Cyclin D1 Induced By Ras/MAPK/ERK Collaborating With MTOR And NF-κB In Liver Tumor

Objective:Hepatocellular carcinoma is one of the top three malignant tumors in China. The mutation of ras gene or the activation of Ras signaling pathway are generally happened molecular events in hepatocellular carcinoma. In addition, the overexpression of cyclin D1, which leading to the shortened cell cycle and increased cell proliferation, is an important feature of hepatocellular carcinoma and other primary tumors and is an important target molecule for diagnosis and prognosis of tumors. Cyclin D1 is mainly regulated by three signaling pathways:(1) p-ERK can promote the m RNA level of cyclin D1 through activating the AP-1 or Ets elements located in the promoter region of cyclin D1;(2) NF-κB can promote the m RNA level of cyclin D1 through binding to the GGG(G/A)NNYYCC sequences located in the promoter region of cyclin D1;(3) m TOR can enhance the stabilities of the m RNA and protein of cyclin D1 to increase the protein level of cyclin D1. However, tumors in different tissues or different sub-type and development stage tumors in the same tissue have different mechanisms for controlling the cyclin D1 levels. Specially,the collaborating mechanisms among ERK, NF-κB, and m TOR still need to be elucidated. In this study, by exploring H-ras12 V transgenic mice and Hep3 B cell lines,the collaborating mechanisms among ERK, NF-κB, and m TOR for controlling the cyclin D1 levels were investigated.Methods:1. In vivo experimentsThe liver tumor tissues and peri-tumor tissues of 9-month-old H-ras12 V transgenic male mice and normal liver tissues of C57BL/6J male mice were sampled.One part of each sample was fixed in 10% formalin and undergone paraffin-embedded tissue section, HE staining, and pathological analysis. Another part of each sample was frozen in liquid nitrogen for extraction of total RNAs and protein. The m RNA level of cyclin D1 was examined by RT-q PCR. The protein levels of cyclin D1, ERK1/2,p-ERK1/2, p-m TOR, and IκB were detected by Western blot.2. In vitro experimentsThe total RNAs and protein were extracted from LO2 and Hep3 B cell lines. The m RNA level of cyclin D1 was examined by RT-q PCR and the protein levels of cyclin D1, ERK1/2, p-ERK1/2, and IκB were detected by Western blot. The inhibitors were used to inhibit the activities of ERK, m TOR, and NF-κB in Hep3 B cells respectively,and the m RNA and protein levels of cyclin D1 were detected.Results:1. In vivo experiments(1) Compared with the liver of non-transgenic mouse, the liver of H-ras12 V transgenic mouse harbored hepatic tumors. The pathology analysis showed that the hepatic tumor tissues exhibited trabecular growth arrangement, atypical liver cell aggregation, cytoplasmic basophila, hyperchromatic nuclei and contained highly undifferentiated cells accompanied by necrosis. The fibrosis and cirrhosis were not observed.(2) Compared with the normal liver tissues and peri-tumor tissues, the m RNA and protein levels of cyclin D1 were significantly increased(P<0.05) in tumor tissues.There was no significantly difference between normal liver tissues and peri-tumor tissues.(3) Compared with the normal liver tissues and peri-tumor tissues, the phosphorylation and total protein levels of ERK and m TOR were significantly increased(P<0.05), and the protein level of IκB was significantly reduced(P<0.05) in tumor tissues. There was no significant difference between normal liver tissues and peri-tumor tissues in ERK level. However, the p-ERK level was significantly increased in peri-tumor tissues(P<0.05) compared to normal liver tissues. There was nosignificantly difference between normal liver tissues and peri-tumor tissues in p-m TOR and IκB levels.2. In vitro experiments(1) Compared to LO2, the m RNA and protein levels of cyclin D1 were significantly increased(115.73 times higher, t=36.617, P<0.001, and 22.60 times higher, t=8.457,P<0.001, respectively).(2) Compared to LO2, the protein levels of p-ERK1, ERK1, and p-m TOR were significantly increased(10.87 times higher, t=5.903, P=0.004; 24.77 times higher,t=23.284,P=0.002; and 20,60 times higher, t=24.492,P<0.001, respectively) in Hep3 B.There was no significant difference between Hep3 B and LO2 in IκBα level.(3) In Hep3 B cells, inhibiting ERK activity by PD184352 treatment had no effect on the protein level of p-m TOR, but the m RNA and protein levels of cyclin D1 were significantly decreased(F=52.53,P < 0.001). Inhibiting m TOR activity by rapamycin treatment significantly elevated p-ERK level, which accompanied with the increased m RNA level of cyclin D1(F=587.47,P = 0.002). However, the protein level of cyclin D1 was significantly decreased. Simultaneously inhibiting ERK and m TOR activities significantly decreased the m RNA and protein levels of cyclin D1. NF-κB activity inhibited by BAY11-7082 had no effect on the m RNA and protein levels of cyclin D1.Conclusions:1. In the development of the liver tumorigenesis induced by H-ras12 V, the activation of MAPK/ERK, NF-κB and m TOR signaling pathways may contribute to the overexpression of cyclin D1.2. The level of cyclin D1 is mainly regulated by m TOR and ERK1 in Hep3 B cells. Specially, ERK inhibited by m TOR may be an important regulation mechanism contributing to maintain high level of cyclin D1.