| Primary liver cancer is one of the highly malignant and life-threatened cancer with poor prognosis. A study showed that the incidence of primary liver cancer ranked the fifth in solid tumors worldwide, and the mortality rate ranked the third. Primary liver cancer includes hepatocellular carcinoma(HCC), cholangiocellular carcinoma(CC), hepatic angiosarcoma(HAS) and hepatoblastoma. Among them the most common one is HCC, which accounts for over 80% of the primary liver cancer. HCC is correlated with a number of risk factors, such as hepatitis virus, smoking, food contaminated by aflatoxin, alcohol abuse, nonalcoholic fatty liver disease, diabetes mellitus, and obesity. These factors are quite different worldwide, therefore, there are difficulties in diagnosis, treatment and prognosis predicting of HCC. Statistics showed that five-year survival rate of HCC patients gradually increased in the recent ten years, but the rate of recurrence and metastasis after resection was about 61.5% within five years. The number of HCC patients of our country accounts for nearly 50% percent of all the world. Every year there are about 230,000 people die of HCC. There are nearly 600,000 people die of HCC worldwide as a result of unfavorable therapeutic effects, and most of HCC patients die after diagnosis within one year, due to diagnosed at advanced stage, drug resistance, recurrence, vascular invasion and multiple intrahepatic metastasis. To those HCC patients with recurrence and metastasis, chemotherapy or oral targeted drug is the main method for treatment, such as arsenic trioxide(ATO) and sorafenib. Although studies showed that some molecular markers involved in the proliferation, apoptosis and metastasis of HCC cells, the concrete mechanism was still unclear. Therefore, clarifying the molecular mechanism in which the pivotal proteins involve in the regulation of proliferation, apoptosis and metastasis of HCC cells is desperately needed. It is of great value and significance in both research and clinics to predict the biomarkers associated to proliferation and drug resistance, search markers for early diagnosis and therapeutic targets.Desmosome is a intercellular junction with intermediate fibers. Recently, it showed that desmosome consisted of a number of proteins, such as glycoproteins and plakins. Glycoproteins include desmoglein(DSG), desmocollin(DSC), both of them are members of cadherin families. Plakins are mainly composed by desmoplakin(DP), plakoglobin(PG) and plakophilin(PP). In addition, some accessory proteins are also components of desmosome, such as desmocalmin and desmoyokin. Desmosomes are fundamental both for intercellular adhesion and for enabling the dynamic rearrangements of epithelia, they are also pivotal regulators for morphogenesis and differentiation of epithelia. Degradation of desmosome structure results in diseases originated from epithelia. In some specific conditions, desmosomes suffer function loss and structure alternation, as a result, the intercellular adhesion reduces and morphological changes occur, which is closely related to proliferation, differentiation, apoptosis, invasion, metastasis and neovascularization of tumor cells. Desmosome-associated protein pinin is a 140 k Da phosphorylated protein, due to its multiple location in the cell, it has a number of biological functions. Pinin was first identified in the intermediate filament of desmosome between cells. Interaction of desmosome-associated proteins enhances intercellular adhesion and reduces cellular migration. In addition to localization to desmosome, pinin also resides in nuclear plague and involves in splicing of pro-m RNA and transportation of m RNA.Pinin could regulate cell cycle, invasion, migration and drug resistance of cancer cells through multiple pathways. In various tumors derived from epithelia, such as renal carcinoma and pulmonary carcinoma, pinin is considered to inhibit the occurrence and development of tumors. Pinin is downregulated in some tumor tissues and tumor cell lines, however, it is unclear that whether pinin could affect the occurrence and development of HCC.In this study, we determined the expression of pinin in both tissue and cell level, and investigated its effects in the proliferation, clone formation and tolerance to glucose deprivation of HCC cells by using molecular biological methods. It is of great value and significance in both research and clinics to predict the biomarkers associated to proliferation and drug resistance, search markers for early diagnosis and therapeutic targets.Objective:Clarifying the function of desmosome-associated protein pinin and molecular mechanisms involved in the carcinogenesis and progression of HCC.Methods: 1. Western blot and real-time PCR were used to detect the difference of protein and m RNA expression of pinin in HCC tissue, peritumoral tissue and normal liver tissue. 2. The immunostaining analysis of pinin protein expression was assessed based on tissue microarrays. 3. Western blot and real-time PCR were used to detect the difference of protein and m RNA expression of pinin in HCC lines and normal liver cell lines. 4. Cell counting and Ed U methods were used to test the proliferation of HCC cells with downregulated pinin. 5. Using crystal violet to test the colony formation of HCC cells with upregulated or downregulated pinin. 6. Using CCK8 to test the cell viability of HCC cells with downregulated pinin in theenvironment of glucose deprivation. 7. Western blot was used to detect the expression of pinin in the environment of glucose deprivation. 8. Using flow cytometry technique and western blot to detect apoptosis of HCC cells with downregulated pinin in the environment of glucose deprivation. 9. Using flow cytometry technique and western blot to detect apoptosis of HCC cells with upregulated pinin in the environment of glucose deprivation. 10. Western blot was used to detect the expression of p-ERK of HCC cells with downredulated or upregulated pinin in the environment of glucose deprivation. 11. After administration of U0126, an inhibitor of p-ERK, flow cytometry technique was used to detect the apoptosis of HCC cells with upregulated pinin in the environment of glucose deprivation. 12. After administration of U0126, an inhibitor of p-ERK, Western blot was used to detect the expression of p-ERK with upregulated pinin in the environment of glucose deprivation.Results: 1. Western blot and real-time PCR showed that protein and m RNA expression of pinin in HCC tissue was much higher than peritumoral tissue and normal liver tissue. 2. By immunohistochemical analysis in 30 human HCC samples, 25(83%) cases show overexpression of pinin in HCC tissues as compared to the corresponding peritumoral tissues. In the clinical samples, moderate to strong cytoplasmic staining was observed in different clinical stages. 3. Western blot and real-time PCR showed that protein and m RNA expression of pinin in HCC cell lines was much higher than normal liver cell line. 4. Cell counting and Ed U methods showed that the proliferation of HCC cells with downregulated pinin reduced. 5. The capacity of colony formation of HCC cells with upregulated pinin enhanced, and the capacity of colony formation of HCC cells with downreguated pinin reduced.6. Cell viability of HCC cells with downregulated pinin reduced in the environment of glucose deprivation. 7. Western blot showed that the expression of pinin increased in the environment of glucose deprivation. 8. Flow cytometry technique and western blot showed that apoptosis of HCC cells with downregulated pinin increased in the environment of glucose deprivation. 9. Flow cytometry technique and western blot showed that apoptosis of HCC cells with upregulated pinin decreased in the environment of glucose deprivation. 10. Western blot showed that downregulation of pinin promoted the decrease of p-ERK in the environment of glucose deprivation, and upregulation of pinin inhibit the decrease of p-ERK in the environment of glucose deprivation. 11. Flow cytometry technique showed that U0126 inhibited pinin upregulation-induced inhibition of apoptosis in the environment of glucose deprivation. 12. Western blot showed that U0126 inhibited pinin-upregulation-induced inhibition of decrease of p-ERK in the environment of glucose deprivation.Conclusions: 1. Protein and m RNA expression of pinin in HCC tissue was much higher than peritumoral tissue and normal liver tissue, protein and m RNA expression of pinin in HCC cell lines was much higher than normal liver cell line, indicating that pinin is involved in the carcinogenesis of HCC. 2. Upregulation of pinin enhanced clone formation and tolerance to glucose deprivation of HCC cells, downreguation of pinin reduced the capacity of proliferation, clone formation and cell viability, suggesting that pinin promotes the carcinogenesis and progression of HCC. 3. Pinin regulates glucose deprivation-induced apoptosis of HCC cells through ERK pathway. |
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