| Background.Prostate cancer(PCa)ranks first among new cases of cancer in men,and about 90.1%of men with prostate cancer and death are diagnosed with bone metastases.Exosomes are extracellular vesicles 30-150 nm in size,which are secreted and released by cells and contain a large number of proteins,DNA,mRNAs and microRNAs(miRNAs).Studies have shown that exosomes also play a key role in the development of disease in bone metastases from prostate cancer.Under normal conditions,the dynamic balance of bone is mainly formed by the balanced activity between osteoblasts and osteoclasts.In cancer bone metastasis,a significant disruption of bone homeostasis occurs,and many studies have shown that prostate cancer bone metastasis is primarily an osteogenic response,and that miRNAs in exosomes secreted by prostate cancer cells among them may play an important regulatory role in bone metastasis.MiRNAs are endogenous RNAs of approximately 22 lengths,and in recent years,many experimental studies have clarified that miR-214-3p has a significant inhibitory effect on osteogenic differentiation.Therefore,we have proposed the conjecture that prostate cancer bone metastasis is caused by a series of osteogenic pathological changes through the regulation of osteoblast differentiation level by miR-214-3p in exosomes secreted by PCa.At present,the mechanism of miR-214-3p in the secreted exosomes of PCa on osteoblasts in bone metastasis is still unclear.Objective.This study investigates the regulatory role of miR-214-3p in secreted exosomes from PC-3 cell line on osteoblast differentiation process in bone metastasis of prostate cancer,which may provide new therapeutic approaches for clinical treatment of bone metastasis of prostate cancer.Methods.1.By culturing three prostate cancer cell lines,DU145,LNCap and PC-3,respectively,the cultures of the above cells were collected and the exosomes were extracted by ultracentrifugation,and the RNA in the exosomes was extracted by trizol method.qRT-PCR was applied to determine the miR-214-3p content in each prostate cancer cell line.2.By culturing PC-3 cell lines,miR-214-3p mimic,inhibitor and NC were transfected into the cells with lipo2000,the culture fluid in each group was collected,and exosomes were extracted from them by ultracentrifugation.The exosomes were observed by transmission electron microscopy.3.Human bone marrow was extracted,bone marrow mesenchymal stem cells were isolated,and PC-3 exosomes of miR-214-3p mimic,inhibitor,NC and blank group were added to the osteogenic culture medium at the time of induction and co-cultured,and the medium was changed every 3 days to induce the mature osteoblasts for 21 days.The morphology of osteoblasts was identified and observed by ALP staining and alizarin red staining.The gene expression levels of miR-214-3p,COL1A1 and OPN were determined by WB and qRT-PCR,and the changes of Nothcl signaling pathway were verified by WB.Results.1.MiR-214-3p levels in exosomes of three prostate cancer cell lines,DU 145,LNCap and PC-3,were determined and analyzed by qRT-PCR.MiR-214-3p levels in PC-3 prostate cancer cell line were significantly reduced compared with the other two cell lines,indicating that miR-214-3p in exosomes secreted by prostate cancer bone metastasis cells PC-3 is significantly regulated by osteoblasts.2.The exosomes of PC-3 prostate cancer cell lines were observed by electron microscopy,and most of the exosomes were lipid bilayer structures with diameters of 40-110 nm,which appeared as concave "cup-like" structures under electron microscopy.3.PC-3 exosomes transfected with miR-214-3p mimic,inhibitor and NC were added to the induction culture of osteoblasts,and ALP and alizarin red staining showed that osteoblast differentiation in the inhibitor group was similar to that in the NC group,and significantly higher than that in the miR-214-3p mimic group and the blank control group.Osteoblast differentiation markers COL1A1 and OPN gene levels showed corresponding results,with osteoblast differentiation in the inhibitor group being similar to that in the NC group and higher than that in the mimic group,while the Notch1 pathway was activated accordingly.Conclusion.1.After cross-sectional comparison of DU 145,LNCap and PC-3 in prostate cancer cell lines,the level of miR-214-3p in the secreted exosomes of PC-3 cell line was significantly reduced compared with other groups.2.By transfecting the PC-3 cell line,it was shown that miR-214-3p in exosomes secreted by PC-3 had a significant enhancement effect on osteogenic differentiation,which increased the expression levels of COL1A1,a marker gene related to osteogenic differentiation,and OPN,and activated the Notchl signaling pathway. |
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