| Objective:Hepatocellular carcinoma(HCC)is one of the most common malignant neoplasms and the third leading cause of cancer mortality worldwide.HCC usually results from chronic liver disease,such as virus(HBV,HCV)infection and chronic liver cirrhosis.The prognosis of HCC patients is poor due to the absence of early diagnosis and treatment.MicroRNAs(miRNAs)are a class of small noncoding RNAs with 18-25 nucleotides and highly conserved,whose function are as regulators of gene expression through specific binding to the miRNA recognition elements(MREs)on the 3' untranslated regions(UTRs)of target mRNAs.More than 60%of human protein encoding genes are under selective pressure to maintain pairing with miRNAs,implying that miRNAs play an important role in the regulation of gene expression at the post-transcriptional level.An increasing number of evidence has indicated that miR-15a and miR-16-1 are highly conserved RNAs that form a cluster at the chromosomal region 13q14.3,which play suppressive.roles in tumor progression of malignancies.The previous experiment on a microRNA chip assay demonstrated that miR-15a/16-1 was down-regulated significantly in hepatocellular carcinoma tissue compared with adjacent tissues,and results showed the frequency of peripheral regulatory B cells of patients with liver cancer is higher than that of the healthy group.Further animal experiments confirmed that the frequency of CD19~+Tim-1+ cells in the spleen from miR-15a/16-1-/-mice is higher than that from the wild type group,and the cells secreted IL-10,however TGF-P has no obvious change.There are following problems remained to be clarified:(1)What are the roles of increased CD19~+Tim-1+cells?(2)CD19~+Tim-1+ cells affect the growth of liver cancer?(3)How CD19~+Tim-1+ cells exert effects.Therefore,miR-15a/16-1-/-mice were introduced for further roles study of CD19~+Tim-1+ B cells and the effects on the growth of liver cancer.We aimed to reveal the mechanism of miR-15a/16 in the liver cancer,elucidate the pathogenesis of liver cancer and provide experimental basis for clinical treatment.Methods:1.To detect the frequency and function of regulatory B cells in the spleen of miR-15a/16-1-/-mice1.1 To detect the frequency changes of B cells in the spleen of miR-15a/16-1-/-miceTesting the frequency of B220+CD19~+ cells in the spleen of KO mice at the age of 8-12 weeks and 15-18 months by FCM.Using ELISA to detect the concentration of IL-10 in the supernatant of CD19~+ cells stimulated by LPS for different hours.Detecting the expression of IL-10 in the CD19~+ cells of KO mice at age of 15-18 months.1.2 To analyze regulatory B cell subsets in the different age of KO miceUsing the FCM to detect the regulatory B cell subsets at different age of KO mice,including CD19~+ Tim-1+,CD19~+ Fc?R?bhi and CD19~+ CD5+ CD1dhi,and detecting IL-10 from splenic CD19~+Tim-1+ cells of miR-15a/16-1-/-mice.1.3 To analyze Breg cell subsets in the young(8-12 weeks)KO mice transplanted with with H22 cellsThe young miR-15a/16-/-mice(8-12 week)were transplanted with liver cancer cells(H22 cells),analyzing Breg cell subsets in the young KO mice and detecting IL-10 secretion in splenic CD 19~+Tim-1+ cells of KO mice 3 weeks later.1.4 To analyze the roles of CD19~+ Tim-1+ cellsThe CD19~+Tim-1+ cells were isolated from the spleen of 15-18 month old KO mice.The CD4+ T cells were selected from the spleen of wild type mice.CD69 expression and IFN-y production were tested when CD 19~+ Tim-1+ cells were incubated with stimulated CD4+ cells for 3 days.Then,IL-10 neutralizing antibodies were added into the B-T cell coculture system,detecting CD69 expression and IFN-y production again.2.The effects of CD19~+ Tim-1+B cells on the growth of liver cancer in mice2.1 To detect the frequency of regulatory B cells in peripheral blood of patients with hepatocellular carcinomaGenerally,the main regulatory B cells in human are CD 19~+C24hiCD38hi cells.The peripheral blood of patients with hepatocellular carcinoma before treatment was collected.Pathological reports confirmed the diagnosis of liver cancer.Then CD19~+C24hiiCD38hi cells was detected by flow cytometry.2.2 To observe the effects of CD19~+ Tim-1+ cells on tumor growth in tumor-bearing miceThe KO mice at the age of 8-12 weeks were subcutaneously transplanted with H22 cells.One weeks later,CD19~+Tim-1+cells selected from aged KO mice were injected into experimental group through caudal vein,the other mice were injected with CD19~+ Tim-1-cells,once injection every 3 days,totally for 3 times.Splenic Breg cells of KO or WT mice were analyzed.Tumor growth was observed and the tumor growth curve was recorded.3.To study the mechanism of the effects of CD19~+ Tim-1+B cells3.1 To detect STAT3 signal pathwaySTAT3 activation is involved in IL-10 production of immune cells.CD19~+ Tim-1+ cells were isolated from spleen of wild type and aged KO mice.Then Western Blot method was used to detect the expression of STAT3,pSTAT3Tyr705and pSTAT3ser727.3.2 To detect the secretion of IL-10 in CD19~+ Tim-1+ cells with StatticCD19~+ Tim-1+ cells isolated from spleen of aged KO mice was treated with the STAT3 inhibitor(Stattic)for 8 hours,then IL-10 production was tested by flow cytometry.3.3 To detect the expression of STAT3 after MiR-16 over-expression in CD19~+ Tim-1+ cellsWe determined whether miR-16 directly downregulated the STAT3 mRNA level,using bioinformatics analysis to find the relationship between miR-16 and STAT3.CD19~+ Tim-1+ cells isolated from spleen of aged KO mice respectively were transfected with miR-16-containing lentivirus,then Western Blot was used to detect the expression of STAT3 again.Results1.Regulatory B cells in spleen of 15-18 month old miR-15a/16-1-/-mice increased and can played a negative regulatory role1.1 The frequency of B cells increased in spleen of 15-18 month old miR-15a/16-1-/-miceFlow cytometry showed that the frequency of B220+CD19~+ cell subsets in spleen of 15-18 month old KO mice was higher than that in WT control group.There was no significant difference in B220+CD19~+ cell frequency between 8-12 week old mice.The concentration of IL-10 in supernatant of stimulated CD19~+ cells was detected by ELISA,the results showed that there was a significant difference in CD19~+ cells of 15-18 month old mice after being stimulated for 48h by LPS.The expression of IL-10 in spleen CD19~+ cells of 15-18 month old KO mice was detected by flow cytometry and the results showed that the IL-10 secretion of KO mice was significantly higher than that of WT mice.These results indicated that IL-10-producing Bregs increased in the spleen of aged KO mice,but the changes in the young miR-15a/16-1-/-mice(8-12 weeks)were not obvious.1.2 The frequency of CD19~+Tim-1+ cells increased in spleen of 15-18 month old miR-15a/16-1-/? miceTo further analyze the specific subsets of regulatory B cells in spleen of 15-18 month old KO mice.The results showed that the frequency of CD19~+Tim-1+ cells in spleen of 15-18 month old KO mice was significantly higher than that in WT group,however,the frequency of CD19~+Fc?R?bhi and CD19~+CD5+CD1dhi cell subsets was not significantly different,and CD19~+Tim-1+ cells also secreted IL-10.These results indicated that CD19~+Tim-1+ cells increased in the aged KO cells in vivo.1.3 The frequency of CD19~+Tim-1+ cells increased in spleen of young miR-15a/16-1-/-mice(8-12 weeks)bearing tumorSubcutaneous implantation of H22 cancer cells in 8-12 weeks old KO mice.The frequency of CD19~+Tim-1+ cells in spleen of 8-12 weeks old KO mice with tumor bearing was significantly higher than that in WT group.Therefore,CD19~+ Tim-1+ cells could be induced in young KO mice transplanted with tumor cells.1.4 CD19~+Tim-1+ cells inhibited the function of T cellsCD19~+ Tim-1+ cells were co-cultured with CD4+ T cells for three days,the consequence showed CD69 expression was significantly downregulated.Moreover,IFN-y production by activated CD4+ cells were inhibited.However,IL-10 neutralizing antibodies were added into the B-T cell co-culture system,the inhibitory effect of CD69 expression was almost reversed,which indicated that Breg cells mediated the regulatory effect via IL-10 secretion.2.CD19~+ Tim-1+ cells promoted the growth of liver tumor2.1 The frequency of regulatory B cells increased in peripheral blood of HCC patientsCompared with healthy group,the frequency of CD19~+C24hiCD38hi cells increased in peripheral blood of HGC patients,which indicated regulatory B cells may play negative immunological roles on the development of HCC.2.2 CD19~+ Tim-1+ cells promoted tumor growthCD19~+ Tim-1+cells were injected into the mice bearing tumor,3 weeks later,the tumor' size of the mice injected with CD19~+ Tim-1+cells were bigger than the control group,the results showed that transfer of CD19~+ Tim-1+ cells significantly promoted tumor growth in vivo.3.MiR-15a/16-1 played negative roles on tumor via STAT33.1 STAT3 increased in CD19~+ Tim-1+ cells of aged KO miceWestern Blot was used to detect the expression of STAT3,STAT3-pY705,and STAT3-pS727,the result revealed all of them were increased in CD19~+ Tim-1+ cells of tumor-bearing KO mice.3.2 CD19~+ Tim-1+ cells secreted IL-10 via STAT3When the STAT3 inhibitor(Stattic)was treated with KO mice-derived CD19~+ Tim-1+ cells,IL-10 production was blocked.The result indicated that STAT3 activation contributed to IL-10 production by CD 19~+ Tim-1+ cells.3.3 MiR-16 over-expression in CD19~+Tim-1+ cells down-regulated STAT3By using bioinformatics analysis,miR-16 was found to directly bind with the 3'-untranslated region(UTR)of STAT3,CD19~+ Tim-1+ cells derived from KO mice were transfected with miR-16-containing lentivirus,then Western blot confirmed that STAT3 was downregulated by transfection of miR-16-containing lentivirus.Therefore,overexpression of miR-16 in KO mice-derived CD 19~+ Tim-1+ cells suppressed STAT3 expression.Conclusion:1.The regulatory B cells in peripheral blood of HCC patients increased;2.CD19~+ Tim-1+ cells promote the growth of liver tumor;3.MiR-16 inhibit CD19~+ Tim-1+ cells secreting IL-10 through STAT3,which exerts negative effects on tumor. |
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