAp-RASSF1A can effectively use that same hypoxia to drive the antitumor activity of RASSF1A.
Our current study is just 'proof-of-principle' and much more work must be done before our results can be translated into clinical trials, such as safety and efficiency of this lentivirus. Our LV-5HRE-CEAp-RASSF1A system may help to solve two key gene therapy problems in cancer, specificity and efficiency. We showed that this system can induce gene expression in CEA-positive gastric cancer cells under hypoxic conditions, further ensuring the cancer cell-specific expression of therapeutic genes. In addition, this study represents the first successful use of the tumor suppressor gene RASSF1A in gene therapy and confirms that the expression of RASSF1A effectively inhibits the growth of gastric cancer cells in vivo and in vitro. More work should be done in the future to verify the persistence and specificity of RASSF1A gene expression using the system described in this study.

Cell lines and cultures
All cell lines were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human gastric adenocarcinoma cell lines (SGC-7901 and MKN28) were grown in RPMI1640 (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal calf serum, 100 units per ml penicillin and 100 units per ml streptomycin in a humidified incubator with 5% CO 2 at 37°C. Human normal mammary epithelial cell line MCF-10A was maintained in Dulbecco's modified Eagle's medium/F12 (1:1) medium (Life Technologies) supplemented with 15% fetal calf serum, 10 μg ml − 1 insulin, 20 ng ml − 1 epidermal growth factor, 100 units per ml penicillin and 100 units per ml streptomycin in a humidified incubator with 5% CO 2 at 37°C. For hypoxic cell culture, cells were incubated with medium containing a hypoxia-mimicking agent, CoCl 2 .

Lentivirus infection of SGC7901 and MKN28 cells
A lentiviral vector carrying RASSF1A, pLV-5HRE-CEAp-RASSF1A or a negative control vector, pLV-5HRE-CEAp-NC, and corresponding viruses (1 × 10 8 pfu) were custom constructed and prepared by GeneChem Co., Ltd. Lentivirus infection was performed in the presence of polybrene (GeneChem Co., Ltd.) according to the manufacturer's protocol. Specifically, cells were seeded in 24-well plates overnight at a density of 5 × 10 4 cells per well and then infected with pLV-5HRE-CEAp-RASSF1A or pLV-5HRE-CEAp-NC lentivirus at a multiplicity of infection of 10 pfu per cell. Three days later, cells were further cultured in a medium containing 2 μg ml − 1 puromycin for SGC7901 and 10 μg ml − 1 for MKN28 for 2 weeks. The drug-resistant clones were used in the following experiments.

Quantitative reverse-transcription PCR
Stably transfected SGC7901/5HC-RASSF1A, SGC7901/NC or MKN28/5HC-RASSF1A cells were subjected to total cellular RNA isolation using an RNA fast 200 kit (Pioneer Biotech, Xi'an, China) according to the manufacturer's instructions. The resulting RNA was reverse transcribed into cDNA using PrimeScript RT Master Mix (Takara, Dalian, China), according to the manufacturer's instructions, and then subjected to quantitative reversetranscription PCR analysis of RASSF1A mRNA using SYBR Premix Ex Taq II (Takara) and specific primers (Table 1). Each reaction was performed in triplicate and the mean RASSF1A mRNA level in each cell line was calculated by the 2 − ΔΔCt method.

Immunocytochemistry
Cells were cultured in 24-well plates with 1 × 10 4 cells per well for 48 h and then washed with phosphate buffered saline and fixed with 4% paraformaldehyde for 30 min. CEA expression was detected by using a CEA polyclonal antibody (1:100, Proteintech, Chicago, IL, USA) and SP assay and DAB detection kits (ZSGB-BIO, Beijing, China). The negative control used an equal volume of phosphate buffered saline to replace the primary antibody. CEA immunostaining quantification was performed using digital image analysis with the Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA) according to the methods described in a previous study. 35 Briefly, images were acquired by a Nikon ECLIPSE Ti-S microscope mounted with a Nikon digital camera (Nikon, Tokyo, Japan). Five randomly selected fields (×400) were acquired per well. The integrated optical density of the positive field and area of interest was measured. The mean density (optical density/area of interest) represented the quantification of the specific protein per unit area.
Tumor cell invasion assay A Transwell system was used to assess tumor cell invasion capacity. In brief, upper chamber filters were coated with 50 μl BD Matrigel (Becton Dickinson) according to the manufacturer's instructions; serum-starved cells were trypsinized and plated at 5 × 10 4 cells per upper chamber of a 24-well plate (8 μm, Corning, New York, NY, USA) and then the lower chambers were filled with 10% fetal bovine serum. For the hypoxia group, CoCl 2 (300 μm l − 1 ) was also added to the upper chamber. The cells were allowed to incubate 30 h at 37°C. After incubation, the non-invasive cells were removed from the upper chamber surface with a cotton swab and the cells under the lower surface were fixed with 4% paraformaldehyde for 20 min and stained with 1% crystal violet solution. Cell invasion was quantified by counting the number of cells in five fields per well under a microscopy.
Nude mouse xenograft assay SGC7901 cells were subcutaneously injected into the right flank of 6week-old BALB/c nude mice at a density of 3 × 10 6 in 100 μl phosphate buffered saline. Tumor volume was calculated using the empirical formula V = 1/2 × ((the shortest diameter) 2 × (the longest diameter)). After xenograft tumors had reached 50-100 mm 3 , mice were randomly divided into three groups: negative solution, LV-NC and LV-5HRE-CEAp-RASSF1A (LV-5HC-RASSF1A). Lentiviruses (1 × 10 8 pfu) in 100 μl serum-free medium were administered intratumorally at three sites per tumor. Three days later, lentivirus administration was repeated. In the negative solution group, 100 μl serum-free RPMI1640 was injected twice instead of the recombinant virus. Tumor size and growth were monitored and measured using a caliper at regular intervals. Mice were killed 2 weeks after the second virus injection and xenograft tumors were collected, measured and photographed. Xenograft tumors were fixed in 10% buffered formalin and embedded in paraffin for sectioning and immunohistochemical staining of RASSF1A expression.

Statistical analysis
All data are representative of three independent experiments and are presented as the mean ± s.d. A two-sample t-test was performed to analyze two independent samples, whereas analysis of variance was conducted for comparison among groups. SPSS 13.0 software (SPSS, Chicago, IL, USA) was used to calculate the P-value and a Po 0.05 was considered to be statistically significant.