| Liver cancer was one of common malignant tumors in China, high mortality, previous studies found that hepatitis virus (HBV, HCV) was the major cause of liver cancer, it had a close relationship with liver cancer. In addition, in signal transduction discovery, Wnt /β-catenin signaling pathway was also closely related with liver cancer, also play an important physiological role in the process of stem cells. Wnt /β-catenin signaling pathway was an extremely conservative in the evolution of the pathway. From lower invertebrates to humans, Wnt /β-catenin signaling molecule to maintain a high degree of homology. In embryonic development, Wnt /β-catenin signaling pathway was involved in many areas of life and growth. Wnt /β-catenin signaling pathway controlled the growth after birth and plays a key role in updating.But the relationship of Wnt /β-catenin signaling and tumor was discovered in the 80s. The role of Wnt /β-catenin signaling in liver cancer has been highly valued, it has been found that Wnt /β-catenin signaling pathway primarily lead to the occurrence and development of liver cancer through the activation of its downstream target genes. The Wnt /β-catenin signaling pathway has become a new treatment for hot spots in recent years.Similar with c-myc and cyclinDl, Lgr5 was also considered to the target genes of WNT signaling pathway. Many studies have also confirmed that Lgr5 increased expression in a variety of tumors such as hepatocellular carcinoma, colon cancer, ovarian cancer and basal cell carcinoma.Leucine rich repeat containing G protein coupled receptor 5 (LGR5) was originally isolated from the colorectal cancer cells. In recent years, researchers found the expression of LGR5 in the liver and ovarian cancer cells, and experiments confirmed that it was the target gene of Wnt signaling pathway. Dutch researchers found that LGR5 expressed only in the small intestine and colon crypt base columnar epithelial cell-specific, the positive cells of gene encoding the product has a stem cell activity, and confirmed that LGR5 was the marker of small intestinal and colorectal stem cell, and speculated that it may also be marker of more kinds of cancer stem cells. Clinical data show that the overexpression of Lgr5 in some patients with hepatocellular carcinoma had a closely related with the gene mutation in exon III ofβ-catenin, witch was an most critical effector in Wnt signaling pathway .Therefore, this study focused on the following aspects: 1. The construction of LGR5 promoter plasmid;2.The expression of LGR5 in a variety of liver cancer cells; 3. The expression of LGR5 in the hepatocellular carcinoma and adjacent tissues. This study aim to confirm, the abnormal activation of Wnt signaling pathway led to the upregulation of Lgr5 ,it may plaied an important role in the abnormal proliferation of liver cells and the happen of liver tumors.It was expected to become a new target for the treatment of hepatocellular carcinomaMethods:1. The construction of LGR5 promoter plasmid: LGR5 promoter fragments of different lengths were amplified by PCR, recycling pGL4.21 fragment inserted into plasmid to construct the 7 different lengths of reporter plasmid, and then were transfected into SW480 and HCT116 cells. The promoter activity and activity in the regulation were detected;2. The expression of LGR5 in a variety of liver cancer cells The expression of LGR5 in 4 kinds of HCC cell (HepG2, Hep3B, Huh7 and PLC) were detected by using western-blot, Real-time PCR..3. The expression of LGR5 in the hepatocellular carcinoma and adjacent tissues The expression of LGR5 in 36 cases of HCC tissue and adjacent tissue were detected by using Immunohistochemistry.Results:1. LGR promoter plasmids were successfully constructed after identified. Transfected cells were detected, LGR5 F1-F6 promoter in SW480 and HCT116 cells, have high activity, and much more active in HCT116.Compared with the rest of the activity, F7 was significantly decreased, indicating that between -645 ~ -330 promoter activity as a key area. In addition, it found that the activity of F1 increased after the co-transfection of AC133 orβ-Catenin, SW480 and HCT116 cells were the same trend, and changes was more obvious in HCT116 cells.2. LGR5 showed low expression in liver cancer cells, compared to normal liver cells L02 (L02: 1.2±0.10), the expression levels of LGR5 protein in four liver cancer cells were (HepG2 = 0.7±0.04, Hep3B = 0.4±0.03, Huh7 = 0.12±0.07, PLC = 0.3±0.04), there were significantly lower (P<0.01). Huh7 cells hardly expressed. However, compared to the other hepatoma cell lines, the expression levels of LGR5 protein in HepG2 cell lines was slightly higher. Compared with normal liver cells L02, the expression levels LGR5 mRNA in four liver cells were (HepG2/L02 = 0.3±0.04, Hep3B/L02 = 0.08±0.03, Huh7/L02 = 0.02±0.01, PLC / L02 = 0.1±0.04), there were significantly lower (P<0.01).The results was same to the result of Western blot. Huh7 cells was most obvious decreased, up to 20 times (P<0.01).3. Compared to the liver adjacent tissues,the expression level of Lgr5 in HCC had an obvious downward trend. In 36 cases of HCC tissue samples, positive was only 6 cases (16.7%) , and negative was 30 cases (83.3%); In liver adjacent tissues, positive was 27 cases (75.0%), and negative was only 9 cases (25.0%). The number of LGR5 positive patients in cancer tissues was significantly reduced than adjacent tissues (P<0.01). In positive cases, lgr5 mainly distributed in the cytoplasm and membrane, very few located in the nucleus.Conclusion:Through the above experimental results showed, CD133, orβ-Catenin levels can up-regulated the expression of LGR5 in the level of promoter. Lgr5 was a target gene of Wnt pathway. Activated Wnt signaling pathway could caused the reduction or increase of Lgr5 expression. It may play a crucial role in cell proliferationa,differentiation and the happen of tumor formation, thus affecting the development of liver cancer. As the latest stem cell markers, Lgr5 could early screening for liver cancer diagnosis, clinical treatment and prognosis of great significance. |
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