The Study For The Functions Of SQLE In The Malignant Behaviors Of Hepatocellular Carcinoma Cells And The Associated Molecular Mechanisms

Primary cancer of liver (PLC) is the third leading cause of cancer related-death in the world. Numerous studies have demonstrated the important roles of cholesterol in the progression of cancer. Statin, the drug for the cholesterol lowering, has shown to inhibit the tumorigenesis of various cancer cells. Stating, the inhibitor of HMGCR, inhibited not only the end-product of cholesterol, but also the intermediates (FPP and GGPP). Therefore, the side effects of Statin could not be ignored. It is very important to identify new therapeutic targets downstream of cholesterol biosynthesis. SQLE is an enzyme involved in cholesterol biosynthesis downstream of HMGCR. It has been reported that the expression of SQLE was dys-regulated in breast cancer, pancreatic cancerr and colon cancer. However, the expression pattern and the function of SQLE in hepatocellular carcinoma remain unknown. In order to study the expression pattern of SQLE hepatocellular carcinoma, using Realtime PCR, western blot and immunohistochemistry, we first examined the mRNA level and protein level of SQLE in HCC tissues and matched normal tissues. It was found that the expression of SQLE were up-regulated in HCC tissues compared with the matched normal tissues. Moreover, the expression of SQLE positively correlated with the malignancy of HCC cell lines with the lowest expression of SQLE in normal liver cells Chang and highest expression of SQLE in Hep3B, MHCC97 and HepG2 cells. These observations indicated that SQLE might play an important role in the tumorigenesis of HCC. Forced expression of SQLE in HCC cells enhanced cell growth, migration and colony formation on the soft agar, while knocking down the expression of SQLE in HCC cells inhibited cell growth in liquid culture, migration, colony formation on the soft agar as well as the metastasis of HCC cells in the nude mice. Molecular mechanism studies showed that SQLE promoted the phosphorylation of ERK, and knocking down the expression of ERK abolished the promoting effects of SQLE on the growth of HCC cells. Collectively, our study suggested that SQLE might promote the growth, migration and metastasis of HCC cells by activating ERK signaling. Our study was divided into the following two parts.Part IThe expression of SQLE was up-regulated in HCC tissues and cell linesObjective:To examine the expression pattern of SQLE in HCC tissues and cell lines and clarify the expression pattern of SQLE in HCC.Methods:The expression of SQLE mRNA was examined in 40 HCC tissues and paired normal tissues using Real-time PCR; the SQLE protein level in randomly chosen HCC tissues and paired normal tissues was examined using immunohistochemistry staining and Western blot analysis; the protein level of SQLE in HCC cell lines and normal liver cells was examined using Western blot analysis.Results:The mRNA level of SQLE was up-regulated in HCC tissues compared with the matched normal tissues. Immunohistochemistry staining Western blot analysis showed that the protein level of SQLE was up-regulated in HCC tissues compared with the matched normal tissues, which was consistent with the observations in Real-time assay. The expression of SQLE was correlated with the malignancy of HCC cells. Lower expression of SQLE was observed in normal liver cells and stronger expression of SQLE was observed in HCC cells Conclusion:The expression of SQLE was up-regulated in HCC, which suggested the important roles of SQLE in the progression of HCC.Part IIThe effects of SQLE on the aggressive behaviors of HCC cells and ERK signalingObjective:To study the functions of SQLE in the growth, migration, colony formation and metastasis of HCC cells, and explore the biological functions of SQLE in HCC.Methods:Constructing the expression vector of SQLE and transfecting the SQLE expression vector to HepG2 and 7404 cells by Lipofectamin. The effect of SQLE on the growth of HCC cells was analyzed using crystal violet assay. The effect of SQLE on the colony formation of HCC cells was analyzed using soft agar assay. The effect of SQLE on the migration of HCC cells was analyzed using Boyden chamber assay. At the same time, the lenti-virus vector to knock down the expression of SQLE in HepG2 and MHCC-97 cells was constructed. The effect of knocking down the expression of SQLE on the growth of HCC cells was analyzed by crystal violet assay. The effect of knocking down the expression of SQLE on the colony formation of HCC cells was analyzed by soft agar assay. The effect of knocking down the expression of SQLE on the migration of HCC cells was analyzed by Boyden chamber assay. The effect of knocking down the expression of SQLE on the metastasis of HCC cells in vivo was analyzed using nude mice. Moreover, the effect of SQLE on the phosphorylation of ERK was examined by Western blot.Results:HepG2 and 7404 stable cell lines over-expressing SQLE were established by stable transfection using Lipofectamin. The crystal violet assay demonstrated that forced expression of SQLE in HCC cells promoted the growth of HepG2 and 7404 cells. The Boyden chamber assay showed that forced expression of SQLE promoted the migration of HCC cells. The colony formation assay showed that forced expression of SQLE in HCC cells enhanced the anchorage-independent growth of tumor cells. On the other hand, we successfully knocked down the expression of SQLE in HepG2 and MHCC97 cells using lenti-virus mediated RNAi system. The crystal violet assay showed that knocking down the expression of SQLE in HCC cells inhibited the growth of HepG2 and MHCC97 cells. The Boyden chamber assay showed that knocking down the expression of SQLE inhibited the migration of HCC cells. The colony formation assay showed that knocking down the expression of SQLE in HCC cells inhibited the anchorage-independent growth of tumor cells. In vivo metastasis assay showed knocking down the expression of SQLE inhibited the metastasis of HepG2 cells in nude mice. Moreover, overexpression of SQLE promoted the phosphorylation of ERK, while knocking down the expression of SQLE inhibited the phosphorylation of ERK. Knocking down the expression of ERK abolished the promoting effects of SQLE on cell growth.Conclusions:SQLE promoted the growth, migration and colony formation of HCC cells, while knocking down the expression of SQLE inhibited the growth, migration, colony formation and metastasis of HCC cells. SQLE played important roles in the tumorigenesis of HCC via activating ERK signaling.