Differential Expression Of Exosomal MiRNAs In The Transformation Of Androgen-Independent Prostate Cancer Cells

Prostate cancer is a highly common cancer of the male reproductive system.Androgens not only play an important role in maintaining the normal growth and function of prostate,but also play an important role in the development of prostate cancer.Drug or surgical castration androgen deprivation therapy(ADT)is still the standard treatment for the advanced metastatic prostate cancer.However,ADT can only relieve and not be cured completely.After a period of effective treatment,most patients develop into androgenindependent prostate cancer.Exosomes are lipid bilayers structure of extracellular vesicles with a diameter ranging from 30 to 150 nm,which are released by any living cells and contain various components such as lipids,proteins,nucleic acids such as messenger RNA(mRNA)and small RNA(miRNA).The contents of these vesicles reflect the source and characteristics of secreted cells and can also serve as important regulators of intercellular signal transduction.At present,the abnormal expression of exosomal miRNAs have been reported in many literatures during the development and progression of prostate cancer.Therefore,we intend to analyze the differential expression of exosomal miRNAs between androgen-dependent prostate cancer cell LNCaP and androgen-independent prostate cancer cell LNCaP-AI+F induced by LNCaP cell.Searching for the differential expression of exosomal miRNAs in the transformation process further,which laid a foundation for the study on the occurrence and development of androgen-independent prostate cancer.Part1: The Function of exosomes in the transformation of androgen-independent prostate cancer cellsObjective: To investigate the role of exosomes in the transformation of androgenindependent prostate cancer cells.Methods: First,exosomes were extracted from the supernatant of LNCaP cells and LNCaP-AI+F cells by ultracentrifugation,and their particle sizes and protein markers Alix and CD63 were identified by transmission electron microscopy and western blot.Then,the extracted exosomes were added into the recipient cells,and the effects of exosomes on the proliferation of the recipient cells in the androgen-deprived environment were detected by CCK-8,the influence of exosomes on the cell cycle of the recipient cells in the androgendeprived environment was detected by flow cytometry and the expression of cell proteins after incubated with exosomes were detected by western blot.Results: Typical lipid-bilayer membrane vesicles with particle sizes of 30-150 nm were observed by transmission electron microscopy,and the expression of exosomal proteins Alix and CD63 were detected by Western blot.Exosome derived from LNCaP-AI+F cells can promote the proliferation rate of LNCaP cells in the androgen-deprived environment,increase the proportion of s-phase cells in the cell cycle and enhance the expression of AR protein and p-Akt protein in LNCaP cells.On the contrary,exosome derived from LNCaP cells can inhibit the proliferation of LNCaP-AI+F cells in the androgen-deprived environment,increase the proportion of G0-G1 phase cells in the cell cycle and reduce the expression of AR protein.Conclusion: Exosome derived from LNCaP-AI+F cells could promote the transformation of the androgen-dependent LNCaP cells,this may be related to the activation of AKT signaling pathway.Besides we found exosome derived from LNCaP cells might reverse the androgen-independent phenotype of LNCaP-AI+F cells.Part2: Differential expression of exosomal miRNAs in the transformation of androgenindependent prostate cancer cells.Objective: To search for exosomal miRNAs that are differentially expressed in the transformation of androgen-independent prostate cancer cells and to lay the foundation for further study on the development of androgen-independent prostate cancerMethods: Exosomes were extracted from the supernatant of LNCaP cells and LNCaPAI+F cells,the particle size and expression of CD63 and CD81 on the surface of exosome were analyzed by zetasizer and flow cytometry.Subsequently,high-throughput sequencing technology was used to screen the differential expression of exosomal miRNAs between the two cells,and quantitative real-time PCR(qRT-PCR)was used to verify the differential expression of miRNAs in cells and their exosomes.Results: Zetasizer indicated that the particle size was between 30 and 150 nm,and the expression of the exosomal marker proteins CD63?CD81 detected by flow cytometry were positive.By screening the results of high-throughput sequencing,we found total 214 differential expression exosomal miRNAs between the two groups,of which 196 were upregulated and 18 were down-regulated.Conclusion: The differential expression exosomal miRNAs might be involved in the transformation of androgen-independent prostate cancer cells.Part3: The function of let-7a-5p in the transformation of androgen-independent prostate cancer cellsObjective: To further explore the role of let-7a-5p in the transformation of androgen independent prostate cancer cellsMethods: The let-7a-5p mimic and inhibitor were transfected into LNCaP cells and LNCaP-AI+F cells respectively.CCK8,flow cytometry and western blot were used to detect the changes of cells proliferation rate,cell cycle and proteins.Results: After the let-7a-5p mimic was transfected into LNCaP cells,the proliferation rate of cells in the androgen-deprived environment was promoted,the proportion of cells in the S phase of the cell cycle and the expressions of AR protein and p-Akt protein were increased.On the contrary,after the let-7a-5p inhibitor was transfected into LNCaP-AI+F cell,the proliferation of cells in the androgen-deprived environment could be inhibited,and the proportion of cells in the G0-G1 phase of the cell cycle was increased.Conclusion: let-7a-5p could promote the transformation of androgen-independent prostate cancer cells,this may be related to the activation of AKT signaling pathway.