| Human hepatocellular carcinoma (HCC) is one of malignant tumors with high incidence in China. Every year there comes out about fifty six thousand new HCC patients in the world, of which about half emerged in China. The death rate of HCC in China jumped from third to second rank among cancer mortality. There exists no effective treatment, even with combined treatment of surgery and chemotherapy, the prognosis is still poor. Three-year relapse rate of small HCC less than 5 cm after sugery was higher than 50%. The main reason is that HCC possesses strong metastasis potential. Metastasis is the most important biological behavior of malignant tumors. It is a multi-stage process, which includes the tumor cells separating with surrounding cells and extracellular matrix, drilling through the vascular basement membrane into the blood circulation, reaching the target organ with the blood flow, colonying to a metastatic tumor. It involved in multiple signaling pathways and a number of gene activation or inactivation. Research on the molecular mechanism of HCC invasion and metastasis will help us to find new targets for intervention to improve the HCC treatment efficacy and prognosis.Aberrant signal transmission between cells, cells and stroma leads to changes in adhesion characteristics, which is an important factor to switch on tumor invasion and metastasis. Connexin (Cx) is a class of membrane proteins encoded by multigene family, including at least 20 kinds in mammals, which is the basic structural unit of gap junction (GJ). Connexin 32 (Cx32) is the major connexin expressing in human liver and main component of hepatocyte gap junctions. Gap junction is a special structure in cell membrane and the only direct channel for material exchange and information trasmission. Cells can transfer energy and information through gap junction intercellular communication (GJIC) to regulate cell growth, differentiation and maintain homeostasis. Recent studies showed that the protein expression of Cx32 was down-regulated in a variety of tumor tissue, moreover, the reduction and the disorder of associated signaling pathway may lead to GJIC dysfunction between cells, which was thought to be an important mechanism of carcinogenesis.Therefore, we chose Cx32 as a potential molecular target to further investigate its effect on HCC invasion and related mechanism in order to provide an experimental basis for the theory of HCC invasion and metastasis. MethodsTwenty cases of HCC resection specimens proven by pathology were collected from liver surgery department of Tongji Hospital and detected mRNA and protein related expression level of Cx32 gene by RT-PCR and Western blot analysis to explore the relationship between Cx32 gene and carcinogenesis of HCC. Three human hepatocellular carcinoma cell lines with different metastatic potential, Hep G2, MHCC-97L and MHCC-97H were cultured and detected mRNA and protein related expression level of Cx32 gene by RT-PCR and Western blot analysis to investigate the correlation between Cx32 gene and metastasis of HCC. The pcDNA 3.1(-)/Cx32 eukaryotic expression vector was constructed and transfected into MHCC-97H cells. Cells with Cx32 overexpression were screened with G418 and validated by Western blot analysis. The gap junction intercellular communication between cells was detected by scrape-loading and dye transfer assay. The invasion of cells was detected by Transwell assay. The Cx32-shRNA eukaryotic expression vector was constructed and transfected into Hep G2 cells. Cells with Cx32 depression were screened with G418 and validated by Western blot analysis. The gap junction intercellular communication between cells was detected by scrape-loading and dye transfer assay. The invasion of cells was detected by Transwell assay. Moreover, the expressions of membrane protein Occludin,Claudin-1,ZO-1 were detected by Western blot assay in cells with Cx32 depression. Furthermore, the Notchl-shRNA was constructed and transfected into MHCC-97H cells. Cells with Notchl depression were screened with G418 and validated by Western blot analysis. Scrape-loading and dye transfer assay and transwell assay were performed on cells with or without gap junction blocker, carbenoxolone.Results1. RT-PCR and Western blot analysis showed that the expression level of mRNA in tumor and adjacent tissue were 0.1600±0.0793 and 0.3169±0.0821, while the expression level of protein in them were 0.5564±0.1735 and 0.8720±0.1321, respectively. Compared with adjacent tissue, the mRNA and protein expression of Cx32 in human HCC tissue was significantly depressed(P<0.05). Furthermore, the expression level of mRNA in three HCC cell lines were 0.3023±0.0401,0.1829±0.02760,0.0746±0.0106 and the protein level in them were 0.5972±0.08831,0.2866±0.03280,0.1544±0.02354, respectively. There were significant difference in mRNA and protein expression of Cx32 gene in human HCC cell lines with different metastatic potential (P <0.05). The expression level was down-regulated with the upgrade of metastatic potential.2. The pcDNA 3.1(-)/Cx32 eukaryotic expression vector was constructed successfully. The protein level of cells transfected with pcDNA 3.1(-)/Cx32 plasmid was 1.2996±0.1641, higher than blank(0.4964±0.08969) and empty vector group(0.5097±0.04927). Compared with blank and cells transfected with empty vector, the gap junction intercellular communication of cells with Cx32 overexpression was enhanced. The migration cell count of Cx32 plasmid group were 159.3±14.0,69.7±6.8, lower than blank (213.3±17.6,102.0±12.5) and empty vector group (209.0±16.5,96.0±10.5).3. We constructed Cx32-shRNA eukaryotic expression vector successfully. The Cx32 expression of cells transfected with Cx32-shRNA plasmid was decreased by 67.4%. Compared with blank and cells transfected with empty vector, the gap junction intercellular communication of cells with Cx32 depression was weaken. The migration cell count of Cx32-shRNA group were 198.3±18.5 and 116.0±10.5, higher than blank (148.7±14.0,79.3±8.5) and empty vector group (137.3±14.6,76.3±6.5).4. Western blot analysis detecting the expressions of membrane proteins including Occludin, Claudin-1 and ZO-1 showed that the expression level of tight junction protein Occludn,Claudin-1 and ZO-1 in cells with Cx32 depression were 0.2675±0.03958, 0.4213±0.02933,0.1319+0.00857, lower than blank (0.414±0.03677,1.0472±0.1095,0.4573±0.03507) and empty vector group (0.4279±0.03958,1.1145±0.1325, 0.4682±0.04186).5. Notchl-shRNA eukaryotic expression vector was constructed successfully and transfected to MHCC-97H to obtain cell line with stable low expression of Notchl which was decreased by 60.78%. Compared with blank and cells transfected with empty vector, the gap junction intercellular communication of cells with Notchl depression was enhanced. The migration cell count were 85.7±6.0,45.7±5.5, lower than blank (215.7±15.6,112.0±12.1) and empty vector group (204.7±15.5,106.0±9.5; P<0.05), while incubating cells with carbenoxolone could increase the cell count to 119.3±10.1 and 63.3±6.1, partially reversed the depression of invasion potential (P <0.05).ConclusionThe expression of Cx32 gene was low in both human HCC tissues and cell lines and was decreased with upgrade of the metastasis potential of cell lines. Cx32 overexpression could promote the gap junction intercellular communication of HCC cells, but reduce the invasion potential of cells. On the other hand, inhibiting Cx32 expression could depress the gap junction intercellular communication, but promote cell invasion. This verified that Cx32 gene may regulate the metastatic potential of human HCC cell lines.Further exploring the molecular mechanism of Cx32 regulating the invasion of human HCC, we found that inhibiting the expression of Cx32 depressed the expression of Occludin,Claudin-1,ZO-1 in the cell membrane, which suggested that Cx32 could control the invasion of human HCC through tight junction. Furthermore, Notchl depression would up-regulate the gap junction intercellular communication and down-regulate the invasion potential of cells, while incubating with gap junction blocker may partially reverse the down-regulation. This suggested that Cx32 may be involved in Notch signaling pathway on the regulation of cell invasion.In summary, Cx32 gene may inhibit the invasion potential of human HCC through gap junction, while it was involved in the regulation of HCC invasion by Notch pathway. It suggested that Cx32 may be an effective molecular target of human HCC treatment.
|
PDF Full Text Request