| Background and aims:Primary liver cancer(PLC)is one of the most common malignant tumors in the digestive system.Epidemiological studies show that PLC is the sixth largest malignant tumor in the world,next to lung cancer.The clinical prognosis of liver cancer is very poor.Tumor recurrence and metastasis are the key factors that restrict the clinical treatment effect.Existing studies have shown that tumor metastasis and recurrence are related to the invasion and metastasis ability of cancer cells,the influence of microenvironment on cancer cells,and the escape of cancer cells from immune cell killing.Among them,how cancer cells escape from immune cell surveillance in the body is an important prerequisite for tumor cell metastasis and survival,which has been the focus of scientific circles.Therefore,it is of great value to study the molecular mechanism of immune evasion in the treatment of HCC.Hypoxia inducible protein 2(hig2)gene is a new lipid drop protein and autocrine growth factor discovered in recent years.Hig2 is widely distributed in many human tissues and cells,and its abnormal expression is related to the state of disease.It was found that hig2 was involved in the regulation of tumorigenesis and development:hig2 could promote the proliferation and metastasis of colon cancer cells;in uterine and renal cell carcinoma,the expression of hig2 was up-regulated abnormally,which was positively correlated with the clinicopathological characteristics of tumor.It has been reported that cytotoxic T cells can recognize the peptide epitopes of hig2 protein,suggesting that hig2 is related to immune response.In the process of the occurrence and development of primary liver cancer,the mechanism of hig2 has not been reported in literature.TCGA database shows that hig2 gene is highly expressed in HCC and negatively correlated with prognosis.If we can clarify the expression,biological function and mechanism of hig2 in primary liver cancer,and clarify its role in immune escape of liver cancer,it has important clinical value for the diagnosis and treatment of liver cancer.In this study,hig2 genes related to the prognosis of HCC were screened through TCGA database.It was confirmed by experiments in vitro that hig2 played the role of oncogenes in the development of HCC,and promoted HCC cells to escape natural killer cell(NK)killing.Natural killer cells are the main effector cells of innate immunity,which play an important role in the inhibition of tumor development.We found that hig2 upregulated the expression of IL-10 in hepatoma cells.This gene was reported to activate STAT3 signaling pathway and play the role of immunosuppression.It was defined as an immunosuppressive factor by scholars,involved in inflammation,autoimmune diseases,and involved in the regulation of tumor biological behavior.Therefore,we propose a hypothesis:in HCC,hig2 promotes the synthesis and secretion of IL10;IL10 promotes the proliferation and metastasis of tumor cells by activating STAT3 signaling pathway;on the other hand,it promotes the immune escape of tumor cells by inhibiting the tumor killing activity of NK cells.This study will further elucidate the regulatory mechanism of hig2 on HCC and provide experimental basis for further understanding of the recurrence and metastasis of HCC.In order to confirm the above hypothesis,firstly,we collected the expression of hig2 in 40 samples of human hepatocarcinoma,analyzed the relationship between the expression of hig2 and the clinicopathological characteristics of patients;then we interfered with and overexpressed the hig2 gene in hepatocarcinoma cell lines,and studied the biological function of hig2 in human hepatocarcinoma cells;we isolated and cultured healthy human peripheral blood NK cells,and studied the hig2 by co culture system The ability of knockout or overexpression hepatocarcinoma cells to escape NK cell killing;the molecular mechanism of hig2 regulating the malignant biological behavior of colorectal cancer cells;finally,the biological function of hig2 was verified in vivo by subcutaneous transplantation tumor model in nude mice.Method:1.The expression level of hig2 gene in liver cancer was correlated with prognosis.Real time PCR(real-time PCR)was used to detect the expression of hig2 in liver cancer tissues and corresponding adjacent tissues,liver cancer cell lines and normal liver cells L02,and to analyze the relationship between the expression and the clinicopathological characteristics of patients.2.Using siRNA interference technology and overexpression of hig2 plasmid to down regulate or up regulate the hig2 gene of hepatoma cell line respectively,CCK-8 cell activity test to detect the change of proliferation ability of tumor cells;Transwell method to analyze the change of metastasis and invasiveness of tumor cells;flow cytometry to detect the change of cell cycle and apoptosis;using hig2 interference hepatoma cell line SMMC-7721 to establish subcutaneous transfer of nude mice The tumor volume and weight of nude mice were recorded.Western blot was used to detect the expression of EMT related proteins.3.NK cells were isolated from peripheral blood of healthy people by magnetic bead sorting.After co culture with hig2 interfering or over expressing hepatoma cells in vitro,apoptosis of tumor cells,phosphorylation of STAT family proteins and expression of surface receptors of NK cells were analyzed by flow cytometry.4.The expression of IL-10 was detected by IHC,the expression of IL-10 was detected by quantitative PCR and ELISA.IL-10 antibody or IL-10 recombinant protein was added to the co culture system of NK cells and hepatoma cells.The apoptosis of tumor cells,the phosphorylation of STAT3 and the expression of receptor on the surface of NK cells were analyzed by flow cytometry.5.Through bioinformatics analysis,the potential transcription factor binding region in IL10 promoter region was screened,and Western blot was used to verify the changes of related transcription factors in hig2 hepatoma cells.After screening the transcription factors with significant differences,the molecular mechanism of IL10 regulated by hig2 was studied by molecular biology technology to recover the expression of transcription factors,and the effects of the transcription factors on hig2 and IL10 were also studied Induced phenotypic effects.Result:1.TCGA database showed that the expression of hig2 in HCC was higher than that in adjacent tissues,but there was no significant difference.Survival analysis showed that the survival time and disease-free survival time of the patients with high expression of hig2 were significantly lower than those of the patients with high expression of dig2.The results of real-time quantitative PCR and IHC showed that the relative expression of hig2 in HCC was significantly higher than that in the adjacent tissues,and was related to the clinicopathological features such as lymph node invasion and stage of HCC;compared with L02,the expression of hig2 in HCC was up-regulated.2.Through siRNA technology to interfere with the expression of hig2 in hepatoma cell lines,the proliferation activity of tumor cells is inhibited,the ability of metastasis and invasion is weakened,G1/S phase is blocked;overexpression of hig2 in hepatoma cells,the proliferation activity of tumor cells is increased,the ability of migration and invasion is enhanced,and G1/S phase transformation is accelerated.3.NK cells from healthy human peripheral blood were successfully isolated by magnetic bead separation technology and cultured in 1640 RPMI cell medium containing IL2.The results of flow cytometry showed that NK cells significantly enhanced the killing effect on hepatoma cells in the hig2 interference group,while NK cells significantly decreased the killing effect on hepatoma cells after overexpression of hig2.At the same time,flow cytometry showed that the supernatant of tumor cells in hig2 interference group could promote the expression of NKG2D,NKp30 and CD 16,and up regulate the expression of granb,perforin,TNF-α and IFN-γ.Further study showed that tumor cells in hig2 interference group significantly decreased the phosphorylation level of STAT3 in NK cells.However,the supernatant of hepatoma cells overexpressing hig2 has the opposite biological function.4.Immunohistochemistry was used to analyze the expression of IL-10 in liver cancer tissues.It was found that the expression of IL-10 in cancer tissues was significantly higher than that in adjacent tissues.At the cell level,the expression of IL10 mRNA in HCC cells decreased significantly after hig2 knockout,but increased after hig2 overexpression.When NK cells were co cultured with the supernatant of tumor cells of hig2 interference group,the killing effect of NK cells on tumor cells was significantly reduced.When NK cells were co cultured with the supernatant of hig2 over expressed tumor cells containing IL10 antibody,the killing effect of NK cells on tumor cells was enhanced.In addition,when IL10 cytokines were added to NK cell culture system,NKp30 and NKG2D on the surface of NK cells were significantly down regulated,and CD 16,granb and TNF-α had no significant changes.5.Combined with bioinformatics and Western blot,we found that CREB,NF-κB,STAT3,STAT1,STAT4,STAT5,STAT6 and p53 can bind to the promoter region of IL10.In hepatoma cells,hig2 can significantly promote the expression of CREB.After overexpression of CREB in the cells of hig2 interference group,its culture supernatant can significantly inhibit the killing of NK cells on hepatoma cells;real-time quantitative PCR detection showed that after the expression of CREB was restored,the expression of IL10 in the cells of hig2 interference group was significantly increased.Western blot was used to further detect other key proteins in CREB pathway,and it was found that hig2 significantly inhibited thr172 phosphorylation of AMPK λ.After adding metformin,an AMPK pathway inhibitor,the expression of IL10 in hepatoma cells of hig2 interference group was significantly restored.Through the double Luciferase Report experiment,we found that after CO transferring CREB and double Luciferase Report plasmids,the fluorescence value increased significantly,which confirmed that CREB can bind to IL10 promoter region.Conclusion:In this project,we combined bioinformatics,molecular biology and cell biology to study the role and mechanism of hig2 in liver cancer at the level of tissue,cell and animal.We confirmed that hig2 plays a role of promoting oncogene in the development of liver cancer Through the activation of AMPK signaling pathway,the expression of CREB is up-regulated,so as to promote the transcription of IL10.The synthesis and secretion of IL10 can down regulate the expression of NKp30 and NKG2D under the activation of NK cells through STAT3 signaling pathway,so as to inhibit the activity of NK cells and lead to the immune escape of liver cancer cells. |
PDF Full Text Request