Effect Of Silencing Notch1 Gene Expression By SiRNA On The Invasive Ability Of Lung Cancer Cells SPC-A-1BM

Objective:To investigate the effects of silencing Notch1 gene on the invasion of human lung adenocarcinoma cells SPC-A-1BM and on the expression of matrix metalloproteinase-9(MMP-9)gene.Methods:1.Cultured human normal bronchial cells 16 HBE and human lung adenocarcinoma cells SPC-A-1BM in vitro until both of them reached the logarithmic growth stage.Then the total RNAs of these two cell lines were extracted and reversely transcribed into c DNAs which were used to detect the m RNA expressions of Notch1 gene in two kinds of cells by Real-time quantitative polymerase chain reaction(RT-PCR).2.Based on the result of the step 1,human lung adenocarcinoma cells SPC-A-1BM were divided into three groups: including blank control group(without si RNA interference),si RNA negative control group(Notch1 non-specific si RNA interference,si RNA-NC)and si RNA experimental group(Notch1 specific si RNA interference,si RNA-Notch1).The small interfering RNA(si RNA)was designed and synthesized by the RNA interference technology to silence the expression of the Notch1 gene via nucleic acid transfection reagent liposome Lipofectamine TM2000(Lipo2000)transfecting SPC-A-1BM cells.At36 h after transfection,we used RT-PCR and Western Blot(BT)technologies to detect the expression levels of Notch1 m RNA and protein in SPC-A-1BM cells of each group.3.The invasive ability of SPC-A-1BM cells was examined by the transwell assay after transfection.4.The expressions of MMP9 m RNA and protein after transfection were measured by RT-PCR and BT experiments.After obtaining the data of each group,the statistical analysis was performed by one-way analysis of variance,with P<0.05 considered to be of statistical difference.Results:1.The expression of Notch1 gene in lung adenocarcinoma cell line SPC-A-1BM:RT-PCR results showed that the m RNA expression level of Notch1 in SPC-A-1BM cellswas 4.121±0.913 times higher than that in 16 HBE cells,with statistical difference(P<0.05).2.The expression levels of Notch1 m RNA and protein in lung adenocarcinoma cells SPC-A-1BM in each group after silencing Notch1 gene : The results suggested that the relative expressions of Notch1 m RNA in the cells of blank control group,si RNA negative control group and si RNA experimental group were respectively 1.000±0.000,0.967±0.026,and 0.347±0.045;meanwhile,the relative expressions of Notch1 protein were respectively 0.551±0.026,0.519±0.060,and 0.242±0.024.Statistical analysis of the experimental data indicted that the expression levels of Notch1 m RNA and protein in the si RNA experimental group were decreased and there was significant difference when compared to si RNA negative control group and the blank control group(P < 0.05).There was no significant difference between the si RNA negative control group and the blank control group(P > 0.05).All of these results suggested that Notch1-si RNA could effectively inhibit the m RNA and protein expression of Notch1 in SPC-A-1BM cell lines.3.Effects of Notch1 gene silencing on the invasiveness of lung adenocarcinoma cells SPC-A-1BM: The lung adenocarcinoma cells SPC-A-1BM were transfected and then cultured for 36 hours before Transwell cell invasion test.The results showed that the number of cells passing through the basement membrane in the blank control group,the si RNA negative control group and the si RNA experimental group were 84.17±5.69,81.67±4.65 and 30.83±3.62,respectively.Statistical analysis suggested that the inhibition rate of the si RNA experimental group was 63% compared with the blank control group,with a statistical difference(P < 0.05).The inhibition rate of si RNA negative control group was 3%,and compared with the blank control group,there was no statistical difference(P > 0.05).These suggested that si RNA targeted siliencing Notch1 could reduce the invasiveness of lung adenocarcinoma cells SPC-A-1BM.4.The m RNA and protein expression levels of MMP-9 in SPC-A-1BM cells in three groups after Notch1 gene was silenced: The results showed that the relative expression levels of MMP-9m RNA in the cells of the blank control group,the si RNA negative control group,and the si RNA experimental group were 1.000±0.000,0.979±0.037 and 0.389±0.025,respectively.The relative expression levels of MMP-9 protein were 0.394±0.058,0.372±0.050 and0.234±0.034,respectively.Statistical analysis suggested that the expression levels of MMP-9 m RNA and protein in si RNA experimental group were lower than those in si RNA negative control group and blank control group(P < 0.05).There was no significant difference between the si RNA negative control group and the blank control group(P >0.05).It suggested that silencing Notch1 gene could down-regulate the expression ofMMP-9.Conclusions:Notch1 was overexpressed in human lung adenocarcinoma cells SPC-A-1BM.Silencing the Notch1 gene expression by si RNA could inhibit the invasion of SPC-A-1BM cells and down-regulate the expression of MMP-9,which suggesting that Notch1 in NSCLC could promote tumor cell invasion and metastasis through MMP-9pathway.