| ObjectiveTo investigate the effects of heparanase(HPSE)gene on invasion and migration of gastric cancer cell lines MGC-80-3 and SGC-7901.MethodsPolymerase chain reaction(PCR)was used to screen the gastric cancer cell lines which expressed HPSE gene.And then four segments of small interference RNAs(siRNAs)targeting heparanase mRNA were designed by bioinformatics technology.The sequences of the designed siRNAs were as follows:5'-GGAGA AGUUACGGUUGGAAUG-3'(labled as siRNA-a),5'-GCUCUGUAGAUGU GCUAUACA-3'(labled as siRNA-b),5'-GCUUUAUGUGGCUGGAUAAAU -3'(labled as siRNA-c),5'-GCAAGUGGAUAAAUACCUUCU-3'(labled as siRNA-d).The no-sense control segment labled as siRNA-NC was also designed,and its sequence was 5'-GUUCUCCGAACGUGUCACGU-3'. Recombination vectors expressing above sequences were constructed and were transfected transiently into two human gastric cancer cell lines MGC-80-3 and SGC-7901.Liposome(Lipofectamine 2000)was used in the tansfection. Forty-eight hours later,Real-time PCR(RT-PCR)was applied to detect the expression of heparanase mRNA.The recombination vector inhibiting HPSE-mRNA expression most significantly and the non-sense vector were used to transfected above gastric cancer cell lines Transwell chamber and Matrigel were used to detect the changes of invasion and migration of these two cell lines.In order to obtain the over expression of HPSE gene simultaneously, pCDH1-MCS1-EF1-copGFP vector with multiple cloning sites was used for the cloning of HPSE coding sequence to construct plasmid vector pCDNA-HPSE-GFP.Plasmid vector pCDNA-HPSE-GFP and empty vector were transfected into two gastric cancer cell lines MGC-80-3 and SGC-7901. Forty-eight hours after transfection,the mRNA expression of HPSE gene was detected by RT-PCR.Transwell chamber and Matrigel were used for the detection of the changes of invasion and migration of these two cell lines.Results1.The gastric cancer cell lines MGC-80-3 and SGC-7901 expressed HPSE-mRNA obviously. 2.The ratio of vector(μg)and lipofectin(μl)was 2:5 in our experiment. The transfection rate was 80%approximately.3.RNA interferencing vectors pGPU6/GFP/Neo/siRNA-a,pGPU6/GFP/ Neo/siRNA-b,pGPU6/GFP/Neo/siRNA-c and pGPU6/GFP/Neo/siRNA-d were constructed successfully.4.In gastric cancer cell line MGC-80-3,the ratio between mRNA expression of HPSE gene and GAPDH(H/G)transfected by pGPU6/GFP/Neo/ siRNA-a,pGPU6/GFP/Neo/siRNA-b,pGPU6/GFP/Neo/siRNA-c,pGPU6/GFP/ Neo/siRNA-d and pGPU6/GFP/Neo/siRNA-NC 48 hours later were 0.035±0.003,0.023±0.000,0.019±0.002,0.032±0.004 and 0.043±0.003,respectively. There were significant differences between the experimental group and the negative control group(P＜0.05).Inhibition rate of interfering groups were 18.6%,46.5%,55.8%and 25.6%,respectively.The most effective RNA interferencing vector was pGPU6/GFP/Neo/siRNA-c.After transfection by pGPU6/GFP/Neo/siRNA-c,invasion assay showed that the ratio between numbers of cells passed through the membrane and not-passed through the membrane were 0.22±0.01 and 0.29±0.01 in interfering group and negative control group,respectivelyo There was significant difference between the interfering group and the negative control group(P＜0.05).In migration experiment,the ratio between numbers of cells passed through the membrane and not-passed through the membrane was 0.61±0.45 and 0.88±0.01 in interfering group and negative control group,respectively.There was no significant difference between these two groups(P＞0.05).5.In gastric cancer cell line SGC-7901,the ratio between mRNA expression of HPSE gene and GAPDH(H/G)transfected by pGPU6/GFP/Neo /siRNA-a,pGPU6/GFP/Neo/siRNA-b,pGPU6/GFP/Neo/siRNA-c,pGPU6/GFP /Neo/siRNA-d and pGPU6/GFP/Neo/siRNA-NC 48 hours later were 0.023±0.001,0.043±0.003,0.002±0.001,0.026±0.000 and 0.027±0.002,respectively.There were significant differences between all of the experimental groups except pGPU6/GFP/Neo/siRNA-b group and the negative control group(P＜0.05).Inhibition rate of interfering group pGPU6/GFP/Neo/ siRNA-a,c,d were 14.9%,92.6%and 3.7%,respectively.The most effective RNA interferencing vector was pGPU6/GFP/Neo/siRNA-c.After transfection by pGPU6/GFP/Neo/siRNA-c,invasion assay showed that the ratio of number of cells passed through the membrane and not-passed through the membrane were 0.48±0.10 and 1.25±0.34 in interfering group and negative control group,respectively.There was significant difference between the interfering group and the negative control group(P＜0.05).In migration experiment,the ratio between numbers of cells passed through the membrane and not-passed through the membrane was 0.29±0.00 and 0.28±0.01 in interfering group and negative control group,respectively.There was no significant difference between these two groups(P＞0.05). 6.Heparanase gene over expression vector pCDNA-HPSE-GFP was constructed successfully.7.In MGC-80-3 cells,the ratio between mRNA expression of HPSE gene andβ-actin(HPSE/β-actin)were 6.1300±0.2546 and 0.0003±0.0000 in experimental group and control group,respectively.There was significant difference between the experimental group and the negative control group(P＜0.05).Invasion assay showed that the ratio between numbers of cells passed through the membrane and not-passed through the membrane were 0.814±0.012 and 0.535±0.010 in interfering group and negative control group,respectively.There was significant difference between the interfering group and the negative control group(P＜0.05).In migration experiment,the ratio between numbers of cells passed through the membrane and not-passed through the membrane was 0.598±0.028 and 0.613±0.018 in interfering group and negative control group,respectively.There was no significant between these two groups(P＞0.05).8.In SGC-7901 cells,the ratio between mRNA expression of HPSE gene andβ-actin(HPSE/β-actin)were 5.9050±0.1061 and 0.0003±0.0000 in experimental group and control group,respectively.There was significant difference between the experimental group and the negative control group(P＜0.05).Invasion assay showed that the ratio between numbers of cells passed through the membrane and not-passed through the membrane were 0.604 ±0.025 and 0.414±0.223 in interfering group and negative control group,respectively.There was significant difference between the interfering group and the negative control group(P＜0.05).In migration experiment,the ratio of number of cells passed through the membrane and not-passed through the membrane was 0.514±0.045 and 0.524±0.034 in interfering group and negative control group,respectively.There was no significant between these two groups(P＞0.05).Conclusions1.In human gastric cancer cell line MGC-80-3,the RNA interferencing vectors pGPU6/GFP/Neo/siRNA-a,pGPU6/GFP/Neo/siRNA-b,pGPU6/GFP/ Neo/siRNA-c and pGPU6/GFP/Neo/siRNA-d can inhibit the expression of mRNA of HPSE gene significantly.In human gastric cancer cell line SGC-7901, all above RNA interferencing vectors except pGPU6/GFP/Neo/siRNA-b can inhibit the expression of mRNA of HPSE gene.2.Among the successfully constructed RNA interferencing vectors, pGPU6/GFP/Neo/siRNA-c is the most effective one,its inhibition rate of HPSE mRNA expression in human gastric cancer cell lines MGC-80-3 and SGC-7901 are 55.8%and 92.6%,respectively.3.Construction of heparanase gene over expression vector pCDNA-HPSEGFP is successful.4.Silencing HPSE gene can decrease the invasion ability of gastric cancer cells,while over expression of HPSE gene can enhance the invasion ability of gastric cancer cells.Neither inhibition of HPSE expression nor over expression of HPSE gene can influence the migration ability of gastric cancer cells.5.Heparanase gene plays definite role in invasion of gastric cancer cells in vitro. |
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