MicroRNA Let-7a Inhibits Proliferation Of Human Prostate Cancer Cells In Vitro And In Vivo By Targeting E2f2 And Ccnd2

MicroRNAs (miRNAs) are endogenous, noncoding small RNAs 20–25 nucleotides in length, which play an important regulatory role through complimentary binding of the 3?untranslated regions (UTRs) of target genes. Many reports have indicated that miRNAs can be useful for cancer diagnosis and therapy. Previous work has shown reduced expression levels of let-7 in lung tumors compared to normal lung tissue. let-7 slows cellular proliferation by down-regulating the oncogenes RAS/c-MYC and HMGA-2 at the translational level. But little is known about the expression or mechanisms of let-7a in prostate cancer. In this study, we used in vitro and in vivo approaches to investigate whether E2F2 and CCND2 are direct targets of let-7a, and if let-7a acts as a tumor suppressor in prostate cancer by down-regulating E2F2 and CCND2.Materials and Methods1. Cell culture and tissue collection Human prostate cancer cell lines LNCap, DU145, PC3, and PrEC (prostate epithelial cells) and human embryonic kidney cells HEK293A were cultured in RPMI-1640 (Gibco) supplemented with 10% fetal-calf-serum and penicillin (100 U/ml). Twenty-six freshly resected prostate cancer specimens and their adjacent non-tumorous specimens were collected from the Department of Urology in Xi'jing Hospital. The specimens were immediately frozen in liquid nitrogen and maintained there until use. All samples were obtained from patients who gave informed consent to use specimens for research purposes. The protocols used in the study were approved by the hospital's Protection of Human Subjects Committee. The use of human tissues in this study was approved by the Institutional Review Board of the xxx University and was in accordance with their guidelines.2. Plasmid construction and cell transfection Let-7a was amplified and purified by miRNA isolation kit (Invitrogen, Carlsbad, CA) according to manufacturer's protocol. Let-7a PCR products were cloned into the EcoRI/PstI cloning site of a pcDNA3 plasmid containing green fluorescent protein (GFP) to form the plasmid pcDNA3-let-7a-GFP. Cell transfection was performed with lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Mixed clones were screened and cultured for an additional 4 weeks. PC3 cells transfected with let-7a were designated as PC3-let-7a-GFP, the negative control (cells transfected with the empty vector) was designated as PC3-GFP. PC3 cells were also transfected with 30 nM of synthetic let-7a (mimics), and synthetic negative control miRNAs (NC). Lastly, a synthetic let-7a-inhibitor sequence and synthetic let-7a-inhibitor negative control (NC inhibitor).3. Total RNA extraction and real-time RT-PCR For let-7a, Total RNA was extracted from samples using a miRNeasy Mini Kit (Qiagen). cDNA was synthesized using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Real-time RT-PCR was performed with TaqMan MicroRNA Assay kit (Applied Biosystems). For E2F2 and CCND2, total RNA extraction and real-time RT-PCR were performed using SYBR? GreenER? Two-step kit (Invitrogen, Carlsbad, CA). All protocols were carried out according to manufacturer protocols. The expression level of let7a was normalized to RNU6B. The expression level of E2F2 and CCND2 were normalized to GAPDH. Real-time RT-PCR was performed by using 7500 Real-time RT-PCR System (Applied Biosystems). PCR was performed under the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 50 cycles at 95°C for 15 s, and 60°C for 1 min. Each sample was run in triplicate.4. MTT Assay PC3 cells and LNCaP cells transfected with either NC or let-7a (mimics), or NC inhibitor and let-7a inhibitor were plated on 96-well plates at 1×104 cells/well. Viable cells were measured 1, 2, 3, 4, and 5 days after plating. After incubation with 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), the cells were lysed in 150?l of 100% dimethylsulfoxide (DMSO) and UV-visible absorbance was read at 490 nm using the 96-well plate reader. Each sample was run in triplicate.5. Soft agar assay Briefly, 2 ml of 0.5% agar was added to each well of a 12-well plate. Detached PC3-let-7a-GFP and PC3-GFP cells were mixed with the topagarose suspension (final concentration 0.3%), and then the cells were layered onto the 0.5% agarose underlay. The number of foci > 100?m were counted after 18 days. Each experiment was performed in triplicate.6. Cell cycle analysis PC3 cells and LNCaP cells transfected with either NC or let-7a (mimics), or NC inhibitor or let-7a inhibitor were harvested 72 h after transfection, washed with cold phosphate buffered saline (PBS), and fixed in 1ml of 70% ethanol. After overnight incubation at 4°C in ethanol, cells were washed in PBS and suspended in 500?l propidine iodide (PI) 30 min before flow cytometry. Populations in G0-G1, S, and G2-M phase were measured by flow cytometry (EPICS XL, Coulter, Miami FL) and the data were analyzed by using Multicycle-DNA Cell Cycle Analyzed Software. The measurement was performed in triplicate.7. Dual-luciferase reporter assay To investigate whether E2F2 and CCND2 expression were regulated by let-7a, a dual-luciferase reporter assay was performed. The 3'-UTR of CCND2 and E2F2 were amplified by PCR respectively. Both of these PCR products contained two binding sites for let-7a and both of these mutated 3'-UTR of E2F2 and E2F2 were synthesized after several point mutations were made in the binding sites PCR products. After restriction digestion, amplified genes were cloned into the corresponding sites of a reconstructed pGL3 expression vector. The pRL-TK control vector expressing Renilla luciferase (Promega) was used for normalization of cell number and transfection efficiency. Cotransfections of synthetic let-7a sequence (mimics) and NC, or let-7a inhibitor and NC inhibitor were also performed in human embryonic kidney cells HEK293A by using lipofectamine 2000 (Invitrogen). Fire?y and Renilla luciferase activity was determined by Pikkagene Dual Luciferase Assay System (Toyo-B-Net).8. Western blotting analysis Twenty-five micrograms of prostate cancer specimens and their adjacent non-tumorous specimens'protein as well as PC3-let-7a-GFP and PC3-GFP protein were electrophoresed in 10% SDS–PAGE minigels and transferred onto Hybond C nitrocellulose membranes (Amersham Life Science, Buckinghamshire, UK). After incubating with primary antibodies at 4°C overnight, the nitrocellulose membranes were washed three times with Tris-Buffered Saline Tween-20 (TBST) and then incubated with fluorecein isothiocyante (FITC)-conjugated, goat anti-mouse or goat anti-rabbit IgG (1:500 dilution, Sigma) for 1h at room temperature. After washing three times with TBST, blots were visualized by using the Odyssey Infrared Imaging system (LI-COR Bioscience, Lincoln, Nebraska USA). Each assay was repeated three times.9. Tumorigenicity Five nude mice were subcutaneously injected with ~1×106 PC3-let-7a-GFP or PC3-GFP cells at a single site of the back. The weight of the tumor and the weight of the mouse were measured at the time of sacrifice (4 weeks after injection). Western blotting was performed to investigate whether E2F2 and CCND2 were down-regulated in the nude-mouse xenograft model. Tumor suspensions were treated with lysis buffer and western blotting was done according to the procedures described above.Results1. Let-7a is down regulated in resected prostate cancer samples and in prostate cancer cells Real-time RT-PCR revealed that the expression levels of let-7a were decreased by ~43% in resected human prostate cancer samples compared to the adjacent non-cancerous samples. The prostate cancer cell lines LNCap expressed only ~70% as let-7a than normal prostate epithelial cells PrEC. Additionally, the amount of let-7a expression in PC3 cells and DU-145 were about 50% of that observed in PrEC. These results indicate that let-7a is decreased in prostate cancer, including the androgen-independent cancers PC3 (* p < 0.05; ** p < 0.01).2. Let-7a inhibits cell growth in vitro and induces cell cycle arrest at the G0-G1 phase After transfection, the expression level of let-7a in PC3-let-7a-GFP was ~300% higher than in PC3-GFP by real-time RT-PCR; A ten-fold increase in expression of let-7a was observed in PC3 cells transfected with let-7a (mimics) versus those transfected with NC (p < 0.01). This indicated that transfection was appropriately performed. Colony formation analysis indicated that the colonies formation ability of PC3-let-7a-GFP cells is ~40% less than that of control cells (p <0.01). MTT growth curves indicated that from the second day, the survival of let-7a transfected PC3 cells and LNCaP cells are significantly less than that of negative control, and the survival of let-7a inhibitor transfected PC3 cells and LNCaP cells are significantly more than that of NC inhibitor transfected cells (p <0.05). Flow cytometry showed that the percentage of let-7a transfected PC3 cells and LNCaP cells in the G0-G1 phase was ~30% (PC3) and 16% (LNCaP) higher than that of negative control, which paralleled with a ~50% (PC3) and 20% (LNCaP) decrease in the S phase. the percentage of let-7a inhibitor transfected PC3 cells and LNCaP cells in the G0-G1 phase was ~23% (PC3) and 19% (LNCaP) less than that of NC inhibitor transfected cells, which paralleled with a ~33% (PC3) and 25% (LNCaP) increase in the S phase (* p < 0.05; ** p < 0.01). These data indicate that let-7a inhibits PC3 proliferation by inducing cell-cycle arrest at G1/S phase.3. Let-7a targets 3?UTR of E2F2 and CCND2 No reduction of luciferase activity was observed in HEK293A cells transfected with let-7a (mimics) and mutated CCND2 or E2F2. But greater than 30% reduction of luciferase activity was observed with wild-type CCND2 and ~50% reduction of luciferase activity was observed with wild-type E2F2 (**p < 0.01). Compared to NC inhibitor, there is no significant difference in luciferase activity when HEK293A cells were transfected with the let-7a inhibitor and wild-type CCND2 or E2F2. These data support that E2F2 and CCND2 are direct targets of let-7a and endogenous let-7a in HEK293A has no interference to our experience.4. Let-7a inhibits expression of E2F2, CCND2 Real-time RT-PCR shows that mRNA relative expression of E2F2 and CCND2 in prostate cancer tissues and in LNCaP, PC3, and DU-145 cells are up-regulated compared with their adjacent non-cancerous specimens and PrEC cells (* p < 0.05; ** p < 0.01). But compared to PrEC, mRNA expression of E2F2 and CCND2 are down-regulated after transfected with let-7a in PC3 and LNCaP cells (* p < 0.05; ** p < 0.01). Western blotting results showed no change of CDK4 protein expression between prostate cancer tissues and their adjacent non-tumorous tissues or between PC3-let-7a-GFP cells and PC3-GFP cells, the expression levels of E2F2, CCND2 increased in prostate cancer tissues compared with their adjacent non-tumorous tissues, but the expression levels of E2F2, CCND2, and k-ras decreased dramatically in PC3-let-7a-GFP cells compared with that in PC3-GFP cells. k-ras, a previously-defined molecular target of let-7a was used as a positive control for these experiments. These data support that let-7a down-regulating the mRNA expression of E2F2 and CCND2 and repress protein translation of them.5. Let-7a inhibits tumor growth in nude mice xenograft model Nude mice bearing PC3-let-7a-GFP or PC3-GFP xenografts were sacrificed 4 weeks after innoculation. Western blotting showed that expression levels of E2F2 and CCND2 decreased dramatically in PC3-let-7a-GFP tumors compared with PC3-GFP tumors. The weight of PC3-let-7a-GFP tumors was ~80% lighter than PC3-GFP tumors (p < 0.01). Furthermore, the ratio of tumor weight/body weight in mice bearing PC3-let-7a-GFP tumors was only ~6% of the ratio from mice bearing PC3-GFP tumors (p < 0.01), which provides strong evidence that let-7a can inhibit tumor growth in vivo.Conclusion1. Real-time RT-PCR demonstrated that decreased levels of let-7a are present in resected prostate cancer samples and prostate cancer cell lines.2. We successfully constructed the expressing plasmid pcDNA3-let-7a-GFP, which containing let-7a.3. Cellular proliferation was inhibited in PC3 cells and LNCaP cells after transfection with let-7a. Cell cycle analysis showed that let-7a induced cell cycle arrest at the G1/S phase.4. A dual-luciferase reporter assay demonstrated that the 3'UTR of E2F2 and CCND2 were directly bound to let-7a and western blotting analysis further indicated that let-7a down-regulated the expression of E2F2 and CCND2.5. Our xenograft models of prostate cancer confirmed the capability of let-7a to inhibit prostate tumor development in vivo.