| Part One:miR-145 Regulates the Differentiation and Mechanism of Spleen Th9 Cells in Hepatocellular Carcinoma Ascites Model MicePurpose:To study the regulatory effect of miR-145 agonist on the differentiation of Th9 cells in spleen of hepatoma ascites model mice and its possible mechanism.Methods:CD4~+T lymphocytes were isolated from the spleen of normal mice by immunomagnetic beads.CD4~+T cells were induced to differentiate Th9cells after transfection with miR-145 mimics or negative control and were induced under TGF-βand IL-4 conditions for 72 hours.The H22 cells were resuscitated and resuspended to 1×10~7/mL with phosphate buffer solution.Ten mice were randomly divided into the miR-145agomir group and negative control group.Each mouse was intraperitoneally injected with 0.5 mL liver cancer H22cells.After 24 hours,mice were intravenously injected with mmu-miR-145-5pagomir or negative control oligonucleotides,3mg/kg,once every 3 days for a total of five times.On day 15,draw ascites from hepatoma for smear and HE staining and the mice were sacrificed and the spleen tissue was isolated.RT-PCR was used to detect the expression of miR-145 and phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin/p70ribosomal protein S6 kinase/HIF-1α(PI3K/Akt/mTOR/p70S6K/HIF-1α)mRNA.Th9 cells were detected by flow cytometry.Enzyme-linked immunosorbent assay was applied to detect the interleukin 9(IL-9)cytokine and hypoxia inducible factor 1 alpha(HIF-1α).Western blotting and immunofluorescence wereperformedtodetecttheexpressionof PI3K/Akt/mTOR/p70S6K/HIF-1α-related proteins.Results:Compared with the negative control group,the expression level of miR-145 in CD4~+T lymphocytes after miR-145mimics intervention was significantly higher(P<0.05),the proportion of Th9 cells,the expression level of IL-9mRNA and IL-9 cytokines were lower(P<0.05),the expression level of HIF-1αmRNA and HIF-1αprotein were down regulated(P<0.05),and the mRNA and protein levels of PI3K,Akt,p70S6K were lower(P<0.05).Compared with the ascites model mice of negative control group,the expression level of miR-145 in the spleen of miR-145agomir group was significantly higher(P<0.05),the proportion of Th9 cells,the expression level of IL-9mRNA and IL-9 cytokines were decreased(P<0.05),the expression of HIF-1αmRNA and HIF-1αprotein was down regulated(P<0.05),and the phosphorylation level of PI3K,Akt,mTOR and p70S6K protein decreased(P<0.05).Conclusion:miR-145 may inhibit Th9 cell differentiation through the PI3K/Akt/mTOR/p70S6K/HIF-1αpathway.Part Two:Study on the Inhibitory Effect of miR-145 on Liver Cancer Ascites ModelPurpose: Measure the expression of IL-9R,JAK,STAT3 and VEGFA in the spleen of mice with hepatoma ascites model,detect peritoneal vascular permeability,observe the volume of ascites and the survival time of mice,and explore the inhibitory effect and possible mechanism of miR-145 agonist on the hepatoma ascites model.Methods: Using liver cancer H22 cells to construct liver cancer ascites model.After 24 hours,mice were intravenously injected with mmu-miR-145-5pagomir or negative control oligonucleotides,3mg/kg,once every 3 days for a total of five times and their daily life was observed.On day15,each group of 5 mice was sacrificed by cervical dislocation,the volume of ascites was measured and the spleen tissue was isolated.Afterwards,5 mice in each group were randomly selected and injected 0.2mL 5% Evan blue solution via tail vein and survival time of remaining mice was observed.miR-145,IL-9R mRNA,JAK1/2/3 mRNA,STAT3 mRNA,VEGFA mRNA in the spleen were detected by real-time reverse transcription PCR.The protein levels of IL-9R,JAK,STAT3,p-STAT3,and VEGFA were assessed by western blot.The absorbance value of Evan blue in the supernatant of ascites of each group was detected by enzyme labeling instrument.Results: Compared with the negative control group,the expression of IL-9R mRNA and IL-9R protein was decreased significantly(P<0.05),the expression of JAK1 mRNA,JAK3 mRNA,STAT3 mRNA and VEGFA mRNA was reduced(P <0.05),and the expression of JAK,STAT3,p-STAT3 and VEGFA protein was downregulated(P<0.05).The absorbance value of ascites supernatant in miR-145 agomir group was 2.38 ± 0.01,which was significantly lower than that in control group(2.51 ± 0.02)(P<0.05).The volume of ascites in miR-145 agomir group and negative control group was 11.16 ± 0.60 mL and13.92 ± 0.70 mL,respectively(P<0.05).The survival time of miR-145 agomir group was 19.20 ± 0.57 days,which was significantly longer than that of the control group 17.20 ± 0.57 days(P<0.05).Conclusion: miR-145 can inhibit the expression of IL-9R in the spleen of mice with hepatoma ascites model,inactivate the JAK/STAT3 pathway downstream,decrease the expression of VEGFA,affect angiogenesis and vascular permeability,thus inhibiting the formation of malignant ascites and prolonging the median survival time. |
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