The present study aimed to investigate the effects of small interfering (si)RNA interference of connexin 37 (Cx37) on subcutaneous gastric tumours in mice. Constructed lentiviruses carrying siRNA against Cx37 significantly knocked down Cx37 mRNA and protein expression
Gastric cancer is the fourth most common type of cancer and the second leading cause of cancer-associated mortality worldwide (
Previously, small interfering RNA (siRNA) has been effective in silencing target genes via RNA interference (RNAi) (
The HEK293 cell line was cultured as previously described (
Three different sequences (sites A–C) of the Cx37 genes in mice were selected as targets for RNAi. The targeted sequences were as follows: mm-Cx37-si-1, 5′-GGUUAACGG UGCUCUUCAU-3′, location 209; mm-Cx37-si-2, 5′-CCAAGG ACCUACAUGUAGA-3′, location 488; and mm-Cx37-si-3, 5′-CAGACCCUUACCCUGAACA-3′, location 841. p3XFLAG-Cx37 and p3XFLAG were used as controls.
The cells were collected following 72 h and were then washed three times with cool phosphate-buffered saline (PBS). Residual PBS was removed by suctioning. Following this, 0.2 ml radioimmunoprecipitation assay lysis buffer containing inhibitors of serine, cysteine and metalloproteinase was added. The resulting mixture was then placed on ice for 30 min. The cells were collected using a cell scraper and placed in 1.5-ml centrifuge tubes on ice. The cells were then ultrasonically lysed for 30 sec at 4°C. The supernate was centrifuged for 30 min at 22,559 × g, transferred to another clean microcentrifuge tube and then stored at −20°C.
The protein concentrations were determined by the bicinchoninic acid (BCA) method using Pierce’s BCA Protein Assay Reagent kit (Pierce Biotechnology, Inc. Rockford, IL, USA). Solutions A and B (50:1) were mixed and the resulting mixture was maintained at a room temperature for 30 min. Following this, 200 μl of the mixture was added to each hole of a 96-well plate, followed by 10 μl of various concentrations of standard bovine serum albumin (dilution, 1:10). A blank sample containing 10 μl of double-distilled water was also placed in a well, adjusted to zero and maintained at 37°C for 30 min in a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The absorbance [optical density (OD) value] of this blank was then determined at 570 nm. The standard value was subsequently used to prepare a standard curve to calculate the total protein concentration. The interference efficiency of siRNA was determined by western blot analysis. SDS-PAGE was performed using a concentration of 10%. The volume of each sample was 30 μg. Anti-tag antibody protein flags were incubated overnight at 4°C and the GAPDH expression in the samples was monitored. The antibody ratios were as follows: flag antibodies: Rabbit pAb = 1:2,000 and GAPDH: Rabbit pAb = 1:10,000.
The transfection reagent Lipofectamine 2000™ was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). A wild-type Lipofectamine 2000 plasmid Cx37 3 interference fragment was used. The negative control and p3XFLAG-Cx37 were co-transfected with the RNAi plasmid and then mixed at a molar ratio of 5:1.
The cells were divided into six groups: The experimental groups (overexpression of empty plasmid group, Cx37 plasmid, Cx37 overexpression of recombinant plasmid + different interference fragment); the transfection reagent group (Lipofectamine 2000); the plasmid group (p3XFLAG and p3XFLAG-Cx37-0.4 μg) and the interference fragment group (50 nM). The transfection time was 72 h. The optimum interference fragment was selected on the basis of the western blotting results.
The lentiviral vectors expressing Cx37 were prepared using the BLOCK-It Lentiviral Pol II miR RNAi Expression system (Yingjun Biological Company, Shanghai, China) and green fluorescent protein (GFP; Catalog no. K4948-00; Invitrogen Life Technologies). A scrambled siRNA sequence (labelled mock-siRNA) with no known homology to mammalian genes served as a control.
SGC-7901 murine gastric cancer cell lines were purchased from the Agricultural Academy (Zhejiang, China). The cells were cultured in an RPMI-1640 complete medium. Sterilised test samples were added to a 96-well plates containing all of the tested cells (1×105 cells/well) to obtain a final concentration of P1 (50, 100 and 200 μg/ml). Dimethyl sulfoxide was used as a negative control. Following cultivation for 24, 48 and 72 h at 37°C in a humidified 5% CO2 incubator, the percentage of viable cells was determined by an MTT assay (Yingjun Biological Company). The reading absorbance was determined at 570 nm using a Benchmark microplate reader (Bio-Rad, Hercules, CA, USA). The inhibitory rate was calculated using the following formula: % inhibitory rate = 1-(mean absorbency in test wells)/(mean absorbency in control wells) × 100.
Female BALB/c-nu/nu mice (age, six weeks) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All of the mice were housed under controlled conditions (12-h light/dark cycle, 60% humidity and 25±1°C) with free access to a standard pellet diet and water. At day 0, 32 mice were randomly divided into three groups and subcutaneously inoculated with 0.2 ml of the murine gastric cancer cell line suspension (2.5×107 cells/ml) at the forelimb, to establish the gastric carcinoma mouse model. Following tumour inoculation, the maximum (a) and minimum (b) diameters of the solid tumours were measured with a vernier calliper every three days. The tumor volumes were calculated using
Data are expressed as the mean ± standard deviation. Continuous variables between the two groups were compared using independent t-tests and χ2 tests. Multiple groups were compared using analysis of variance. All of the analyses were conducted using the SPSS 17.0 software for Windows (SPSS, Inc., Chicago, IL, USA). The proportions were compared using Fisher’s exact test when the expected frequency was <5; otherwise, the χ2 test was used. P<0.05 was considered to indicate a statistically significant difference.
The HEK293 cell line was transfected with lentivirus-based vectors expressing three different Cx37 siRNA. Gene silencing analysis demonstrated that the Cx37-site 2 and 3 lentivirus was the most effective vector in blocking Cx37 expression. Therefore, Cx37-site 3 lentiviruses were selected for the subsequent
Previous local viral delivery to subcutaneous gastric cancer in mice resulted in an efficient transfection. GFP expression is used as an efficient and convenient monitoring tool for determining the transfection efficiency of the lentiviruses. In the present study, GFP fluorescence was detected in the gastric cancer samples one week following transfection to confirm siRNA transfection. Increased fluorescence was detected two weeks following transfection. A total of six weeks after the transfection, at the end of the study, a weak GFP remained visible (
To examine the efficacy of the lentivirus-mediated gene silencing
The apoptotic index of the Cx37-RNAi groups was higher than that of the mock-siRNA and control groups (19.7±5.1 vs. 9.8±6.4 vs. 10.5±7.2; 11.1±6.9%; P<0.05;
In the development of genetic information in various organisms, homeobox genes encode and determine the location of information and are thus crucial in normal cellular control (
In conclusion, the present study demonstrated that lentivirus-mediated RNA interference effectively knocked down Cx37 genes in mice, which resulted in reduced Cx37 expression and an increased apoptosis index. Cx37 was demonstrated to be correlated with gastric cancer and this association may be used to develop novel therapeutic approaches for gastric cancer.
Western blot analysis of three different Cx37 siRNAs silencing
Lentiviral transfection efficiency observed by green fluorescent protein in gastric cancer cells at (A) 1 week; (B) 2 weeks; and (C) 6 weeks following transcfection.
Electrophoresis results of Cx37 mRNA expression levels in gastric cancer cells in the three groups. siRNA, small interfering RNA; Cx37, connexin 37; bp, base pairs; M, marker.
Cx37 mRNA expression analysis in gastric cancer cells in the three groups. siRNA, small interfering RNA; Cx37, connexin 37.
Cx37 protein level analysis in gastric cancer in the three groups. Data are expressed as the mean ± standard deviation. siRNA, small interfering RNA; Cx37, connexin 37.
Apoptotic index in gastric cancer in the three groups. Data are expressed as the mean ± standard deviation. siRNA, small interfering RNA; Cx37, connexin 37.
Apoptotic tumor cells in gastric cancer in the three groups (haematoxylin and eosin staining; magnification ×10): (A) Cx37 siRNA;(B) mock siRNA; (C) saline. siRNA, small interfering RNA.