| Prostate cancer(PCa)is a common malignant tumour of the male urogenital system,the second leading cause of cancer mortality in men worldwide and a significant cause of death in elderly men.In recent years,with the aging of population and the change of diet structure,the incidence of PCa has been increasing in china.The occurrence of bone metastases is an important clinical feature and cause of death in PCa.Cancer-derived exosomes have been revealed to play an increasingly important role in microenvironment transformation.Cancer cells are reported to secrete a large number of exosomes,which are rich in miRNAs that can integrate into target cells and affect their physiological function.Most importantly,exosomes can enter the blood circulation and reach metastatic loci before cancer cells,thereby stimulating the microenvironment and promoting metastasis.Our previous work demonstrated that exosomal miR-141-3p is upregulated in the serum of metastatic PCa patients,which is related to bone metastasis.However,the role and regulatory mechanism of miR-141-3p in the bone metastasis microenvironment remains poorly understood.Based on this,this paper attempts to study from the following five parts:Part 1 Characterization of exosomes and miR-141-3p expressionObjective: To isolate and identify exosomes from cell culture medium,then to investigate the biological characteristics and miR-141-3p expression of prostate cancer exosomes.Methods: We incubated PCa cell line in exosomes-free medium generated from exosomes-free FBS.Exosomes were isolated from cell culture medium after 48 h by ExoperfectTM.To examine the size distribution,morphology and marker protein expression of the isolated exosomes,the exosome pellets were examined by nanoparticle tracking analysis,transmission electron microscopy,flow cytometry and western blotting.We examined the expression of miR-141-3p in exosomes from the cell supernatant using real-time PCR.The NGS differential expression analysis of miRNAs in exosomes was measured with high expression miR-141-3p and low expression miR-141-3p cell line.Results: 1.Characterization of exosomes :The isolated exosomes appeared as uniformly round or oval membrane vesicles with sizes within the characteristic diameter range of 30-150 nm(maximum peak distribution of 72 nm).The isolated exosomes were positive for the exosomal marker CD63,CD81,CD9,Hsp70,Alix.2.miR-141-3p expression of exosomes: The levels of miR-141-3p were high expression in exosomes from MDA PCa 2b cells,and low expression in exosomes from the normal prostate epithelium cells RWPE-1.3.Analysis of difference expression of NGS: The deep sequencing results showed there were 50 miRNAs differentially expressed in exosomes from MDA PCa 2b cells.miR-375,miR-200c-3p,miR-141-3p were progressive high expressed,and miR-100-5p,miR-584-5p,miR-125b-1-3p were progressive low expressed.Conclusion: These results confirmed that the vesicles isolated from the conditioned media were exosomes based on their morphology,size and marker protein expression.Compared with exosomes from other cells and RWPE-1 cells,in exosomes from MDA PCa 2b cells,the levels of miR-141-3p were increased significantly.Part 2 Exosomal miR-141-3p promote the activity of osteoblasts in vitroObjective: To determine whether exosomal miR-141-3p from MDA PCa 2b cells regulates osteoblasts activity in vitro.Methods: We constructed a lentiviral vector system containing cytomegalovirus(CMV)-driven red fluorescence protein(RFP)-tagged CD63(CMV-RFP-CD63)to label the exosomes derived from MDA PCa 2b and RWPE-1 cells,and observed by confocal imaging.Real-time PCR was applied to confirm whether the MDA PCa 2b-derived exosomal miR-141-3p was responsible for the elevated miR-141-3p in osteoblasts.The effects of miR-141-3p on osteoblasts activity were observed after we transfected osteoblasts with miR-141-3p mimics to alter the level of miR-141-3p by CCK-8 test,alkaline phosphatase activity,extracellular matrix mineralization and rhodamine-phalloidin and DAPI double-staining.Results: 1.Transfer of miR-141-3p from donor cells to recipient cells through exosomes: We co-cultured either MDA PCa 2b or RWPE-1 cells with osteoblasts in a transwell system.Numerous RFP+ particles were present within the osteoblasts after 72 h of co-culture with MDA PCa 2b cells,and its miR-141-3p level was significantly higher than that in osteoblasts alone or in osteoblasts co-cultured with RWPE-1 cells at 72 h after co-culture.2.Exosomal miR-141-3p promote the activity of osteoblasts in vitro: CCK-8 test results showed that miR-141-3p significantly promoted osteoblasts proliferation.Alkaline phosphatase staining showed that the vast majority of osteoblasts contained black-grey particles after miR-141-3p mimic transfection,with significant differences in the number of positive cells and the level of alkaline phosphatase activity.The level and quantity of calcium nodule deposition were significantly higher than the level and quantity in non-transfected cells.We then observed rhodamine-phalloidin and DAPI double-staining of actin microfilaments and nuclei,respectively,by confocal microscopy to evaluate osteoblasts adhesion.Osteoblasts showed good morphology and obvious actin microfilaments,and the cell area was significantly increased after miR-141-3p mimic transfection.Conclusion: Exosomal miR-141-3p can significantly increase the proliferation of osteoblasts,alkaline phosphatase activity,extracellular matrix mineralization and cell adhesion,suggesting that it promotes osteoblasts activity.Part 3 Exosomal miR-141-3p promotes osteoblasts activity in vivoObjective: To study the organ-target specificity and the role of osteoblasts activity of exosomal miR-141-3p from the bone metastatic cell of prostate cancer MDA PCa 2b.Methods: Six-week-old male BALB/c nude mice were intravenously injected with Di D-labelled exosomes(100 ?g per mouse)isolated and purified from the supernatant of MDA PCa 2b or RWPE-1 cells;equal volume of phosphate-buffered solution(PBS)was used as a negative control.The distribution of Di D-exosomes was evaluated by biophotonic imaging at 8h after injections.At 4 weeks after the injections,the expression level of miR-141-3p in bone tissue was detected by PCR;the status of osteoblasts was observed in vivo by HE staining.Results: 1.Organ-target specificity of exosomal miR-141-3p: Fluorescence quantification determined significant accumulation of exosomes from MDA PCa 2b in the bone at 8h after injections.Exosomes from MDA PCa 2b and RWPE-1 cells showed no significant differences at 8h after injections,although the liver and stomach had fluorescent expression.2.Exosomal miR-141-3p promotes osteoblasts activity in vivo: At 4 weeks after the injections,real-time PCR analysis showed a significant increase of the level of miR-141-3p in the distal femur of mice treated with MDA PCa 2b exosomes.HE staining of bone showed that a large number of osteoblasts were active in bone tissue of mice treated with MDA PCa 2b exosomes.Conclusion: Exosomal miR-141-3p from MDA PCa 2b cells had bone-target specificity and promoted osteoblasts activity.Considering the role of exosomal miR-141-3p derived from MDA PCa 2b cells in regulating osteoblasts activity in vivo and vitro,it is necessary to further explore its role of the bone metastasis of prostate cancer.Part 4 Identification of miR-141-3p target genes and the role of DLC1 related signaling pathwayObjective: According to the results of the gene expression analysis of the dual luciferase reporter system,the targeted negative regulation of miR-141-3p on DLC1 was verified.The changes of DLC1 related signal pathway was further investigated.Methods: We build pmiR-RB-REPORT? dual luciferase reporter carrier,and the interaction between miRNA and target genes was verified by the relative fluorescence of the reported gene.Sh RNA was synthesized to knock out DLC1 in vitro,and DLC1 related signal pathway was observed.Results: 1.MiR-141-3p negatively regulates DLC1 expression :The expression analysis using the dual luciferase reporter system showed that miR-141-3p mimics lowered the luciferase activity of the DLC1-WT plasmid.Luminescence intensity recovered in the mutant plasmid in which the first predicted site(mut1)was disrupted,whereas the luminescence intensity of the mutant plasmid containing a mutation at the second predicted site(mut2)was obviously down-regulated.2.Changes of DLC1 related signal pathway: DLC1 KD activated Rho A and Cdc42 but had no effect on total protein levels;Activation of p38 MAPK pathway is closely related to the expression of osteoblasts activity-related marker genes(Runx2,Opn,Bsp and Opg).Conclusion: MiR-141-3p negatively regulates DLC1 expression by binding the mut1 site.miR-141-3p activated p38 MAPK signalling by inhibiting DLC1 and promoted osteoblasts activity and increased the expression of osteoblasts activity-related marker genes.These changes favour the formation of a bone metastasis microenvironment and lay a foundation for the formation of PCa bone metastases.Part 5 Exosomal miR-141-3p promotes osteoblastic metastasisObjective: To build tumorigenesis model of animal,and investigate whether exosomal miR-141-3p from prostate cancer cell can promote bone metastasis of prostate cancer.Methods: Six-week-old male BALB/c nude mice were divided into three group and respectively intravenously injected with exosomes transfected miR-141-3p mimics,exosomes transfected miR-141-3p inhibitor,and exosomes only from PCa cells,three times a week.At 3 weeks after the injections,the animals respectively received 2.0×106 MDA PCa 2b cells transfected with miR-141-3p mimics /inhibitor and only MDA PCa 2b cells via intraosseous injection.Results: 1.Exosomal miR-141-3p promotes osteoblastic metastasis: Mice injected with miR-141-3p mimics developed apparent bone metastases compared with those injected with the miR-141-3p inhibitor and the control at 4 weeks after injection.Micro-CT showed that the right femur/tibia formed visible metastases in mice injected with miR-141-3p mimics,with increased shadow density on the bone background;the bone shape had no obvious change.HE staining of bone showed osteogenic changes,and there was a large amount of osteoblasts and osteogenesis with tumour characteristics in the limbic cortex.The tumour cells were closely aligned,with loss of polarity and an imbalance of the nuclear to cytoplasmic ratio,and there was increased pathological nuclear fission.There was a significant increase in the level of miR-141-3p and OPG and a significant decrease in DLC1 in the distal femur of mice treated with miR-141-3p-mimic exosomes.2.The influence of exosomal miR-141-3p on survival time: The median survival times in the three groups(miR-141-3p mimics,miR-141-3p inhibitor and control)were 42,60,51,and the log-rank method test showed significant differences in the survival times of the three groups(?2 = 9.701,P = 0.02).The mice injected with miR-141-3p mimics exhibited a significantly shorter survival time.Conclusion: MDA PCa 2b cells-derived exosomal miR-141-3p transfers to osteoblasts and regulates its activity to promote osteoblastic metastasis. |
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