| Background:Hepatocellular carcinoma(HCC)is a highly invasive cancer with high morbidity and mortality.Our previous research found that cyclin B2(CCNB2)is closely related to the occurrence and development of HCC.According to bioinformatics analysis,mi R-670-3p may specifically bind to the3?untranslated region(3?UTR)of CCNB2.More and more evidence shows that mesenchymal stem cell(MSc)-derived exosomes(MSc-exos)have great potential in cancer treatment by transporting mi RNAs.However,the effect of exosomal mi R-670-3p on hepatocellular carcinoma is unclear.Objective:This study intends to mediate the transport of mi R-670-3p through adipose-derived mesenchymal stem cell exosomes(AMSC-exos),and to explore the effect and potential mechanism of exosomal mi R-670-3p on the biological function of hepatocellular carcinoma cells.Methods:1.AMSCs were isolated and cultured from human adipose tissue.The expression of surface markers CD90,CD44,CD34 and CD45 were detected by immunofluorescence double labeling experiment to identify AMSCs.2.AMSCs were transfected with mi R-670-3p mimic and mimic NC respectively,and the transfection efficiency was detected by real-time quantitative PCR(q PCR).Exosomes were extracted from AMSC supernatant by ultracentrifugation,and AMSC-exos were identified by transmission electron microscopy,nanoflow technology and Western blot.The expression level of mi R-670-3p in AMSC-exos was detected by q PCR experiment.3.AMSC-exos(mi R-670-exo and NC-exo)were co-cultured with Huh-7cells,and the expression level of mi R-670-3p in Huh-7 cells was detected by q PCR.The uptake of PKH67-labeled exosomes by Huh-7 cells was observed by confocal laser scanning microscope.4.The effects of exosomal mi R-670-3p on the proliferation,invasion,cell cycle and mitotic catastrophe of hepatoma cells were investigated by CCK-8 assay,Transwell assay,flow cytometry and immunofluorescence assay,respectively.5.The target genes of mi R-670-3p were predicted by the bioinformatics analysis Target Scan,and the regulation effect of mi R-670-3p on CCNB2 was verified by dual luciferase assay and Western blot.6.Functional recovery experiment to study whether CCNB2 mediates the inhibitory effect of mi R-670-3p on the proliferation and invasion of hepatoma cells.Results:1.Immunofluorescence double-labeling experiments of cells isolated and cultured from adipose tissue showed that CD44 and CD90 were positively expressed,while CD34 and CD45 were negatively expressed,which was in line with the characteristics of AMSCs.2.The extracted exosomes showed a typical " cup and plate shape" under transmission electron microscope.The particle size distribution of exosomes analyzed by nanoflow cytometry was 30-150 nm,with an average particle size of 75.74 nm.Western blot showed that the expression of CD63,CD81 and TSG101 was positive,while the expression of calnexin was negative.It shows that AMSC-exos extraction is successful.The q PCR results showed that the expression level of mi R-670-3p in both AMSCs and AMSC-exos transfected with mi R-670-3p mimic group was significantly increased compared with the control group(p < 0.001).3.The q PCR results showed that the expression level of mi R-670-3p was significantly increased in Huh-7 cells co-cultured with mi R-670-exo compared with the control group(p < 0.001).Confocal laser microscopy showed that there was obvious green fluorescence of PKH67 in Huh-7 cells,indicating that AMSC-exos could be effectively taken up and internalized by Huh-7 cells.4.CCK-8 showed that exosomal mi R-670-3p inhibited the proliferation of HCC cell line Huh-7(p < 0.01).Transwell experiment showed that exosomal mi R-670-3p inhibited the invasion of Huh-7 cells(p < 0.01).The results of flow cytometry showed that exosomal mi R-670-3p could block the G2 phase of hepatoma cells.Immunofluorescence assay showed that exosomal mi R-670-3p could induce mitotic catastrophe in Huh-7 cells.5.Bioinformatics analysis predicted that mi R-670-3p could specifically bind to the 3?UTR of CCNB2.The results of the dual luciferase reporter assay showed that mi R-670-3p mimic could significantly reduce the activity of CCNB2 wild-type 3?UTR luciferase(p < 0.001),but had no significant effect on the CCNB2 mutant 3?UTR luciferase activity.Western blot showed that overexpression of mi R-670-3p could significantly reduce the expression level of CCNB2 protein(p < 0.05)6.The results of CCK-8 and Transwell experiments showed that compared with the control group,overexpression of mi R-670-3p could significantly inhibit the proliferation and invasion of Huh-7 cells(p < 0.01),while overexpression of CCNB2 could partially restore the inhibitory effect of mi R-670-3p mimic on the proliferation and invasion of hepatoma cells.Conclusions:1.Exosomes derived from AMSCs can effectively mediate the transmission of mi R-670-3p to hepatoma cells.2.Exosomal mi R-670-3p inhibits the proliferation and invasion of HCC cells,resulting in cell cycle arrest and mitotic catastrophe.3.CCNB2 is the target gene of mi R-670-3p and is negatively regulated by mi R-670-3p in HCC cell lines.mi R-670-3p inhibits the proliferation and invasion of hepatoma cells by regulating CCNB2. |
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