| Objective:1. To research the mRNA expression level of PI3K/Akt/mTOR key signaling molecule (PI3K, AKT, mTOR, P70S6K, PTEN,4EBP) between gastric cancer tissue and adjacent non-turner tissues.2. To research the mRNA expression level of PI3K/Akt/mTOR key signaling molecule (PI3K, AKT, mTOR, P70S6K, PTEN,4EBP) in different degree of differentiation in gastric cancer cells(Poorly differentiated gastric cancer cell lines MGC-803, BGC-823 Moderately differentiated gastric cancer cell lines SGC-7901 Highly differentiated gastric cancer cell lines MKN-45, MKN-28) and Normal gastric mucosa cells GES-1. To screen out abnormal activation of gastric cancer cell line in PI3K/Akt/mTOR signaling pathway for subsequent experiments.3. To observe activity of PI3K/AKT/mTOR signal transduction pathway between gastric cancer cells and normal gastric mucosa cells by using inhibitors of key molecules LY294002 and Rapamycin. To explore the effects of key signaling molecule (mRNA and protein) expression on PI3K/Akt/mTOR signaling pathway by LY294002 and Rapamycin.4. Rapamycin can reduce the gastric cancer cells MGC-803, BGC-823 and SGC-7901 proliferation rate.Methods:1. This study raised gastric carcinoma and adjacent non-tumer tissues from 62 patients with primary gastric cancer. Real-time PCR were carried to detect the Ct value of PI3K, AKT, mTOR, P70S6K, PTEN,4EBP, Actin in gastric cancer tissue and adjacent non-tumer tissues. Count the ?Ct value, ACt=Cttarget gene-CtActin, Using case group and control group for paired t-test, compared the key signaling molecule's mRNA expression difference between gastric cancer tissue and adjacent non-tumor tissues.2. Real-time PCR were carried to detect the key signaling molecule's expression level of PI3K/Akt/mTOR include five strains of gastric cancer cells(Poorly differentiated gastric cancer cell lines MGC-803, BGC-823 Moderately differentiated gastric cancer cell lines SGC-7901 Highly differentiated gastric cancer cell lines MKN-45?MKN-28) and a control group of normal gastric mucosa cells (GES-1).3. LY294002 and Rapamycin which are the PI3K/Akt/mTOR signaling pathway inhibitors LY294002 and Rapamycin were used to gastric cancer cells and normal gastric mucosa cells in 24h and 48h levels. To observe the activity of PI3K/AKT/mTOR signal transduction pathway between gastric cancer cells and normal gastric mucosa cells. Real-time PCR and Western blot were used to detect the change of key signaling molecules involved in PI3K/Akt/mTOR signaling pathway.4. To research the pathway inhibitors LY294002 and Rapamycin on biological behavior of gastric cancer cells and normal cells. MTT assay was applied for investigating the effects of pathway inhibitor on the activity of cell proliferation for 24h,48h.. PI staining and Annexin V-FITC/PI were used to detect the cell cycle and apoptosis between gastric cancer cells and normal gastric mucosa cells.Results:1. PCR showed that:PI3K, AKT, mTOR, P70S6K, PTEN,4EBP between gastric cancer tissue and adjacent non-tumor tissues were correlated with statistical significance (P<0.05). The fold-change 2-??Ct were 2.259,4.234,2.709,2.713,0.623. The expression of P70S6K has no statistical significance (P>0.05).2. In the PI3K gene, the mRNA's expression of gastric cancer cells (MGC-803, BGC-823, SGC-7901, MKN28) of PI3K was significantly higher than normal gastric mucosal cells (P<0.05). The fold-changes 2-??Ct were 2.468,9.388,4.18,1.842. The expression of MKN-45 was lower than GES-1 (P<0.05), and its 2-??Ct was 0.372.3. In the AKT gene, the MGC-803's expression has no statistical significance (P>0.05), compared with normal gastric mucosal cells. The mRNA's expression of gastric cancer cells (BGC-823, SGC-7901) was significantly higher than normal gastric mucosal cells (P<0.05). The fold-changes 2-??Ct were 5.870,1.268. MKN-45, MKN-28 expression was lower than GES-1 (P<0.05), and its value of 2-??Ct were 1.268 and 0.309.4. In the mTOR gene, the mRNA expression of gastric cancer cells (MGC-803, BGC-823, SGC-7901, MKN28) of mTOR was significantly higher than normal gastric mucosal cells (P<0.05). The fold-changes 2-??Ct were 1.537,9.388,2.241,1.842. The MKN-45's mTOR expression has no statistical significance (P>0.05).5. In the P70S6K gene which was in the downstream of mTOR pathway, the mRNA's expression of gastric cancer cells (MKN-28, SGC-7901, BGC-823) was significantly higher than normal gastric mucosal cells (P<0.05). The fold-change 2-??Ct were 32.323,2.142, 6.638. The MKN-45, MGC-803's expression was lower than GES-1 (P<0.05), and the value of 2-??Ct were 0.052 and 0.468.6. In another mTOR downstream gene 4EPB, the expression of mRNA in gastric cancer cells (MGC-803, BGC-823, SGC-7901, MKN28 of 4EBP) was significantly higher than normal gastric mucosal cells (P<0.05). The fold-changes 2-??Ct were 5.682,8.535,5.357. The expression of highly differentiated gastric cancer cell lines (MKN-45, MKN-28) was lower than GES-1 (P<0.05), and their value of 2-??Ct were 0.049 and 0.122.7. In PTEN, the tumor suppressor gene, the expression of mRNA in gastric cancer cells (BGC-823, SGC-7901, MKN-28, MKN-45) of PTEN was significantly higher than normal gastric mucosal cells (P<0.05). The fold-changes 2-??Ct were 7.539,1.687,28.840,42.224. The expression of MGC-803 was lower than GES-1(P<0.05), and its 2-??Ct was 0.466.8. LY294002 can inhibit the expression of the key signaling molecules of PI3K/Akt/mTOR in gastric cancer cells (MGC-803?BGC-823?SGC-7901). After 24h and 48h, the mRNA's expression of PI3K, AKT, mTOR, P70S6K, PTEN,4EBP in experimental group and control group were correlated with statistical significance (P<0.05). The expression of protein on P-AKT (Ser473), p-P70S6K (Thr389), p-4EBP (Thr37/46) is inhibited.9. Compared with the control group, the proliferation rate of human gastric cancer cells could be significantly inhibited by LY294002 with the concentration increased.10.50?M LY294002 has significantly effect on cell cycle of gastric cancer cells (MGC-803, BGC-823, SGC-7901) and normal gastric mucosal cells GES-1. With LY294002 for 48h, it can block MGC-803 at S phase. S phase fraction has a significant difference in the experimental group (60.00±4.17)% and the control group (33.70±0.62)%. It can block MGC-803 at S phase. LY294002 shortened SGC-7901 S phase cell cycle process. S phase fraction has a significant difference in the experimental group (37.64±1.07)% and the control group (33.81±1.15)%. LY294002 decreased the rate of G1 phase in GES-1, G1 phase fraction has a significant difference between the experimental group (49.26±2.76)% and the negative control group (64.38±1.78)%.11. 50?M LY294002 can promote the apoptosis rate of gastric cancer cells (MGC-803, BGC-823, SGC-7901) and normal gastric mucosal cells GES-1. The cell apoptosis rate of MGC-803, BGC-823, SGC-7901, GES-1 in control group respectively were (3.2±0.4)%, (4.4±1.7)%,(5.3±0.1)% and (5.8±0.4)%. The four cell apoptosis in experimental group respectively were (55.9±7.0)%, (13.8±1.4)%,(10.2±0.9)% and (16.6±2.5)%. MGC-803 cells were more sensitive to LY294002.12. Using 50nM Rapamycin to MGC-803, BGC-823, SGC-7901 at 48h, the expression of mRNA in PI3K, PTEN, AKT, mTOR,4EBP and P70S6K of experimental group was significantly different compared to the control group. p-P70S6K (Thr389), p-4EBP (Thr37/46) protein expression is inhibited.13.50nM Rapamycin has an obvious influence on cell proliferation rate to MGC-803, BGC-823, SGC-7901 in 48h. Cell cycle were arrested at G1 phase in experimental groups of MGC-803, BGC-823 and SGC-7901. Only the apoptosis rate of BGC-823 cells was increased. Compared with the control group, the normal cells GES-1 Rapamycin group had no significance effect on proliferation, cycle and apoptosis.Conclusion:1. The PI3K/AKT/mTOR signal transduction pathway of gastric cancer patients activated abnormal.2. Compared with gastric mucosa cells GES-1, poorly differentiated gastric cancer cells MGC-803, BGC-823 and moderately differentiated gastric cancer cell SGC-7901, whose mRNA expression of PI3K, AKT, mTOR, P70S6K, PTEN and 4EBP increased significantly, PI3K/AKT/mTOR signaling pathway activated abnormal. However, the highly differentiated gastric cancer cell lines (MKN-45, MKN-28), whose key signaling molecule the mRNA of AKT. mTOR, P70S6K.4EBP was lower than GES-1.3. The proliferation rate, cell cycle ratio, and apoptosis were significantly affected by LY294002 in MGC-803, BGC-823and SGC-7901 and normal GES-1.4. Rapamycin can reduce gastric cancer cell lines (MGC-803, BGC-823 and SGC-7901) proliferation rate. Cell cycle are arrested in G1 phase. Rapamycin can promote the apoptosis of BGC-823 cells. Rapamycin has no significant effect on proliferation of GES-1 cells, apoptosis ratio. Treatment of Rapamycin on gastric cancer has important significance. |
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