RNA Interfering Effect Of Nucleostemin On Signal Molecule Of PI3K/AKT Pathway In Human Embryonic Esophageal Carcinoma Epithelial Cells

Objective:To detect the expression of NS and PI3K/AKT/m TOR signal transduction pathway critical molecules PI3 K, AKT, m TOR in immortalized esophageal epithelial cell line SHEE and its malignant cell line SHEEC cells. Then use the RNA interference(RNAi) technology silencing NS gene in SHEEC cells, to explore the effect of NS was down-regulated to the PI3K/AKT/m TOR signal pathway related molecules expression and the influence on SHEEC cells apoptosis and proliferation, preliminary exploration the role of NS gene in the development of esophageal cancer and its effects on PI3K/AKT/m TOR signal transduction pathway. Methods:1. SHEE and SHEEC cells continuous subculture, select the logarithmic growth phase cells, and using Real-time PCR and Western Blot methods to detect the expression of NS and PI3K/AKT/m TOR signal transduction pathway critical molecules PI3 K, AKT, m TOR m RNA and proteins in SHEE and SHEEC cells.2. RNA interference technology was used to silence the expression of NS gene in SHEEC cells, and the transfection efficiency was detected by inverted fluorescence microscope and flow cytometry after transfection 6h, and the expression level and inhibition rate of NS gene was detected by RT-PCR after transfection 24 h.3. After transfection of 24 h and 48 h, the expression of m RNA and proteins of PI3K/AKT/m TOR pathway related factors after down regulated NS gene in SHEEC cells were detected by using RT-PCR and Western Blot methods.4. The apoptosis rate and proliferation activity of SHEEC cells were detected by flow cytometry and MTT method before and after NS RNA interference.5. SPSS 21.0 statistical software was applied to statistically analyze. Data were used X ±S, t-test and ANOVA were used to compare the quantitative data, significant level is 0.05. Results:1. The expression of NS, PI3 K, AKT and m TOR m RNA in SHEEC cells were significantly higher than in SHEE cells, the amount of expression in the SHEEC were SHEE 2.047±0.507、2.13±0.262、1.614±0.256 、2.072±0.746 �'� 2.00±0.432 times respectively(P<0.05). The expression of NS, PI3 K and p-AKT proteins in SHEEC cells were apparently higher than in SHEE(P<0.05).2. RNAi agents successfully transfected to SHEEC cells of fluorescence microscope observed after transfection 6h, and the flow cytometry detected the transfection efficiency was 87.12%. According to the results of RT-PCR, the expression level of NS was apparently decreased, and the inhibition rate of NS was about 47.8% in SHEEC cells.3. RT-PCR results showed that the expression of PI3 K, AKT and m TOR m RNA were apparently decreased after inhibit NS gene in SHEEC cells, and the comparison of NS-RNAi and SHEEC group, NC, MOCK group differences were statistically significant. Western Blot results showed that the level of PI3 K, p-AKT were 0.353±0.361 �'� 0.312±0068 after inhibit NS gene in SHEEC cells, and compared with SHEEC group, NC, MOCK group differences were statistically significant.4. The apoptosis rate of NS-RNAi group was significantly higher than that of SHEEC, MOCK, NC group(35.487±1.631 vs. 0.227±0.114、6.683±0.486、 11.580±2.24,P<0.05). Compared with SHEEC, MOCK, NC group, the cell proliferation ability of NS-RNAi group was apparently decreased, the differences were statistically significant. Conclusion:1. NS and PI3K/AKT/m TOR signal transduction pathway related factors is highly expression in SHEEC cells, but low expression in SHEE cells.2. The expression of NS and PI3K/AKT/m TOR signal pathway related factors were significantly decreased with NS down-regulation in SHEEC cells, and the PI3K/AKT/m TOR signal pathway may be a kind of NS p53-independent pathway.3. Inhibition of NS gene expression can significantly reduce SHEEC cells proliferation and promote cells apoptosis, suggests that NS may be closely related to the malignant proliferation and apoptosis of tumor cells.