| Hepatocellular carcinoma（HCC）is one of the most common and highly malignant tumors.Cancer cell metastasis is the main cause of mortality,postoperative recurrence and poor prognosis.Tumor metastasis is closely related to the internal and external environment of tumor cells.On the one hand,the high expressed tumor metastasis-related genes,such as Osteopontin（OPN）,can alter the inherent adhesion and migration ability of tumor cells.On the other hand,exosomes released from tumor cells carrying various proteins,messenger RNAs（m RNAs）,micro RNAs（miRNAs）and other signaling molecules,are involved in cell-cell communication and tumor cell growth and metastasis.However,the interaction between tumor metastasis-related genes and tumor-derived exosomes remains to be clarified.Previously,we found that miRNAs expression levels were significantly altered in exosomes secreted from SMMC-OPN cells with OPN overexpression using transcriptional sequencing.To explore the role of exosomal miRNAs in OPN promoting HCC metastasis,we validated the sequencing results and investigated the effect of exosomal miR-4660 on OPN-mediated hepatoma cell migration.This study includes four parts as follows:Validation of differentially expressed Exosomal miRNAs and screening of their target genes.According to the sequencing results,the expressions of 79 miRNAs were significantly altered in exosomes of SMMC-OPN cells（36 up-,43 down-）.Firstly,exosomes were isolated from SMMC-7721,SMMC-P,and SMMC-OPN cells and characterized by their morphology,particle size and specific surface markers respectively.The quantitative real-time PCR（q RT-PCR）was conducted to examine the expression 6 up-and 9 down-regulated candidate miRNAs in exosomes,which showed that expressions of miR-133-3P（up）and miR-4660（down）were consistent with the sequencing results.Then,total 10 candidate target genes of miR-4660 involved in the cell adhesion and migration were predicted by software.We detected these genes expressions in cells and exosomes by q RT-PCR and demonstrated that LGALS3BP expression was up-regulated in both SMMC-OPN cells and their exosomes,indicating that LGALS3BP cou Ld be a target gene of miR-4660.These results suggested that OPN may regulate the expression of LGALS3BP through the exosomal miR-4660 and promote the hepatoma cells migration.The regulatory relationship between miR-4660 and its target gene LGALS3BP.After transfection of miR-4660 mimics（miR-mimic）,inhibitors（inh-4660）and negative control（miR-NC）into SMMC-7721 cells,the expression of LGALS3BP was detected by RT-q PCR and Western Blot.The results showed that miR-4660 could significantly inhibit the expression of LGALS3BP.According to Targetscan analysis there were two binding sites of miR-4660 in the 3’-untranslated region（3’UTR）of LGALS3BP gene.We constructed the wild-type（W-3’UTR）and two-site mutant（M-3’UTR）recombinant luciferase reporter vectors of LGALS3BP by overlapping PCR and site-directed mutagenesis PCR techniques and co-transfected them with miR-4660mimics or miR-NC into HEK-293T cells respectively.The double fluorescent reporter gene assay displayed that miR-4660 could significantly reduce the relative luciferase activity（RLA）of wild-type vector,and partially inhibit the RLA of the single-point mutation vectors,however,there was no effect on RLA of the vector with double site mutation.Accordingly,our finding revealed that miR-4660 could directly bind to the3’UTR of LGALS3BP,and thus negatively regulate the expression of LGALS3BP.The role of exosomal miR-4660 in OPN promoting hepatoma cells migration.Firstly,the effect of OPN on SMMC-7721 cells migration was verified.Wound healing and Transwell TMassays showed that SMMC-OPN cells had more better migration abilities compared to SMMC-7721 cells.Simultaneously,the expression levels of m RNA and protein of LGALS3BP in SMMC-OPN cells were also significantly higher than that of SMMC-7721.Thus,it appeared that the OPN-mediated hepatoma cells migration was related to the expression of LGALS3BP.To demonstrated the role of exosomal miR-4660 in the above migration process,we carried out studies from three levels:（1）SMMC-7721 cells migration ability was significantly enhanced after interfering with SMMC-OPN cells supernatant;（2）The exosomes isolated from SMMC-OPN cells supernatant were labeled with PKH67 and then co-cultured with SMMC-7721 cells.The results displayed that SMMC-7721 cells cou Ld effectively uptake the exosomes and their migration ability was also significantly enhanced after co-culturing with these exosomes.（3）miR-4660 overexpression in SMMC-OPN cells could inhibit the expression of LGALS3BP and limited the cell migration ability.The miR-4660 expression in exosomes was also increased after transfection of miR-4660 mimics into SMMC-OPN cells.Through co-culturing with these miR-4660 high expressed exosomes,the expression of miR-4660 was increased in SMMC-7721 and cells migration was inhibited.Thus,these findings provide evidence that the exosomal miR-4660 inhibits OPN-mediated hepatocarcinoma cell migration.Mechanisms of exosomal miR-4660 inhibiting OPN-mediated hepatoma cell migration.We detected LGALS3BP expression levels in SMMC-7721 cells after treating with SMMC-OPN cell supernatant and exosomes secreted,and found them up-regulated in both treating group.However,the exosomes from SMMC-OPN cell with miR-4660overexpressed could inhibit the LGALS3BP expression in SMMC-7721 cells.Combined with the above migration experiments,miR-4660 could inhibit the migration of hepatoma cells mediated by OPN by regulating the target gene LGALS3BP.Taken together,we identified a novel molecular in exosomes,miR-4660,and selected one of its key target gene,LGALS3BP.Subsequent data revealed that exosomal miR-4660 could inhibit OPN-mediated hepatoma cells migration through direct targeting to LGALS3BP in target cells.Our findings discover the interaction between internal and external factors in tumor cell migration,which provide ideas for screening anti-tumor drugs by targeting components in tumor microenvironment and have potential application value. |
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