| Objective:To investigate the expression of Slug, the effect of Slug on growth by degradation of cell cycle regulator Cyclin D1 and on metastasis in prostate cancer via in vitro and in vivo studies on prostate cancer cell lines PC-3, LNCaP, DU-145,22RV1. Our findings should encourage the development of Slug as the novel agent for the prevention or delay of prostate cancer progression.Material and Methods:Cell culture of PC-3, LNCaP, DU-145,22RV1; The plasmids pMIGW-Cyclin Dl-HA and pMIGW-Cylin D1-HA T286A were constructed; The protein expressions of Slug, Cyclin D1, Cyclin D2, HA,α-Tubulin,β-actin were measured by SDS-PAGE and Western Blot; The retrovirus with pMIGRI vector expressing Slug, Cyclin D1-HA, Cylin D1-HA T286A and with pMIG vector were prepared. The lentivirus with pLK0.1 vector harboring Slug-shRNA was prepared. PC-3 and DU-145 were infected by virus prepared previously; Total RNA was extracted from prostate cancer cells and reverse-transcribed to cDNA; mRNA expressions of Snaill, Snail2/Slug, Snail3, Scratchl, Scratch2, Cyclin D1 were measured by RT-PCR and Real-Time PCR.DNA content of PC-3 cells with pMIGRI and pMIGRI-Slug vector were measured separately by flow cytometry with propidium iodide (PI) staining to analyze the change of cell cycle. The growth of PC-3 cells with pMIGRI and pMIGRI-Slug vector was measured separately by MTT assay. The growth of PC-3 cells with pMIGRI, pMIGRI-Slug and pMIGW-Cyclin D1-HA vectors, and the growth of DU-145 cells with pLKO. land pLK0.1-shSlug vectors were measured separately by quick cell proliferation assay. The two kinds of PC-3 cells with pMIGRI and pMIGRI-Slug vector were injected to NCR-nu/nu (nude) mice's subcutaneous tissue separately to construct in vivo tumor model for the evaluation of their growth. 0μl,10μl,20μl,40μl adenovirus expressing human Slug were added to DU-145 cells separately, protein and mRNA expressions of Slug and Cyclin Dl were measured by Western Blot and Real-Time PCR respectively four days after infection. 0μl,40μl,80μl,120 u 1 adenovirus expressing human Slug were added to PC-3 cells with pMIGW-Cyclin Dl-HA and pMIGW-Cylin D1-HA T286A vector separately, protein expressions of Slug and Cyclin D1 were measured by Western Blot five days after infection; 30 u 1,60μl adenovirus expressing human Slug were added to PC-3 cells with pMIGW-Cyclin D1-HA vector and those treated by 0.1μM/L MG-132(inhibitor of ubiquitination pathway) separately, and protein expression of Cyclin D1 was measured by Western Blot three days after infection; The binding of Slug protein and Cyclin D1 protein in U20S cells transfected by pMIGW-Slug-HA vector were analyzed by co-immunoprecipitation. The binding of Slug protein and Cyclin D1 protein in U20S cells co-transfected by pcDNA-Cyclin D1-HA and pMIGW-Slug-GFP vector were analyzed by cofocal microscopy at 10μm interval.The invasion of PC-3 cells with pMIGRI and pMIGRI-Slug vector was analyzed by scratch assay and matrigel invasion assay in vitro. Results:Slug protein was highly expressed in prostatic tumor samples from transgenic adenocarcinoma of the mouse prostate (TRAMP), compared to that of wide-type ones. Slug protein was highly expressed in PC-3, LNCaP, and 22RV1, which is the highest in LNCaP followed by PC-3 and 22RV1. The expression of Slug protein is the lowest in DU-145. Among Snaill, Snail2/Slug, Snail3, Scratchl, and Scratch2 five Slug/Snail superfamily members, only Slug transcripts were evident in prostate cancer cell lines, the level of which is the highest in PC-3 followed by DU-145, LNCaP, and is the lowest in 22RV1. There is distinct statistic significance among the four cancer lines for Slug transcript level (P<0.05). There is no correlation between the level of protein and transcript in the four cancer cell lines. And, Slug protein expression level was not reversely correlated with p53 status. PC-3 and DU-145 with pMIGRI-Slug vector grew slower, compared to their negative controls. There is statistic significance between their growth curves (P<0.05). PC-3 cells with pMIGRI-Slug vector involving G0/G1 phase increased and those involving S phase decreased, compared to their negative control. There is statistic significance between cell numbers involving each cell cycle phase of two groups. PC-3 with pLK0.1-shSlug vector grew faster, compared to their negative controls. There is statistic significance between their growth curves (P<0.05).The protein expression of Cyclin D1 in PC-3 carrying pMIGRI-Slug vector was reduced, compared to their negative control. And, the protein expression of Cyclin D1 in PC-3 carrying pLK0.1-shSlug vector was increased, compared to their negative control. The protein expressions of Cyclin D1 were all reduced four days after infection by adding 10μl,20μl,40μl adenovirus expressing human Slug to DU-145 carrying pMIGRI-Slug vector compared to negative control, which is dose-dependent. However, there is no statistic significance between their mRNA expressions (P>0.05). The protein expressions of HA-Cyclin Dl were all reduced five days after infection by adding 40μl,80μl,120μl adenovirus expressing human Slug to PC-3 carrying pMIGW-Cyclin Dl-HA vector compared to negative control, which is dose-dependent. However, there is no change for HA-Cyclin D1 expression in PC-3 with pMIGW-Cyclin D1-HA T286A. The protein expressions of HA-Cyclin Dl were all reduced three days after infection by adding 30μl,60μl adenovirus expressing human Slug to PC-3 carrying pMIGW-Cyclin D1-HA vector compared to negative control, which is dose-dependent. However, there is no change for HA-Cyclin Dl expression in PC-3 with pMIGW-Cyclin D1-HA treated by 0.1μM/L MG-132(inhibitor of ubiquitination pathway). Co-immunoprecipitation showed that HA-Slug protein and Cyclin D1 were both pulled down at the same time. Co-colonization showed that green fluorescent protein (GFP) binding to Slug and red fluorescent protein (RFP) bound to HA-Cyclin Dl overlapped in part. PC-3 carrying both pMIGRI-Slug and pMIGW-Cylin Dl-HA grew faster, compared to those with pMIGRI-Slug. There is statistic significance between their growth curves (P<0.05). However, There is no statistic significance between their growth curves in PC-3 cells carrying both pMIGRI-Slug and pMIGW-Cylin Dl-HA and those with pMIGRI (P>0.05).Scratch assay showed that PC-3 cells with pMIGRI-Slug exhibited a significant increase in motility capacity, compared to negative control. However, the motility capacity in PC-3 with pLK0.1-shSlug was decreased. Matrigel invasion assay showed that there were more PC-3 cells with pMIGRI-Slug involving invasive capacity than their negative control. There is statistic significance between their invasive cells number (P<0.05).Conclusion:Slug protein was highly expressed in the mouse prostate cancer tissues. And, Slug protein was selectively expressed in PC-3, LNCaP, DU-145,22RV1; its expression may be subjected to regulation at transcription or post-translation modification. Slug expression in prostate cancer cells is not regulated by p53. The up-regulation of Slug expression contributed to the degradation of cell cycle regulator Cyclin D1, which leading to G0/G1 phase arrest and S phase prolonged, to inhibit the growth of prostate cancer cells in vitro and in vivo. The degradation of Cyclin Dl by up-regulation of Slug was performed primarily via the binding of Slug protein and Cyclin Dl protein under ubiquitination-dependent pathway, not by the inhibition of Slug protein to Cyclin D1 gene transcript. The up-regulation of Slug contributed to metastasis of prostate cancer cells in vitro. These findings show that the regulation of Slug to signaling pathway can provide the important target for prostate cancer therapy. Key words:Slug, prostate cancer, Cyclin D1, growth, metastasis Chinese Library classification:R6...
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