| With the development of society, diabetes mellitus(DM) and malignant disease have been the global issueses that threaten human life and health. In recent years, more and more epidemic and clinical dataes have shown that diabetic patients were liable to malignant disease. Some researches have found that digestive cancer was at the most particular risk when the malignant disease was simultaneous with DM. Hepatocullular carcinoma(HCC) was one of the most popular digestive cancer in the world with a high-grade malignancy, and incidence and mortality rate of HCC were respectively fifth and third high-grade of all these cancer. There were the most HCC patients in China and about50%patients died of HCC were in China, so HCC have been making a threat to peoples’health and life, and also be focused by reseachers at home and abroad. At present, the infection of hepatitis B virus,hepatitis c virus and alcohol have close relations with HCC, but the pathogenesis of some kinds of HCC are still undefined. What is the relationship of type2diabetes mellitus and HCC? What is the molecular mechanism? We need to explore. At the same time, the documents of relative research were infrequent and limited to epidemiological investigation and case control study.According to the physiopathologic change of type2DM, we divide course of this disease into four stages with the different change of glucose and insulin. The change of levels of blood glucose and insulin run through the overall process of type2DM. The relationship between HCC and type2DM implied that the change of levels of blood glucose and insulin might influence the biological characteristics of HCC. As these relative researches are relatively rare, further and deeper studies have been in desired. The generation and development of HCC underwent multi-gene, multi-factor and muti-stage process, which involved activation of oncogenes and inactivation of tumor suppressor genes, and induced to change of biological behaviour. Cyclooxygenase-2(COX-2) frequently participate in the pathological process of inflammation and tumor, and was expressed in80%turner samples, but did not express in normal tissues and cells. COX-2could promote the activation of oncogenes and inactivation of tumor suppressor genes, followed by cell proliferation and/or cell apoptosis, tumor angiogenesis, invasiveness and metastasis of cancer cells, immune tolerance,and so on. So to speak that COX-2could promote the generation and development of cancer. Vascular endothelial cell growth factor(VEGF) was a kind of specific mitogen in endothelial cells. VEGF could regulate the generation ofblood and lymphatic vessel with the most effective function in angiogenic factor. The expression of VEGF was regulated by many elements and had a close relation with cancer and other disease. HCC was a kind of solid tumor with abundant blood supply, and the generation of blood vessel played an important role in the tumor angiogenesis, invasiveness and metastasis of cancer cells. In this study, we imitated the change of different levels of glucose and insulin in type2DM, detected the growth influence of HCC cell line SMMC-7721, and the expression regulation of COX-2and VEGF genes. The results of this study could preliminary expound the relationship between HCC and type2DM, and provide new thinking and theory of clinial study for HCC.Methods1. Case control study of HCC with risk factors1350cases of HCC patients and non-cancer patients (control) from the first affiliated hospital of Zhengzhou university were selected, and the relative data was then collect and classified. The difference of demography characteristic and the correlation of type2DM with HCC were evaluated by using chi-square test, and the odds ratio(OR) of risk factors and95%confidence interval(95%CI) for HCC were evaluated by Logistic regression analysis.2. Optimizations of concentration and time for HCC SMMC-7721cell cultureOptimizations of concentration and time for HCC SMMC-7721cell culture were confirmed by observation from inverted microscope and using MTT assay to evaluate cell quantity and condition.3. Proliferation of HCC SMMC-7721cells treated with different levels of glucose and insulinHCC SMMC-7721cells were treated with different levels of glucose(5mmol/L, lOmmol/L,20mmol/L), insulin(10μIU/ml,50μIU/ml,100μIU/ml) and combination of glucose and insulin (5+10,5+100,20+100,20+10) for48hours, and MTT assay was used to detect the proliferation capacity.4. Apoptosis of HCC SMMC-7721cells treated with different levels of glucose and insulinHCC SMMC-7721cells were treated with different levels of glucose(5mmol/L,10mmol/L,20mmol/L), insulin(10μIU/ml,50μIU/ml,100μIU/ml) and combination of glucose and insulin (5+10,5+100,20+100,20+10) for48hours, and flow cytometry (FCM) was used to assay the apoptosis rate and analysis the apoptosis condition of cells.5. Expression of COX-2and VEGF mRNAReverse transcript PCR(RT-PCR) was applied to detect the expression of COX-2and VEGF mRNA in HCC SMMC-7721cells were treated with different levels of glucose(5mmol/L, lOmmol/L,20mmol/L), insulin(10μIU/ml,50μIU/ml,100μIU/ml) and combination of glucose and insulin (5+10,5+100,20+100,20+10) for48hours.6. Expression of COX-2and VEGF proteinWesternblot was used to evaluated the expression of COX-2and VEGF mRNA in HCC SMMC-7721cells were treated with different levels of glucose(5mmol/L,10mmol/L,20mmol/L), insulin(10μIU/ml,50μIU/ml,100μIU/ml) and combination of glucose and insulin (5+10,5+100,20+100,20+10) for48hours.Statistical AnalysisStatistical analysis was performed with chi-square test, Logistic regression analysis and ANOVA analysis of variance. Statistical significance was set at α=0.05.Results1. Results showed that infection of hepatitis B virus and hepatitis C virus, alcoholomania, type2DM and family history of HCC were morbidity risk factors of HCC(P<0.05), and type2DM could increase the onset risk of HCC(odds ratio(OR)=1.881,95%CI:1.314～2.671), while smoking and fat were independent to HCC(P>0.05)2. Optimizations of concentration and time for HCC SMMC-7721cell culture was proceeded to avoid the influence by the cells’growth factors, results showed that the optimum concentration was2×105cells/ml, and cells were appropriately cultured for48h.3. In this part, we found that the proliferation capacity of SMMC-7721cells was enhanced gradually following by the concentration increasing of glucose(5mmol/L,10mmol/L,20mmol/L) and insulin(10μIU/ml,50μIU/ml,100μlU/ml). The proliferation of cells in100μIU/ml insulin group was the highest of all groups, but the combination groups did not showed the synergistic effect to proliferation capacity. The group(20+100) cells had a better proliferation compared with other combination groups (P<0.05)4.The apoptosis cells of SMMC-7721decreased gradually following by the concentration increasing of glucose(5mmol/L,10mmol/L,20mmol/L) and insulin(10μIU/ml,50μIU/ml,100μIU/ml). The apoptosis rate of cells in100μIU/ml insulin group was the lowest of all groups, but the combination groups did not showed the synergistic effect to cells apoptosis.5. With the increasing of glucose concentration(5mmol/L, lOmmol/L,20mmol/L), expression of VEGF mRNA in SMMC-7721cells was increased obviously:the relative VEGF mRNA expression of HCC cells in20mmol/L glucose group was2.78±0.07, which was higher than other groups (P<0.05), while the relative COX-2mRNA expression change did not keep track of VEGF mRNA, that is the highest expression of COX-2was in10mmol/L group(0.57±0.05)(P<0.05). Unlike to glucose group, With the increasing of insulin concentration (10μIU/ml,50μIU/ml,100μIU/ml), expression of VEGF and COX-2in SMMC-7721cells was increased obviously, relatively expression of VEGF and COX-2mRNA were respectively1.71±0.03and1.74±0.05, which were higher than other groups (P<0.05) Meanwhile, relatively expression of VEGF of cells in the combination groups was increased obvious (P<0.05), but COX-2did not changed evidently.6. With the increasing of glucose concentration(5mmol/L,10mmol/L,20mmol/L), expression of VEGF protein in SMMC-7721cells was increased obviously:the relative VEGF protein expression of HCC cells in20mmol/L glucose group was2.53±0.11, which was higher than other groups (P<0.05), while the relative COX-2mRNA expression change did not keep track of VEGF protein, that is the highest expression of COX-2was in10mmol/L group(0.74±0.08)(P<0.05). With the increasing of insulin concentration (10μIU/ml,50μIU/ml,100μIU/ml), expression of VEGF and COX-2protein in SMMC-7721cells was increased obviously, relatively expression of VEGF and COX-2protein were respectively2.05±0.14and0.92±0.15, which were higher than other groups (P<0.05). Meanwhile, relatively expression of VEGF protein of cells in the combination groups was increased obvious (P<0.05), but COX-2did not changed evidently.Conclusions1. As a risk factor, type2DM could increase the onset risk of HCC. The positive correlation was type2DM and HCC. Type2DM was a independent risk factor and exerted synergistic effects with other risk factors such as HBV/HCV, alcoholic consumption on the development of HCC。2. High concentration of glucose and insulin could increase proliferation ability and decrease the apoptosis of HCC cells, and the effect of insulin was stronger than glucose, but combination of glucose and insulin did not show any synergistic effect.3. High concentration of glucose could increase expression of VEGF, but had no effect to COX-2expression; while high concentration of insulin could increase both VEGF and COX-2gene expression, but anyhow combination of glucose and insulin had little synergistic effect to these two genes expression. |
PDF Full Text Request