The Mechanism Of Tumor-derived Exosomal MiR-6756-3p Induces Osteoclasts Differentiation

Part ? Tumor-derived exosome induces osteoclasts differentiationObjective To investigate the phenomenon of osteoclast differentiation induced by tumor-derived exosome.Methods Hepatoma cell lines LM3,LM3-BM1,HLF and HLF-BM1 were cultured,and the cell supernatants were collected.The purified exosomes were extracted from cell supernatants by differential centrifugation and ultracentrifugation in order,and exosomes were exterminated by transmission electron microscopy,western blot,and nanosight.After identification,the exosomes were co-cultured with macrophage cells RAW264.7 to observe the differentiation of osteoclasts.In addition,the macrophage RAW264.7 was co-cultured with LM3-BM1 and HLF-BM1 cell exosome which were pretreated by inhibiting the secretory of exosome by the drug GW4869,and the recovery ability of various indicators was performed by the above experiments respectively.Results Compared with the control group,exosomes secreted by LM3-BM1 and HLF-BM1 cells can obviously induce osteoclast differentiation.Compared with the non-intervention group,the cell exosome collected after inhibiting secretion of exosomes can significantly inhibit the differentiation of osteoclasts.Conclusion Exosomes secreted by liver cancer cells can obviously induce osteoclast differentiation.Part ? Tumor-derived exosomal miR-6756-3p induces osteoclasts differentiationObjective To study which miRNAs derived from the exosome of liver cancer cells induce osteoclast differentiation,and to explore the mechanism of osteoclast differentiation.Methods LM3 and LM3-BM1 cells were sequenced by miRNA sequencing technology to preliminarily screen differentially expressed miRNAs.Then the total RNA was extracted from purified exosomes collected from LM3 and LM3-BM1 cell supernatants to screen out the candidate miR-6756-3p;Cy3 fluorescently labeled miR-6756-3p was transient transfected with donor cells LM3-BM1 and HLF-BM1 and the latter was co-cultured with recipient cell RAW264.7.After 24 hours,the fluorescence of the recipient cell was detected to reflect the present ability of miR-6756-3p from donor cells to recipient cells.Macrophage cells RAW264.7 were transiently transfected with miR-6756-3p mimic to observe the differentiation of osteoclasts.Results Compared with the control group,Cy3 labeled miR-6756-3p was presented from donor cells to recipient cells by exosome;miR-6756-3p could obviously induce the RAW264.7 differentiated to osteoclasts by transiently transfected with miR-6756-3p mimicConclusion Tumor-derived exosomal miR-6756-3p can induce osteoclast differentiation.Part ? Exosomal miR-6756-3p induces osteoclasts differentiation through the target gene-NF-?B pathwayObjective To study which target genes of miR-6756-3p derived from liver cancer cells induce osteoclast differentiation,and to explore the mechanisms by which target genes mediate osteoclast differentiation.Methods The target genes of miR-6756-3p were predicted using three major websites of Targetscan,Tarbase and DIANA micro T.Mi R-6756-3p mimic was transiently transfected,or small interfering RNA(si RNA)was transiently transfected or combined with miR-6756-3p knockdown lentivirus to stably transfect into RAW264.7 macrophages and then q RT-PCR and Western blot methods was uesd to detect the target genes expression,and the genes of CALN1,IP6K1 and ANKRD52 were screened according to the differentiation of osteoclasts.In addition,each of the three target genes CALN1,IP6K1 and ANKRD52 was constructed with respective mutants.Mi R-6756-3p mimic combined with the target gene wild-type or mutant plasmid was transfected into macrophage cells RAW264.7,and the dual luciferase reporter gene assay was used to detect the regulation of miRNA to target genes.Further,Western blot and dual luciferase reporter gene assay were used to detect NF-?B expression in LM3-BM1 and HLF-BM1 exosomes,or miR-6756-3p mimic after transient transfection of RAW264.7 macrophages.Results Compared with control group,miR-6756-3p obviously induced macrophages to differentiate into osteoclasts through the target genes CANN1,IP6K1 and ANKRD52;miR-6756-3p was able to inhibit the expression of target genes CALN1,IP6K1 and ANKRD52,and the latter activates the NF-?B signaling pathway,and further promotes osteoclast differentiation.Conclusion Exosomal miR-6756-3p derived from liver cancer cells induce the differentiation of osteoclasts through the target genes CANN1,IP6K1 and ANKRD52.And the latter promotes the activation of NF-?B pathway.Part ? Exosomal miR-6756-3p mediates bone colonization of liver cancer cells and its expression in clinical patients' blood and clinical significanceObjective To study the bone colonized cell lines LM3-BM1 derived from liver cancer cells LM3 on osteoclast differentiation and bone tissue destruction in the bone microenvironment;and to explore the expression level of serum-derived exosomes miR-6756-3p from patients and its relevance to clinicopathology.Methods LM3-BM1 cells constructed with miR-6756-3p overexpression lentivirus were injected into mice with left ventricle to detect tumor growth,osteoclast differentiation and local bone tissue destruction in bone tissue;Exosomes were extracted from the serum of the patients.After exosomes were identified by transmission electron microscopy and nanosight method,miR-6756-3p was extracted from the exosomes and its expression was detected by q RT-PCR,and the correlation between the expression level of miR-6756-3p and clinicopathology was analysed.Results Compared with the control group,after miR-6756-3p was over-expressed,LM3-BM1 cells significantly enhanced the local colonization ability of mouse bone tissue,the number of osteoclasts around the tumor was increased significantly,and the damage degree of local bone was significantly increased;In addition,compared with normal individual or hepatitis B patients,the expression level of miR-6756-3p in liver cancer patients was significantly increased,and its level was positively correlated with clinical stage(Barcelona stage);Compared with the serum of non-bone metastatic patients,the expression level of miR-6756-3p was significantly increased in the blood exosomes of patients with bone metastases.Conclusion The bone colonized cell lines LM3-BM1 derived miR-6756-3p can promote tumor local osteoclast differentiation and promote bone tissue destruction;The expression of-6756-3p was highly expressed in the blood exosome of liver cancer patients,and its expression was positively correlated with the clinical pathological stage of patients.