| Prostate cancer,as a common malignant tumor in elderly men,ranks second in the number of deaths after lung cancer in Europe and the United States.In recent years,with the improvement of people’s living standards and the gradual aggravation of population aging,the incidence rate of prostate cancer in China has increased year by year.Therefore,research on the examination and treatment of prostate cancer has gradually attracted attention.Currently,the most common treatment methods for early prostate cancer include radical resection of prostate cancer,particle implantation,and radiation therapy.For locally advanced and systemic multiple metastatic prostate cancer,endocrine therapy and chemotherapy can be combined with other treatment methods.Although there are various treatments,the prognosis of patients with advanced prostate cancer,especially hormone resistant prostate cancer is poor.With the continuous updating of various tumor biology,gene targeted therapy,and immunotherapy research,more and more targeted genes,immune proteins,and cytokines have been discovered,and have achieved certain results in clinical or animal experimental treatment research of tumors.As a class of short stranded non coding RNAs,mi RNAs have approximately 22 nucleotides and can be found in all eukaryotic cells in nature.They are often used as key gene regulators to inhibit certain gene translation.More and more research evidence indicates that some mi RNAs play a crucial role in tumor genesis and development through regulating downstream targeted genes,such as tumor cell proliferation,differentiation,apoptosis,and metastasis.Among cancer related mi RNAs,research on mi R-187 has also increased in recent years.Currently,a large number of studies have demonstrated that mi R-187 is involved in the occurrence and development of various diseases.Mi R-187 can exert its function of regulating tumor genesis and development by regulating a variety of target genes,including affecting the proliferation,differentiation,and invasion of malignant tumor cells.Its related functions may become a hot spot in tumor treatment and prevention in the future.Part One Expression of mi R-187 in prostate cancer and its effect onbiological behavior of prostate cancer cellsObjective: To study the expression of mi R-187 in prostate cancer tissues and cell lines,and the relationship between its expression and prognosis.Overexpression of mi R-187 was studied to investigate its effects on biological behaviors such as proliferation,invasion,migration,and apoptosis of prostate cancer cells.Methods:1.Quantitative real-time PCR(q RT-PCR)was used to detect the expression of mi R-187 in prostate cancer cells,adjacent tissue cells,prostate cancer cell lines(22Rv1,LNCa P,Du145,PC-3),and normal prostate epithelial cell line RWPE-1.The relationship between mi R-187 and patient prognosis was evaluated by Kaplan-Meier curve analysis using the average value.2.CCK-8 was used to test cell proliferation,flow cytometry was used to detect apoptosis,and Transwell and scratch tests were used to detect cell invasion and migration capabilities.3.WB was used to detect the expression of proliferation and apoptosis related genes(Ki-67,Bcl-2,Bax)and EMT related genes(E-cadherin,N-cadherin,Slug)in the cells of NC minimal group,mi R-187 minimal group,NC inhibitor group,and mi R-187 inhibitor group to determine the relationship between mi R-187 and tumor cell apoptosis.Results:1.The low expression of mi R-187 in prostate cancer,and the lower the expression,the poorer the prognosis of patients,suggesting that mi R-187 may have a cancer inhibitory effect.2.After overexpression of mi R-187,the proliferative activity of PC-3cells significantly decreased,the rate of apoptosis significantly increased,and the ability to invade and migrate significantly decreased.3.After knocking down mi R-187,Du145 cell viability significantly increased,the rate of apoptosis significantly decreased,and its invasion and migration abilities were significantly enhanced.Conclusions: The expression of mi R-187 is low in prostate cancer,and the lower the expression,the worse the prognosis of patients.Overexpression of mi R-187 can inhibit the proliferation,invasion,migration,and promote apoptosis of prostate cancer cells.Part Two Mi R-187 targets down regulation of CD276,thereby downre-gulating the proliferation,invasion,migration,andpromoting apoptosis of prostate cancer cells by inhibiting theJAK3-STAT3 Slug pathwayObjective: On the basis of the previous study,we first searched for the correlation between mi R-187 and the target gene CD276 and how it regulates the biological function of prostate cancer cells through the JAK3-STAT3-Slug signaling pathway.Methods:1.Using RNA22,mi RSearch,mi RDB,Tar Base,and DIANA databases to predict mi R-187 target genes,and comparing the predicted results of the target genes with GSE30994 differential genes,CD276 was selected,and its expression in prostate cancer was analyzed through the Star Base Pan-Cancer Analysis Platform.2.The luciferase activity of CD276 wild type 3 ’-UTR and mutant type 3’-UTR in the NC-mic and mi R-187-mic groups were detected by the dual luciferase reporter gene assay.3.After overexpression of mi R-187 in PC-3 cells and interference with mi R-187 expression in Du145 cells,the m RNA and protein expression of CD276 were detected by q RT-PCR and WB.4.Immunohistochemistry(IHC)was used to detect the expression of CD276 in prostate cancer tissue and its corresponding adjacent tissues,and WB was used to detect the expression level of CD276 in prostate cancer cell lines.5.Using q RT-PCR and WB to detect the overexpression efficiency of CD276 in PC-3 cells,and after combined intervention with mi R-187 and CD276,the expression of CD276 and mi R-187 in each experimental group was detected again.6.CCK-8,flow cytometry,Transwell,and cell scratch assay were used to determine the biological function of prostate cancer cells after overexpression of CD276 and mi R-187,respectively.7.WB was used to detect the expression of proliferation and apoptosis related genes and JAK3-STAT3-Slug pathway related genes(JAK3,STAT3,p-STAT3,Slug,E-cadherin,Fibronectin,MMP2)in prostate cancer cells in each group.Results:1.Various gene databases have screened that CD276 may be a downstream target gene for mi R-187.GSE30994 microarray and cancer related platform analysis also indicate that CD276 is highly expressed in prostate cancer tumors.2.The results of the dual luciferase reporter gene experiment showed that the luciferase activity of CD276 wild-type 3 ’-UTR in the mi R-187 mic group was significantly inhibited by mi R-187 compared to the NC mic group,while the luciferase activity of mutant 3’-UTR was not inhibited.This result indicates that mi R-187 targets the 3 ’-UTR binding to CD276.3.QRT-PCR and WB analysis showed that the expression level of CD276 in PC-3 cells significantly decreased after overexpression of mi R-187,The expression of CD276 in Du145 significantly increased after interfering with mi R-187 expression,indicating that mi R-187 can target to inhibit CD276.4.IHC detection found that the positive rate of CD276 in prostate cancer tissue was as high as 59.65%,while the positive rate near cancer was 14.04%.WB analysis showed that compared with normal prostate epithelial cell line RWPE-1,CD276 was highly expressed in prostate cancer cells,and the expression level was relatively highest in PC-3 cell line.The above results showed that CD276 was highly expressed in prostate cancer,and mi R-187 could target to inhibit CD276.5.Compared with the NC mic+oe NC group,the expression of mi R-187 in the mi R-187 mic+oe NC group significantly increased,while the expression of CD276 significantly decreased;Compared with the mi R-187 mimic+oe NC group,the expression of CD276 in the mi R-187 mimic+oe CD276 group was significantly increased,while the expression of mi R-187 had no significant change.6.After overexpression of mi R-187,the proliferation,migration,and invasion abilities of cells were significantly reduced,while apoptosis was significantly enhanced;Overexpression of mi R-187 and overexpression of CD276 can reverse the effect of overexpression of mi R-187.7.Compared with the NC mimic+oe NC group,the expression of Ki-67,Bcl-2,JAK3,p-STAT3,Slug,N-cadherin,Fibronectin,and MMP2 in the mi R-187 mimic+oe NC group was significantly decreased,while the expression of Bax,E-cadherin was significantly increased;Overexpression of mi R-187 and overexpression of CD276 can reverse the effect of overexpression of mi R-187.Conclusions: mi R-187 inhibits the JAK3-STAT3-Slug pathway by targeting down regulation of CD276,thereby inhibiting the proliferation,invasion,migration,and promoting apoptosis of prostate cancer cells.Part Three Molecular mechanism of inhibitory effect of bone marrow-derived mesenchymal stem cell derived exosomes mi R-187in prostate cancerObjective: To further study the mechanism of bone marrow-derived mesenchymal stem cell-derived exosomes carrying mi R-187 in combination with the previous study on the role and mechanism of mi R-187 in prostate cancer cells,and to provide evidence through animal experiments.Methods:1.Human bone marrow mesenchymal stem cells were isolated from human bone marrow,and secretions were isolated from these cells.HBMSCs were observed using transmission electron microscopy and analyzed for particle size and concentration using Zetasizer Nano ZS.2.Using q RT-PCR to detect the expression of mi R-187 in HBMSCs and their secretions after overexpression of mi R-187.3.Co-culture PKH67 labeled HBMSCs exo with PC-3,and observe the uptake of exosomes by cells under a fluorescence microscope.4.The expression of mi R-187 and CD276 in PC-3 cells at different time points(0h,12 h,24h,and 48h)was detected by q RT-PCR and WB.5.Extract HBMSCs secretions that have overexpressed mi R-187,and co culture the extracted HBMSCs with PC-3.CCK-8,flow cytometry,Transwell,and scratch test were used to detect the proliferation,invasion,migration,and apoptosis of cancer cells in each group.6.WB was used to detect the expression of E-cadherin,Bax,CD276,Bcl-2,and JAK3-STAT3-Slug pathway related genes in each group.7.Subcutaneously or caudally injected prostate cancer cell PC-3 into nude mice to establish a subcutaneous tumor transplantation model and a tumor metastasis model in nude mice;Extracts were extracted from HBMSCs transfected with mi R-187 in each group and injected into nude mice through the tail vein.The formation of transplanted tumors was observed and recorded in real time.8.The expression of related proteins in the transplanted tumor tissues of nude mice in each group was detected by q RT-PCR and WB.Results:1.The exosomes isolated from HBMSCs were observed by transmission electron microscopy,and it was found that the exosomes were a group of round or elliptical membranous vesicles with significant heterogeneity in size,with a diameter of between 40 and 150 nm,and a basically uniform morphology.The membranous structure was visible around the vesicles,with a low electron density component in the center,with a diameter of62.58 nm.2.The overexpression of mi R-187 in HBMSCs cells was significantly higher,and the expression level of mi R-187 in their exocrine bodies was significantly increased.3.With the increase of time,the uptake of PC-3 into the exocrine body also increased significantly.4.The expression of mi R-187 was upregulated with time,while the expression of CD276 was downregulated with time.5.Compared with the Control group,the proliferation,invasion,and migration abilities of cells in the Exo group were significantly reduced,and apoptosis was significantly increased;Compared with the Exo NC minimal group,the proliferation,invasion,and migration abilities of cells in the Exo mi R 187 minimal group were further significantly reduced,and the level of apoptosis was significantly increased;Compared with the Exo-mi R-187-mic+oe-NC group,the Exo-mi R-187-mic+oe-CD276 group significantly enhanced the ability of cell colonization,invasion,and migration,and significantly reduced cell apoptosis.6.Compared with the Control group,the expression of E-cadherin and Bax in the Exo group significantly increased,while the expression of CD276,Bcl-2,and JAK3-STAT3-Slug pathway related genes significantly decreased;Compared with the Exo NC mimic group,the expression of E-cadherin and Bax in the Exo mi R-187 mimic group was further significantly increased,and the expression of CD276,Bcl-2,and JAK3-STAT3-Slug pathway related genes was significantly decreased;Compared with the Exo-mi R-187-mic+oeNC group,the expression of E-cadherin and Bax in the Exo-mi R-187-mic+oeCD276 group was significantly reduced,and the expression of CD276,Bcl-2,and JAK3-STAT3-Slug pathway related genes was significantly increased.7.Compared with the Control group,the tumor volume,weight,and number of metastatic nodules on the lung surface of the xenograft tumor in the Exo group were significantly reduced in nude mice;Compared with the Exo NC agomir group,the tumor volume,weight,and number of metastatic nodules on the lung surface of the xenograft tumor in the Exo mi R-187 agomir group were further reduced in nude mice;Compared with the Exo-mi R-187agomir+oe-NC group,the tumor volume,weight,and number of metastatic nodules on the lung surface of the xenograft tumor in the Exo-mi R-187agomir+oe-CD276 group were significantly increased in nude mice.8.Compared with the Control group,the expression of mi R-187,E-cadherin,and Bax in the transplanted tumor tissue in the Exo group significantly increased,while the protein expression levels of CD276,Bcl-2,and JAK3-STAT3-Slug pathway related genes were significantly decreased;Compared with the Exo NC agomir group,the expression of mi R-187,E-cadherin,and Bax in the transplanted tumor tissue of the Exo mi R-187 agomir group further increased,and the protein expression levels of CD276,Bcl-2,and JAK3-STAT3-Slug pathway related genes further significantly decreased;Compared with the Exo-mi R-187 agomir+oe-NC group,the Exo-mi R-187 agomir+oe-CD276 group significantly decreased the expression of E-cadherin and Bax in the transplanted tumor tissue,and significantly increased the protein expression levels of CD276,Bcl-2,and JAK3-STAT3-Slug pathway related genes.Conclusions: HBMSC-exos inhibits the activity of JAK3-STAT3-Slug pathway by targeting the expression of CD276 with mi R-187,thereby inhibiting the tumorigenesis and metastasis of prostate cancer cells in vivo. |
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