The Role Of MiR-17-92 Gene Cluster In The Tumorigenesis And Drug Resistance Of Prostate Cancer

Objective This study is to clarify the effect of miR-17-92 gene cluster overexpression on the biological characteristics of DU145 prostate cancer cells and explore the internal mechanism of these effects.Methods miR-17-92 overexpression vectors were constructed.DU145 cells were infected with the virus supernatants produced by Phoenix A packaging system to establish the stable cell line with miR-17-92 overexpression(DU145-17-92).The cell growth,migration and invasion abilities were measured by a real-time x Celligence system.Proliferation and apoptosis of the DU145-17-92 and DU145-control cells were examed using Ki-67 and TUNEL assay.Phases of cell cycle of DU145-17-92 and DU145-control cells were detected by flow cytometry.Expression of apoptosis-related protein and proteins in Akt-pathway in DU145-17-92 and DU145-control cells were determined by Western blotting.The scratch healing assay was carried out to investigate the migration abilities.The expression of Integrin ?1 was detected by Western blotting,and the activities of MMP-2 and MMP-9 were measured by gelatin zymography experiment.The mRNA expression of ERCC1 was measured by qRT-PCR.Western blotting was conducted to investigate the protein expressions of ERCC1,ERK1/2 and p-ERK1/2.Results The cell growth were monitored for 72 h with the xCelligence RTCA instrument.DU145-17-92 cells growed much faster than DU145-control cells since 24 h with significant difference(P<0.05).The frequencies of Ki-67-positive cells in the DU145-control group were(56.57±1.68)%,(85.48±0.26)%,(90.85±2.08)%,while those of ki-67-positive cells in the DU145-17-92 group were(73.64±0.68)%,(93.43±1.23)%,(97.36±0.86)% at 24,48 and 72 h respectively.Cellular proliferation activity of DU145-17-92 was much higher than that of DU145-control cells,with significant difference at 24 h.The percentages of apoptotic cells in the DU145-control group were(6.76±0.09)%,(14.51±0.86)%,(20.73±1.64)%,respectively,while those of apoptotic cells in the DU145-17-92 group were(1.86±0.15)%,(7.90±0.40)%,(4.92±0.48)at 24,48 and 72 h,respectively.The apoptotic frequencies were increased gradually in a time dependent manner,and the frequencies of apoptotic cells in DU145-17-92 group were much lower than those in DU145-control group.The frequencies of the cells in G0-G1,S,and G2-M phase in DU145-control group were(65.89±0.47)%,(23.14±0.63)% and(10.54±0.59)%,while those of the cells in G0-G1,S,and G2-M phase in DU45-17-92 group were(64.61±0.83)%,(22.86±0.55)% and(11.30±0.61)%,respectively.There was no significant difference between the DU145-control cells and DU145-17-92 cells in any of the three phases.The expression levels of pro-apoptotic protein BIM and tumor suppressor PTEN in DU145-17-92 cells were much lower than those in DU145-control cells.miR-17-92 overexpression led to a clear induction in the expression level of p-AKT(Ser473)with no affection in that of p-AKT(Thr308).The expression level of ERK1/2 in the whole cell extract of the DU145-control and DU145-17-92 cells was close to each other.miR-17-92 overexpression led to a significant induction in the expression level of p-ERK1/2.DU145-17-92 cells migrated faster than DU145-control cells during the 24-h continuous monitoring(P<0.01).The scratch healing assay indicated that DU145-17-92 cells migrated from the edge towards the scratch center faster than DU145-control cells.DU145-17-92 cells invaded through matrigel markedly faster than DU145-control cells during the 24-h continuous monitoring(P<0.01).The protein expression level of Integrin ?1 and the MMP-9 activities in DU145-17-92 cells were increased than those in DU145-control cells.After the treatment of cisplatin,DU145-17-92 cells grew faster than DU145-control cells,presenting cisplatin resistance(P<0.01).The phosphorylation of ERK1/2 in DU145-17-92 cells was constantly at a high level regardless of the treatment of cisplatin.Compared with DU145-control cells,the expression of drug-resistance-related gene ERCC1 was dramatically increased in DU145-17-92 cells after the treatment of cisplatin.Conclusions 1.miR-17-92 plays a pivotal role in cell growth of DU145 cells due to reg?lation of proliferation and apoptosis.Repressed expression of pro-apoptotic protein BIM,activation of AKT and ERK1/2 signaling pathway were involved in these processes.And it can be used as a reference to determine the invasion and metastasis of prostate cancer and cisplatin resistance.2.miR-17-92 overexpression increases the migration and invasion abilities of DU145 cells,which is associated with the upregulated expression of Integrin ?1 and the increased activity of MMP-9.3.miR-17-92 overexpression enhances the cisplatin-resistance of DU145,which is correlated with the increased phosphorylation level of ERK and the upregulated expression of ERCC1 at both the mRNA and protein level.4.miR-17-92 gene cluster plays a cancerous protein-like role in prostate cancer,which can be used as a reference index of cancer invasion,metastasis and cisplatin resistance.