| Part one: Screening of target miRNA according to different expression of miRNA clinical specimensObjective: To investigate the significantly different expression of miRNA existing in these specimens which include prostatic hyperplasia, Pericarcinoma, Benign prostatic hyperplasia, castration-resistant prostate cancer. Finially, the target miRNA was sreened according to data of clinical specimens.Methods: The total RNA was extracted from the cell lines which included Benign prostatic hyperplasia extract samples, 18 cases of primary prostate cancer specimens and 10 cases of benign prostatic hyperplasia CRPC tumor samples as well as 22 cases in the sampleof castration-resistant prostate cancer. MiRNA microarray V2 chips were used to analyze miRNA of total RNA and target miRNA according to the data of different miRNA expression was selected.Results:339 miRNA(47%) included in detected 723 individual miRNA were significantly different. Of these, miRNA-32, miRNA-590-5p and miRNA-21 significantly overexpressed, but mi RNA-99 a, miRNA-221 and miRNA-26 ab significantly suppressed.MiRNA-26 ab, miRNA-21 and mi RNA-221 was found in CRPC specific expression but there was no significant difference in the PC.Conclusions: Since miRNA-26 ab, mi RNA-21 and miRNA-221 was found to have specific expression and no significant differences in the PC in CRPC, so this can be determined that miRNA-26 ab, miRNA-21 and miRNA-221 expression is associated with CRPC. As has been shown to regulate hormones associated with male and CRPC has also been involved in before miRNA-21 and miRNA-221. Finally, miRNA-26 ab is selected for further study large.Part II: A preliminary study of mi RNA identification and mechanism of action in the AR signaling pathwayObjective: By transfected LNCa P cell line to explore the role of mi RNA in prostate cancer cell growth and cell cycle, as well as to determine the target genes of mi RNA-26 ab, finally a clear relationship between mi RNA and the AR signaling pathway.Methods:â‘ First,fluorescence detection was used to identify mi RNA-26 ab transfected LNCa P cells,followed by flow cytometry analysis which was utilized to detect the number of cells in various stages of the growth cycle.â‘¡The second step, the mi RNA-26 ab was combined with PTEN gene and the new gene was quantified by quantitative PCR, mi RNA-26ab-PTEN was cloned into plasmid vectors p SGG-3UTR and luciferase expression was performed by enzymatic detection.â‘¢Finally, the influence of western-blot technique for detection of mi RNA-26 ab AR signaling pathway has been used.Results: â‘ mi RNA-26 ab of mi RNA precursors were transfected into the parental LNCa P cells, and significantly reduced the growth rate of LNCa P cells(P <0.05) mi RNA-26 ab in a time-dependent manner.â‘¡Precursor mi RNA-26 ab transfected LNCa P cells were used for m RNA microarray analysis, the results of five genes(PTEN, FBN1, REBB, TPM1, E2F1) shows the change of approximately 5 multiples. Of these, PTEN is predicted as the most promisingly functional target gene for mi RNA-26 ab and in precursor mi RNA-26 ab transfected into LNCa P cells, the expression of PTEN protein increased as well. Meanwhile luciferase results clearly show the direct regulation of PTEN by mi RNA-26 ab.â‘¢In LNCa P cells, mi RNA-26 ab can block DHT-induced expression of PSA and PSA-LUC receptor gene activity, but the mi RNA-26 ab AR expression did not change, indicating that the precursor mi RNA-26 ab is a direct signal path to reduce the AR target gene transcription and expression. |
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