1.                   Introduction

Gastric Cancer (GC) is the fourth most common malignancy and the second most common cause of cancer-related mortality [1]. Gastric cancer is a multi-factorial disease and causes continuous cell damage due to a lifetime of exposure to different carcinogens. Gene therapy as a novel treatment method has achieved improvements over past decades, particularly for treating malignant cancer [2-6]. However, there are still some unsolved problems, limiting the further development of gene therapy for malignant cancer. Recently, several preliminary studies of targeted genes in gastric cancer have been performed [7-11].

The translocation t (11;19) (q23; p13.1) is a recurring abnormality in gastric cancer. In the study, to investigate the effect of t (11; 19) (q23; p13.1) gene silence on proliferation and apoptosis 0f SGC 7901 gastric cancer cell line．

2.                   Material and Methods

2.1.              Designing and Constructing Expression Plasmid

To find the gene sequence of chromosome t (11; 19) (q23; p13.1) from NCBI Gen Bank, using siRNA design software to find the target sequence with characteristics in the downstream of the starting codon, and manually designing and synthesizing the siRNA oligonucleotide sequence, End plus corresponding enzyme cut point and protection base, PCR amplification, reaction conditions: 94℃ pre-degeneration 5 min,94℃ degeneration 30s, 65℃ annealing 30s, 70℃ extended 110s, 25 cycles, 70℃ extended 7 min after the end amplification reaction, the product was recycled and purified by glue. Negative control siRNA is a universal unrighteous sequence, and all siRNA chains are purified by high performance liquid phase to remove unpaired single chains.

2.2.              Cell Culture and Cell Gene Transfection

SGC-7901 cells were cultured continuously under the condition of RBMI1640 culture solution containing 10% fetal bovine serum, 37cc, 5%CO and saturated humidity environment. 1days before transfection, select logarithmic growth SGC-7901 cells. Inoculation of appropriate density cell suspension in cultured utensils (24-hole culture Plate: 2.5x10 cell/fl; 96 Hole culture Plate: 0.4xl0 cell/fl; 6 Hole culture Plate: 1.2x10 cell/fl; 6cm Petri dish: 2.5x10; 10cm petri dish: 7x10), culture overnight up to 40%~50% Fusion, the next day for instantaneous transfection. The basic operation refers to the cationic liposome Meta fectenetm (Biontex Company) Manual. Experimental group:

① negative control group: only inoculated cells, not transfected plasmid;

② Air Carrier control group: transfection Pegfp-c1;

③ Experimental Group: Transfection pegfp-c1-t (one;) (q23; p13.1). Continue to be cultured to 24, 48, 72h, respectively.

2.3.              RT PCR Detects mRNA Expression

After transfection, collected each group of cells from the 6-hole culture plate at 24, 48 and 72h, extracted the total RNA, ultraviolet spectrophotometry and agarose gel electrophoresis to detect its purity and concentration in accordance with the Trizol reagent (Invitrogen Company) manual, according to reverse transcription System Kit Fpromega Company operated synthetic cDNA for PCR amplification. Reaction conditions: 94℃ Pre-degeneration 5 Min,94℃ degeneration S, 65℃ annealing 3O S, 70℃ extended S, 25 cycles, 70℃ extended 7 rain after the end amplification reaction, amplification products by 1.5% agarose gel electrophoresis detection.

2.4.              MTT Assay for Cell Proliferation

After transfection, MTT assay was applied to evaluate the proliferation of both introduced MGC-803, AGS, and SGC-7901 cells and normal GES-1 cells. A total of 2 × 104 cells per well were plated in 96-well plates that were cultured for 24, 48, and 72 h. Before removal from incubation, the cells were incubated with 200 μl of 0.5 mg/ml MTT (Sigma, Guangzhou, China) for an additional 4 h. The medium was then replaced with 200 μl of dimethyl sulfoxide (Sigma-Aldrich, Guangzhou, China) to resolve the crystals. The absorbance at 490 nm was then measured for each well.

2.5.              TUNEL Assay

Apoptosis cells were detected in a MEBSTAIN Apoptosis Kit Direct (MBL International, Woburn, MA, USA) for Tunel assay. After transfection for 24 to 72h, POD-horseradish peroxidase was added. Then cells were incubated with TdT buffer for 1h. The apoptosis cells were counted under an Olympus fluorescent microscope (Olympus Corp). The Apoptotic Index (AI) was (number of apoptotic cells/total number) × 100%.

2.6.              Statistical Analysis

The paired two-tailed Student t test was used to analyze the results by Clinical Physician Statistics Assistant 5.0 (Book Sword Software Online, hangzhou, china) and significance was assumed at P < 0.05.

3.                   Results

3.1.              Expression of Transfection Cell mRNA

The cells of transfection t (11; 19) (q23; p13.1) siRNA group and other groups of cells were collected at 24,48 and 72h respectively. The total RNA were extracted. RT-PCR test results showed the relative expression of t (11; 19) (q23; p13.1) mRNA was lower than that of the control group, respectively, the difference was statistically significant (P<0.05); In the transfection siRNA group, the relative expression of mRNA at different points of time was also statistically significant, and the expression of mRNA decreased with the prolongation of transfection time (P<0.05). There was no statistically significant difference in the expression of mRNA between control groups at each point in time (P>0.05). This showed that the role of siRNA is specific and time-dependent (Table 1).

3.2.              Expression of t (11; 19) (q23; p13.1) gene inhibits SGC -7,901 Cell Proliferation

T (11; 19) (q23; p13.1) after transfection into SGC-7901 cells for 24, 48and 72h, the proliferative ability of cells decreased significantly compared with the control group (P<0.05), and the growth inhibition rate increased with the prolongation of transfection time. There was no statistically significant difference in cell growth rate between points in the control group (P>0.05), which suggested that siRNA might have the ability to inhibit cell proliferation (Table 2).

3.3.              Detection of Apoptosis by Tunel Method

Positive staining was in the nucleus (Figure 1). After transfection of 24,48 and 72 the number of apoptosis of SGC-7901 cells increased significantly with time. The apoptosis index of 24h group was 4.95±0.29%. The apoptosis index of 48h group was 16.97±0.34%. The apoptosis index of 72h Group was 27.56±0.39%, and the difference was significant (P<0.05) after examination.

4.                   Discussion

Gastric Cancer (GC) is the fourth most common malignancy and the second most common cause of cancer-related mortality. Gastric cancer is a multi-factorial disease and causes continuous cell damage due to a lifetime of exposure to different carcinogens. Gene therapy as a novel treatment method has achieved improvements over past decades, particularly for treating malignant cancer.

However, there are still some unsolved problems, limiting the further development of gene therapy for malignant cancer. Recently, several preliminary studies of targeted genes in gastric cancer have been performed. Some studies that the t (11;19) (q23; p13.1) translocation results in many cancers [12-13]. The translocation t (11;19) (q23; p13.1) is a recurring abnormality in gastric cancer. In the study, to investigate the effect of t (11; 19) (q23; p13.1) gene silence on proliferation and apoptosis 0f SGC 7901 gastric cancer cell line．

This study showed the relative expression of t (11; 19) (q23; p13.1) mRNA transfection t (11; 19) (q23; p13.1) siRNA group was lower than that of the control group, respectively, In the transfection siRNA group, the relative expression of mRNA at different points of time was also statistically significant, and the expression of mRNA decreased with the prolongation of transfection time. This showed that the role of siRNA is specific and time-dependent.

This study showed after transfection into SGC-7901 cells for 24, 48and 72h, the proliferative ability of cells decreased significantly compared with the control group, and the growth inhibition rate increased with the prolongation of transfection time. I t suggested that siRNA might have the ability to inhibit cell proliferation. After transfection into SGC-7901 cells for 24, 48and 72h, the AIs decreased with the prolongation of treatment time.

5.                   Conclusion

We found that t (11;19) (q23; p13.1) gene can inhibit gastric cancer cell proliferation and improve apoptosis of gastric cancer cell.

6.                   Acknowledgments

Thank the financial support of the Research Project on the Application of Public Welfare Technology, Zhejiang Provincial Science and Technology Department, China, No. 2015C33105.

Figure 1: Apoptotic cells as shown by the TUNEL assay. Positive staining located in the nucleus. Magnification × 200.

Table 1: Expression of t (11;19) (q23; p13.1) mRNA in each group of cells.

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