动物造模,肝肿瘤 z-bio 2021-02-22 268

　　Introduction: Chronic liver disease and hepatocellular carcinoma (HCC) are increasingly becoming a global human problem. Hepatocellular carcinoma is the most deadly and common form of primary liver cancer. It is not an accidental event, but the slow progression of chronic liver disease, and it is still one of the most deadly cancer types. Every year, more than 800,000 people worldwide are diagnosed with liver cancer, and there is no successful treatment plan. The incidence and mortality of liver cancer in men worldwide are almost three times that of women. Although HCC is a multifactorial disease, including genetic factors, environmental factors, lifestyle and dietary factors, the vast majority of cases usually progress from liver cirrhosis. Considering the increased risk of death from liver cancer and the limitation of clinical treatment options, it is urgent to understand the cancer-promoting mechanism of liver cancer. In the past few decades, a large number of mouse models have been established to study the pathogenesis of liver cancer. Diethylnitrosamine (DEN) is a well-known liver chemical carcinogen. It can cause liver cancer similar to human liver cancer by producing reactive oxygen species and forming mutagenic DNA adducts. C57BL/6 mice were injected with a single dose of 25 mg/kg DEN intraperitoneally on the 14th day, and tumor nodules formed in the liver 9 months later. Liver cancer is usually associated with cirrhosis secondary to chronic liver disease. Domenicali et al. established a mouse model of advanced liver cirrhosis after continuously exposing mice to carbon tetrachloride (CCl4) for about 12 weeks. As a facilitating factor, taking carbon tetrachloride every week and drinking alcohol, liver cancer occurred after 104 weeks. The combined mouse model of single DEN and intraperitoneal injection of CCl4 showed that the incidence of liver tumors at 5 months of age was 100%. Some molecular pathways and cellular events have been shown to be related to the progression of liver cancer. SIRT6 is a member of the NAD+-dependent protein deacetylase in the sirtuin (SIRT) family. It has been identified as a key regulator of basic human processes (including lifespan, metabolism and inflammation). SIRT6 knockout mice worldwide Death within. Many molecular pathways in the aging process also help to suppress tumors, and more and more evidences show that SIRT6 has a tumor-inhibiting effect in human cancers. SIRT6 plays an important role in chronic liver disease, because the loss of liver-specific SIRT6 accelerates fatty liver and liver steatosis. However, the role and mechanism of SIRT6 in the occurrence of liver tumors induced by DEN and CCl4 are still unclear. In view of the key role of SIRT6 in liver function, we established a mouse model of liver cancer induced by DEN and CCl4 in liver-specific SIRT6-deficient mice. The effect of SIRT6 deficiency on liver injury and chemically induced liver cancer in mice was studied by activating the ERK1/2 pathway.

　　 SIRT6 gene knockout contributes to chemically induced liver cancer: SIRT6-deficient mice worldwide die on the 24th day after birth. Therefore, in order to understand the impact of SIRT6 deficiency on liver tumorigenesis, we used alb-cre transgenic mice to specifically knock out SIRT6 in the liver. The transgenic mice mainly mediate cre/loxp recombination in liver cells. Diethylnitrosamine (DEN) is a widely accepted genotoxic agent that induces liver cancer. After 45-104 weeks, the incidence of male and female is 100% and 30%, respectively. Continuous tetrachloride Carbon (CCl4) can promote the progress of liver fibrosis. In order to study whether SIRT6 knockout would affect the development of DEN and CCl4-induced liver cancer, 14-day-old female and male mice were injected with a single dose of 25 mg/kg DEN as an initiator, and repeated injections of CCl4 (dissolved in corn oil) Acts as an accelerator for up to 14 weeks. At the end of the 7-month-old age, tumor development was visible to the naked eye, and more HCC nodules were found in both female and male SIRT6 knockout mice, while only 1 of 8 mice in the WT female group had a tumor nodule . Chemically induced liver development did not affect their body weight. Quantitative analysis showed that compared with wild-type, SIRT6 knockout increased the liver weight ratio of female HKO mice by 16%, and the liver weight ratio of male HKO mice by 11%. "The liver-specific knockout of SIRT6 promotes liver cancer in mice induced by DEN and CCl4. (A) Experimental design of a mouse model of chemically induced liver cancer. The mice were given 25mg/kg of DEN on the 14th day after birth, and then CCl4 was injected twice a week starting from the age of 8 weeks for 14 consecutive weeks, and the mice were sacrificed at 28 weeks. (B) Representative western blot image showing knockout of SIRT6 in liver/liver tumors of HKO mice. (C) H&E and Masson trichrome stained liver sections of 7-month-old untreated normal WT and HKO mice. HKO mice showed more steatosis and fibrosis without induction. (D) Representative microscope image of H&E stained liver section. Histopathological examination showed that HKO mice were more prone to balloon, steatohepatitis and inflammatory infiltration. Scale bar: 500μm. (E) HKO mice show more severe liver fibrosis. (F-I) Mouse serum ALT, AST, TC and TG levels. (J) The gross morphology of the liver of the four groups of animals at the time of execution. Even though female mice are not prone to tumor formation, SIRT6 knockout showed more HCC nodules in both female and male groups. Blue arrows indicate tumor nodules in female mice. (K) Tumor nodules (J), total body weight (BW), liver weight (LW) and the ratio of LW to BW in the 4 groups of mice at the age of 7 months. In order to clarify the effect of SIRT6 loss on the recovery of liver injury induced by DEN and CCl4, we performed H&E and Masson trichrome staining on liver sections, and found that male and female HKO mice are more prone to ballooning, steatohepatitis and inflammatory infiltration than WT mice. , The incidence of liver injury in male mice is higher. Use the METAVIR Liver Fibrosis Score to measure the range of fibrosis/cirrhosis, from 0 (no fibrosis) to 4 (cirrhosis). Quantitative analysis showed that the fibrosis score was severe. After exposure to DEN and CCl4, serum liver injury markers ALT, AST, TC and TG in HKO mice increased significantly. In summary, it is the first study that SIRT6 deficiency leads to DEN and CCl4-induced liver cancer in mice. "SIRT6 is upregulated in tumor tissues and restricts HCC cell proliferation and tumor growth: It is reported that mouse liver-specific SIRT6 deletion develops 90% fatty liver in approximately 7.5-13 months. H&E and Masson's tricolor staining found that the 7-month-old HKO mice showed more severe steatosis than the normal control group without chemotherapy. In view of the discovery that SIRT6 is a tumor suppressor, and the lack of SIRT6 can lead to liver damage and cancer, we checked the expression of SIRT6 in tumor tissues and found that compared with non-tumor, even in the tumors of HKO mice, SIRT6 Expression is also up-regulated. Western blotting analysis showed that SIRT6 was also up-regulated in human HCC cell lines compared with normal human hepatocytes. SIRT6 may be a compensatory increase in tumors to inhibit the development of cancer. In order to evaluate the role of SIRT6 in cell proliferation in vitro, we successfully knocked out SIRT6 in the human HCC cell line HuH7 by shRNA, and overexpressed SIRT6 by pcDNA3.1-SIRT6 transfection. Cell clone formation experiments showed that overexpressed SIRT6 inhibited cell clone formation in HuH7 cells, while knocking out SIRT6 significantly improved the clonal formation ability. Taken together, these data indicate that SIRT6 is compensatory over-expression in tumors and plays a role in limiting cell clone formation in vitro. "SIRT6 is up-regulated in tumor tissues, promoting HCC cell proliferation and tumor growth inhibition. (A) Representative western blot image showing the expression of SIRT6 in human HCC cell lines and normal human liver cell lines. (B–C) RT-qPCR and western blotting were used to detect the knockout effect of shRNA on SIRT6 in HuH7 cells. (D-E) RT-qPCR and Western blot analysis confirmed the overexpression of SIRT6. (F) Overexpressed SIRT6 inhibits cell clone formation in HuH7 cells, while knocking out SIRT6 significantly promotes clone formation. (G) Quantitative analysis of cell clone formation. (H) In vivo anti-tumor efficacy of SIRT6 in xenograft tumor models, implanted with HuH7 cells expressing pcDNA3.1 or pcDNA3.1-SIRT6. (I) The tumor size is measured every four days until 34 days after injection. (J) Western blot analysis of SIRT6 in four representative xenograft tumors in each group. (K) Representative images of Ki-67 expression in xenograft tumor tissues analyzed by immunohistochemistry.

　　 After confirming the effect of SIRT6 deletion on the development of liver cancer in mice and human cell lines, the overexpression of SIRT6 also showed limited cell proliferation. To study whether the overexpression of SIRT6 affects the biological behavior of HCC tumors. Therefore, we used stably transfected HuH7-pcDNA3.1 or HuH7-pcDNA3-SIRT6 cells to establish a xenograft mouse model. Animal experiments show that SIRT6 has a certain inhibitory effect on the formation of xenografts and tumor growth. At the same time, compared with the pcDNA3.1-HuH7 negative control, in pcDNA3-SIRT6 xenografts, the level of SIRT6 protein remained significantly higher. We also stained xenograft tumor tissue with the proliferation marker Ki67 and found that in the SIRT6 high expression group, Ki-67 expression was down-regulated. Overall, these data indicate that SIRT6 plays a tumor suppressor effect in HCC tumor growth.

　　SIRT6 exerts a tumor suppressor effect by inhibiting the ERK1/2 pathway: To study how SIRT6 inhibits liver tumorigenesis in mouse and human HCC cell lines. Serine and threonine kinases ERK1/2 (extracellular signal-regulated kinases 1 and 2) are members of the mitogen-activated protein kinase (MAPK) family, whose dysregulation contributes to the development of many cancers, including HCC. According to reports, the MAPK signaling pathway is an important pathway related to SIRT6 and cancer. It was found that SIRT6 gene knockout activated the ERK1/2 pathway, while overexpression of SIRT6 or activation of the selective SIRT6 activator MDL-800 significantly inhibited the phosphorylation of ERK1/2 in HuH7 cells. The abnormal activation state of ERK1/2 in HKO mice and tumor tissues was also studied. It is speculated that SIRT6 may inhibit the occurrence of liver cancer by inactivating the ERK1/2 pathway. "SIRT6 deficiency induces activation of the ERK1/2 pathway. (A-D) Quantitative Western blot analysis showed the expression of p-ERK and ERK in the group. Group 1: HuH7 cells were stably expressed in the negative control group or SIRT6 shRNA. Group 2: 72h after transfection with pcDNA3.1 or pcDNA3.1-SIRT6, cells were taken for Western blotting. Group 3: After 48 hours of treatment with 25 μM MDL-800 or vehicle control, HuH7 cells were collected for Western blotting. (E-F) Western blot analysis was performed using liver extracts from normal WT and HKO mice and a chemically induced mouse model of hepatocellular carcinoma (isolated tumor nodules). "In summary", studies have shown that mouse liver-specific knockout of SIRT6 promotes the occurrence of liver tumors and anti-tumor effects by inhibiting the ERK1/2 signaling pathway. The combination of activating SIRT6 and inhibiting ERK1/2 activity can significantly improve the treatment of liver injury and liver cancer.