Research On Expression And Function Of PACE4(One Precursor Protein-processing Enzyme) In Prostate Cancer

Objective:To explore expression of PACE4in BPH, PIN, and prostate cancer, and the relationship of clinical staging, PSA level and Gleason’s scores. Furthermore, we go on study the relationship between expression of PACE4and level of malignancy and invasivene in prostate cancer cells in vitro. MiRNAs targeting PACE4were predicted, validated and further-corroborated using bio-software, real time-PCR, luciferase reporter assay and transfection of miRNA mimics and inhibitor, it can be provide a new marker for the diagnosis, monitoring and followed-up of PCa.Methods:Clinically pathological analysis of immunohistochemistry/in situ hybridization was carried out to detect the relationship between PACE4expression/miRNAs and the malignancy of prostate mass. Prostate cell lines (DU145, C4-2and BPH-1) were cultured for growth curve, immunocytochemistry analysis, colony formation, Matrigel invasion and transcriptional/translational expression assay of PACE4-related signaling molecules for confirming the relationship. MiRNAs targeting PACE4were predicted, validated and further-corroborated using bio-software, real time-PCR, luciferase reporter assay and transfection of miRNA mimics and inhibitor.Finally, these results were analyzed statistically.Results:(1) Expression of PACE4was a small amount expressing in benign prostate hyperplastic tissue, and express large number of PACE4in PCa, PACE4expression positive rates in prostate cancer, PIN and benign prostate hyperplastic tissue were192%、80%and20%, these results showed they were statistically significantly different in prostate cancer and benign prostatic hyperplasia; All cases are divided into three groups:less than10ng/ml,10±20ng/ml, higher than20ng/ml, PACE4expression positive rates are respectively54.55%,90.91%,97.44%, PACE4expression is positively correlated with PSA levels (P<0.05). PACE4expression positive rate is respectively45%,75%,91.3%,100%, according to different clinical stages, PACE4expression in prostate cancer is positively correlated with clinical stage (P<0.05);(2) Prostate cell lines (DU145, LNCap, PC3, C4-2and BPH-1) were cultured and cell imaging showed that DU145seemed to be of higher amplification activity than C42and BPH-1did. Soft agar cloning assay and Matrigel migration assay showed that DU145possessed a significantly greater colony-forming ability and migration/invasion ability that other prostate cell lines. In invasion experiments it indicating the strongest aggression and malignancy of DU145.PACE4expression in DU145was significantly higher than the other cell lines no matter at the transcriptional or translational levels, and it was supported by related signaling molecule expression, including a lower mRNA level of IGFBP3and THBS1,a higher mRNA level of Furin, Seprine2, MUC, CDK6and a higher protein level of P53, Muc1and PTEN. On the contrary, BPH-1was manifested the lowest level of PACE4expression. Immunohistochemical staining showed that cellular localization of PACE4was the cytoplasm and corroborated the above results in situ. The results suggested that PACE4expression level was closely associated with aggression and malignancy of PCa, involving some crucial signaling molecules during its regulation processing.(3) Using Software miRanda v3.3with PACE4mRNA sequence, five miRNAs were detected out to have a potentially biological interaction with3’UTR of PACE4mRNA. prostate cell lines were selected to validate the practical significance of the five candidate miRNAs. Real-time PCR showed miR-124presented with the most obvious changes among the three prostate cell lines. Luciferase reporter assay showed that fluorescence intensity decreased only after co-transfection of miR-124and miR-21, further results showing miR-124was of stronger biological association with PACE4. Furthermore, the result of in situ hybridization indicated that more miR-124expression was found in normal prostate tissue than that in PCa tissue. After transfection of miR-124oligo into DU-145, C4-2and BPH-1cells with corresponding inhibitor and NC RNA fragment as control, PACE4expression level in DU145decreased most significantly among groups, but miR-124inhibitor in C4-2and BPH-1caused a more obviously increased PACE4expressionthan that in DU-145. Cell cycle analysis by flow cytometry indicated that transfection of miR-124into DU145resulted in a elevated proportion of G0-G1phase. Similarly, MUC1expression evaluated by immunocytochemistry was also inhibited greatly in DU145by miR-124supporting that miR-124exhibited antiproliferative and antiaggressive effectes on proatate cancer cells.