| Objective:MiRNA plays an important role in the occurrence and development of liver tumors.The elevated expression levels of mi R-183 had been detected in human hepatic tumors and thought to be a characteristic of hepatic tumorigenesis.But the mi R-183 related molecular mechanisms are largely unknown.The aim of this work is to investigate the expression of mi R-183 and the related upstream regulatory mechanisms and downstream target proteins in human hepatoma Hep3 B cells and hepatic tumor cells in H-ras12 V transgenic mice.Methods: 1.The proliferation activities of the normal human hepatocytes LO2 and human hepatoma cells Hep G2 and Hep3 B were assayed by MTT.2.Total RNA and protein were extracted from the cells and hepatic tumor tissues.The mi R-183 levels were detected by RT-q PCR and protein levels of p-ERK1/2,ERK1/2,p-PI3 K,p-AKT,IκBα,PDCD4 were detected by Western blotting.3.The activities of ERK,PI3 K,AKT,and NF-κB signaling molecules were inhibited in Hep3 B cells,respectively,and the corresponding expression levels of mi R-183 and protein levels of PDCD4 were detected.Results: 1.Cell proliferation was detected by MTT experiment.The results showed that the fold increases of proliferation cells of LO2,Hep G2,and Hep3 B at 48 h were 8.76±0.22,16.61±1.59,and 19.86±0.69,respectively(F=159.90,P<0.001).2.Compared to LO2(41.68±9.62)and Hep G2(41.53±1.20)cells,the expression level of mi R-183 was significantly increased in Hep3B(69.15±11.02)cells(F=10.250,P=0.012).3.The expression levels of signaling molecules were detected by RT-q PCR.The experimental results showed that,in Hep3 B cells,the protein levels of p-ERK1,ERK1,p-AKT,and IκBα increased 10.87,24.68,6.67 and 1.92 times of LO2 cells,respectively.The protein levels of PDCD4 decreased 0.14 times of LO2 cells.In Hep G2 cells,the protein levels of IκBα and PDCD4 increased 4.46 and 7.90 times of LO2 cells,respectively.4.Anti-ERK(PD184352),Anti-PI3K(LY294002),Anti-AKT(MK2206),AntiNF-κB(BAY11-70829)significantly inhibited ERK、PI3K、AKT、NF-ΚB signaling activity in Hep3 B cells,respectively.At 12 h and 24 h,the expression level of mi R-183 was detected by RT-q PCR.The results showed that inhibition of ERK activity significantly induced the expression levels of mi R-183 at 24 h(F=215.459,P<0.001).Inhibition of PI3 K activity significantly reduced the expression levels of mi R-183 at 12 h and 24 h(F=80.215,P<0.001).Inhibition of AKT activity significantly reduced the expression levels of mi R-183 at 12 h and 24 h(F=101.947,P <0.001).Inhibition of NF-κB activity had no effect on the m RNA levels of mi R-183 at 12 h and 24 h(F=1.826,P=0.216).5.Anti-ERK(PD184352),Anti-PI3K(LY294002),Anti-AKT(MK2206),AntiNF-κB(BAY11-70829)significantly inhibited ERK、PI3K、AKT、NF-ΚB signaling activity in Hep3 B cells,respectively.At 24 h and 48 h,the expression level of PDCD4 protein was detected by Western blot.The results showed that inhibition of ERK and NF-κB activity had no effect on the protein levels of PDCD4.Inhibition of PI3 K signaling molecule,the expression level of PDCD4 protein increased significantly in 24 h,but had no significant change in 48 h.Inhibition of AKT signaling molecules in 24 h and 48 h,the expression level of PDCD4 protein was significantly increased.6.The results of high-throughput mi RNA expression sequencing and RT-q PCR showed that the expression level of mi R-183 in hepatic tumor tissue was significantly higher than that in normal liver tissue and peri-tumor tissue(P <0.05).7.The results of Western blotting showed that the expression of P-ERK,P-PI3 K and P-AKT proteins in hepatic tumor tissue were significantly higher than that in normal liver tissue and peri-tumor tissue,while the expression of IκB and PDCD4 protein was significantly decreased.Conclusions: The activation of PI3K/AKT signaling pathway significantly induces the expression levels of mi R-183 in Hep3 B cells,and PDCD4 is an important downstream target protein of mi R-183.The above phenotype was also validated in H-ras12 V transgenic mice. |
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