STAT3 Involved In The Liver Tumor Development Of Transgenic Mice Induced By H-ras12V

Objective: To investigate the functions of STAT3 in ras oncogene-induced liver tumor.Methods:1. The tumor tissues(T) and peri-tumor tissues(P) of 9-month-age H-ras12 V transgenic male mice and the liver tissues of 9-month-age non-transgenic male mice(Wt) were collected. Pathological analysis was performed and total proteins and RNAs were extracted. The total RNAs and proteins were extracted from human normal liver cell lines(L-O2) and human hepatoma cell lines(Hep3B). The protein levels of p-ERK,ERK, p-PI3 K, PI3 K, p-AKT, AKT, p-m TOR, m TOR, p-STAT3, STAT3 in the liver tissue and cell samples were detected by western blot. The m RNA level of pik3r1, the target gene of STAT3, in the liver tissue and cell samples was detected by Fluorescence quantitative PCR.2. The inhibitor LY294002, PD184352 and Rapamycin(RAPA) were used to inhibit the activities of PI3K/AKT, ERK, and m TOR in Hep3 B respectively, and the protein level of STAT3 and the m RNA level of pik3r1 were detected.Results:1. The protein level of p-ERK in T tissues was 22.09±6.57, significantly increased than that in Wt(0.08±0.02), t=5.80, P=0.004, and P(8.60±3.23), t=3.19, P=0.033, the protein level of p-ERK in P tissues was significantly increased than that in Wt, t=4.57,P=0.045; the level of p-ERK in Hep3 B was 10.87 times higher than that in L-O2. The protein level of ERK in T tissues was 16.39±3.40, significantly increased than that in Wt(5.27±1.43), t=4.20, P=0.025, and P(4.76±1.79), t=5.24, P=0.006; the level of ERK in Hep3 B was 24.77 times higher than that in L-O2. The protein level of p-PI3 K in T tissues was 0.45±0.04, significantly increased than that in Wt(0.06±0.00), t=8.12,P=0.004, and P(0.23±0.04), t=4.12, P=0.015, the protein level of p-PI3 K in P tissues was significantly increased than that in Wt, t=3.32, P=0.045; there was no significant difference between Hep3 B and L-O2 for p-PI3 K level. The protein level of PI3 K in T tissues was 0.87±0.13, significantly increased than that in Wt(0.03±0.00), t=10.56, P<0.001, and P(0.28±0.09), t=3.96, P=0.029; the protein level of PI3 K in P tissues was significantly increased than that in Wt, t=3.43, P=0.019; the level of PI3 K in L-O2 was500 times higher than that in Hep3 B. The protein level of p-AKT in T tissues was0.69±0.04, significantly increased than that in Wt(0.10±0.02), t=22.90, P<0.001, and P(0.08 ± 0.03), t=21.58, P < 0.001; the level of p-AKT in Hep3 B was 6.77 times higher than that in L-O2. The protein level of AKT in T tissues was 0.19±0.04,significantly increased than that in Wt(0.08±0.05), t=2.96, P=0.041, the protein level of AKT in P tissues was 0.25±0.04, significantly increased than that in Wt, t=4.38,P=0.012; the level of AKT in Hep3 B was 6.75 times higher than that in L-O2. The protein level of p-m TOR in T tissues was 0.44±0.02, significantly increased than that in Wt(0.14±0.03), t=14.36, P<0.001, and P(0.16±0.04), t=10.51, P<0.001; the level of p-m TOR in Hep3 B were 21.74 times higher than that in L-O2. There is no significant differences in the m TOR level of among Wt(0.18±0.06), P(0.35±0.27), T(0.61±0.67); the level of m TOR in L-O2 was 5 times higher than that in Hep3 B. The protein level of p-STAT3 in Wt tissues was 42.87±9.36, significantly increased than that in T(4.27±0.72), t=4.119, P=0.015, the protein level of p-STAT3 in P tissues was32.47±3.21, significantly increased than that in T, t=8.73, P=0.001; the level of p-STAT3 in L-O2 was 10 times higher than that in Hep3 B. There is no significant differences in STAT3 level among Wt(25.81±8.97), P(27.81±0.61), T(26.67±0.43);the level of STAT3 in L-O2 was 2 times higher than that in Hep3 B. RT-q PCR results showed that, compared to Wt, the level of pik3r1 was significantly decreased in P and T(P < 0.01); the level of pik3r1 was significantly decreased in Hep3 B than that in L-O2(P < 0.001).2. Inhibition experiments showed that the level of p-AKT was gradually decreased with time by using Y294002 and followed by the increased p-STAT3 level. The level of p-ERK was gradually decreased with time by using PD184352 and followed by the decreased p-STAT3 level. The level of p-m TOR was increased at 3 h by using RAPA and followed by the decreased levels of p-STAT3. However, the level of p-m TOR was decreased at 12 h by using RAPA and followed by the increased levels of p-STAT3. By using LY294002 and PD184352, the m RNA levels of pik3r1 increased at 6 and 12 h and returned to normal levels at 18 h when compared with the control group. By using RAPA, the m RNA level of pik3r1 increased at 12 and 18 h.Conclusions: In vivo experiments confirmed that the activation of ras signaling pathway reduces the protein level of p-STAT3 and the m RNA level of pik3r1,indicating that STAT3 may act as a tumor suppressor in oncogene ras induced liver cancer. In vitro experiments also confirmed that STAT3 may act as a tumor suppressor in Hep3 B cells. The in vitro inhibition experiments showed that ERK pathway may play roles in the promotion for the phosphorylation of STAT3. However, PI3K/AKT and m TOR pathways may play roles in the inhibition in the phosphorylation of STAT3.