| The China National Cancer Registry published an article in the journal Cancer J Clin in 2016.After they analyzed the incidence and death of cancer in China from 2009 to 2011,they estimated that the incidence of liver cancer in China in2015 was ranked fourth in malignant tumors,and malignant tumor-related mortality ranked third.Although with the advancement of medical research and the advancement of medical equipment and technology,the medical level has made great progress,but there is no effective clinical treatment for liver cancer so far.A large number of malignant ascites(MA)occur in advanced liver cancer patients,and a large amount of MA severely affects the quality of life of patients.There is currently no effective treatment for MA.Angiogenesis and increased vascular permeability are considered to be the main mechanisms of MA formation.The drugs used to treat MA are mainly to reduce the formation of MA by inhibiting the expression of factors such as vascular endothelial growth factor(VEGF)and matrix metalloproteinase-2(MMP-2),but the clinicalefficacy of these drugs is limited.Need to find other more effective drugs for treatment.At present,studies have found that immune cells and immune factors are involved in all aspects of the pathogenesis of liver cancer,and have a significant impact on the growth and prognosis of cancer.In addition,multiple signaling pathways are known to play an important role in the development of liver cancer.STAT3 is an important member of signal transduction and activators of transcription(STATs).Current research indicates that STAT3 signaling pathway is abnormally expressed in various tumor tissues and cell lines such as lung cancer,breast cancer,and liver cancer.Activating of STAT3 was involved in tumor proliferation,differentiation,apoptosis and other processes.Interleukin(IL)-9 is an important immune factor in the body and can be secreted by various immune cells such as Th2,Th9,and Th17,and exerts a series of biological effects through binding to IL-9R.IL-9 has a variety of immune effects,the current research shows that it exists in the presence of pro-inflammatory effects,and aslo anti-inflammatory effects.However,there is less research on IL-9 in tumors,and the relationship between IL-9 and tumors is not yet clear.Studies have found that the use of IL-9 to interfere with melanoma cells HTB-72 can inhibit the growth of tumor cells,and in the study of lung cancer found that IL-9 can promote tumor cell proliferation.In our previous study,the proportion of Th9 cells and IL-9 levels in MA of MA patients with liver cancer were higher than their peripheral blood levels and ascites levels incirrhosis,and the proportion of Th9 cells and IL-9 levels were negative with the survival time of patients.So what is the effect of Th9/IL-9 on the biological behavior of liver cancer cells? What mechanism does Th9/IL-9 affect the liver cancer cells? Is Th9/IL-9 a target for liver cancer MA intervention? In order to explore the above issues,we will study the following two aspects:Part one: IL-9 Promotes Proliferation and Metastasis of Hepatocellular Cancer Cells by Activating JAK2/STAT3 PathwayOBJECTIVE:To study the changes in the biological behaviors of hepatocellular carcinoma cells such as proliferation,migration,and invasion after interleukin(IL)-9 intervention,and to explore the possible mechanisms of the effects of IL-9 on the biological behavior of hepatoma carcinoma cell.METHODS:SMMC-7721 cells were cultured in vitro to the logarithmic growth phase.SMMC-7721 cells were divided into experimental groups and control groups.The experimental groups were treated with 12.5 ng/m L,25 ng/m L,and 50ng/m L rh IL-9.The control group was not gave any treatment.24 h after intervention,CCK8 assay was used to detect the proliferation of the tumor cells.Flow cytometry was used to detect the apoptosis rate the tumor cells.Transwell migration and invasion assay was used to detect the cell migration and invasion ability.The culture medium supernatant was used to detect the levels of MMP-2,MMP-9 and VEGF by using enzyme linked immunosorbent assay.After extraction of cellular RNA,MMP-2,MMP-9 and VEGF m RNA levels were detected by RT-PCR.To investigate the underlying mechanism of IL-9 affecting SMMC-7721 cells,50 ng/m L IL-9,50 ng/m L IL-9+80?mol/L AG490 were administered to the experimental groups,no treatment were administered to the control group.24 h after intervention,CCK8 assay was used to detect the proliferation of tumor cells.Transwell migration and invasion assay was used to detect the cell migration and invasion ability.The expression of STAT3 and p-STAT3 in cells was detected by Western-blot.The relative expreesion levels of MMP-2,MMP-9 and VEGF m RNA levels were detected by RT-PCR.RESULTS:After intervention of SMMC-7721 cells with 0 ng/m L,12.5 ng/m L,25ng/m L,and 50 ng/m L IL-9 for 24 h,the proliferation rate of SMMC-7721 cells increased significantly(p <0.05).At the same time,the migration and invasion ability of SMMC-7721 cells were also increased(p <0.05),and the promoting effect of IL-9 on the proliferation and metastasis of SMMC-7721 cells was enhanced with the increase of IL-9 concentration.However,there was no significant change in the cell apoptosis rate after IL-9 intervention(p >0.05).The ELISA assay of the culture supernatant revealed that compared with the control group,the levels of MMP-2,MMP-9 and VEGF in the supernatant of the experimental group after IL-9 intervention were significantly higher(p <0.05).The results of RT-PCR also showed that compared with the control group,the expression of MMP-2,MMP-9 and VEGF m RNA in the cells of the experimental group after IL-9 treatment was increased(p <0.05).Compared with the control group,the level of JAK2 and STAT3 protein in SMMC-7721 cells was not significantly changed after IL-9 intervention(p >0.05),but the expression level of p-JAK2 and p-STAT3 was significantly higher(p <0.05).After treatment with AG490,the promotion of proliferation and metastasis of SMMC-7721 by IL-9 was weakened(p <0.05),and the expression levels of p-STAT3,MMP-2,MMP-9 and VEGF m RNA were also significantly reduced(p <0.05).CONCLUSION:IL-9 promoted the proliferation,migration and invasion of hepatoma cells in a concentration-dependent manner,but had no significant effect on apoptosis.The promotion of IL-9 on hepatoma cells is achieved by activating STAT3 phosphorylation and promoting the expression of MMP-2,MMP-9 and VEGF.Part two: Effects and mechanism of anti IL-9 antibody on malignant ascites of hepatic carcinomaOBJECTIVE::The concentrations of IL-9 in peripheral blood and malignant ascites(MA)of hepatocellular carcinoma with malignant ascites were measured.The ratio of Th9 cells in spleen of hepatocellular carcinoma with MA was aslo measured.The present study was designed to investigate the effect of inhibition of IL-9(by intraperitoneal injection anti-IL-9 antibody)in the mice with malignant ascites.And to explore the underlying mechanism of anti-IL-9 antibody inhibit malignant ascites(MA).METHODS:One mouse with H22 hepatocellular carcinoma ascites was passaged on the7 th day,and the mice's peritoneal milk white tumor fluid was taken.After that,the concentration of the cell suspension was adjusted to 1×107/m L with PBS.Then,a mouse model of hepatoma H22 ascites was prepared by intraperitoneally injecting 0.2 ml of tumor cell suspension(corresponding to 2×10*6 tumor cells per mouse)into each mouse.After 24 hours of modeling,60 mice were randomly divided into experimental group,negative control group and blank control group,with 20 mice in each group.The experimental group was given intraperitoneal injection of anti-IL-9 antibody,the negative control group was given the same type of Ig G antibody injection,and the blank control group was given normal saline injection every other day for a total of 5 injections.Each group of mice was weighed before each injection and the daily activity of the mice was observed.At the end of 24 h after the last administration,5 mice were sacrificed in each group.The volume of MA was measured.MA supernatants were collected and the expression levels of VEGF,IL-9 and IFN-? in MA were detected by ELISA.The mouse spleen was taken and the lymphocytes in the spleen were isolated and pretreated with a stimulatory blocking agent for 4 hours.The surface was then stained for CD4 antigen,and then the membrane was broken for intracellular IL-9 and IL-? staining.After staining,the flow cytometry was used to measure the proportion of Th9 cells and Th1 cells in the spleen.Five mice were taken from each group 24 hours after the end of the last drug treatment.Each mouse was administered intravenously with 0.2 ml Evans blue at a concentration of 50 mg/ml.2 hours later,ascites fluid was collected and the supernatant was removed after centrifugation.Then Spectrophotometry was used to measure the concentration of Evans blue in the supernatant.The remaining mice were observed for their survival time.RESULTS:The volume of MA in the experimental group,negative control group and blank control group after intraperitoneal injection of anti-IL-9 antibody was(6.83±1.46),(10.50±1.58),(10.42±2.24)m L,respectively.Compared with the control group,the MA volume in the experimental group was significantly reduced,and the difference was statistically significant(p<0.05).The median survival time of mice in the experimental group was 18 days,which was significantly longer than the negative control group and the blank control group(p<0.05).Compared with the negative control group and the blank control group,the levels of VEGF and IL-9 in MA of the experimental group were significantly decreased(p<0.05),but the levels of IFN-? in the three groups had not statistically significant(p>0.05).The mean absorbance values ??of the three groups of ascites fluid were 1.82±0.14,1.78±0.08 and 1.25±0.09.Compared with the negative control group and the blank control group,the concentration of Evans blue in the ascitic fluid of the experimental group decreased significantly.Flow cytometry test results show that compared with negative control group and blank control group,the proportion of Th9 cells and Th1 cells in the experimental group decreased,the difference was statistically significant(p<0.05).CONCLUSION:The level of IL-9 in malignant ascites in mouse with H22 hepatocellular carcinoma ascites increased.And intraperitoneal injection of anti-IL-9 antibody can effectively inhibit the production of ascites in mice with H22 hepatocellular carcinoma ascites and prolong the survival time of mice.It is achieved by inhibiting the expression of VEGF in ascites and decreasing the peritoneal permeability of mice. |
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