MicroRNA-125a-5p regulates cancer cell proliferation and migration through NAIF1 in prostate carcinoma

Abstract

Background: We investigated the functional roles of microRNA-125a-5p in regulating human prostate carcinoma.

Methods: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to evaluate the gene expression levels of miR-125a-5p in eight prostate cancer cell lines and nine biopsy specimens from patients with prostate cancer. miR-125a-5p was genetically knocked down in prostate cancer cell lines, DU145 and VCaP cells by lentiviral transduction. The effects of miR-125a-5p downregulation on prostate cancer cell proliferation and migration were evaluated by MTT assay and transwell assay, respectively. Direct regulation of miR-125a-5p on its downstream targets, NAIF1, and apoptotic gene caspase-3 were evaluated through dual-luciferase reporter assay, qRT-PCR, and Western blot, respectively. NAIF1 was then ectopically overexpressed in DU145 and VCaP cells to modulate prostate cancer cell proliferation and migration. Finally, the effects of miR-125a-5p downregulation or NAIF1 overexpression on the growth of in vivo prostate cancer xenograft were evaluated.

Results: miR-125a-5p was upregulated in prostate cancer cell lines and human prostate carcinomas. Lentivirus induced miR-125a-5p downregulation in DU145 and VCaP cells inhibited prostate cancer cell proliferation or migration. NAIF1 was the direct target of miR-125a-5p, as both gene and protein expression levels of NAIF1, as well as caspase-3 were upregulated by miR-125a-5p. Forced overexpression of NAIF1 had similar antitumor effects as miR-125a-5p downregulation on prostate cancer cell proliferation and migration. In vivo prostate xenograft assay confirmed the tumor-suppressive effect of miR-125a-5p downregulation or NAIF1 overexpression.

Conclusion: miR-125a-5p regulates prostate cancer cell proliferation and migration through NAIF1.

Keywords: NAIF1; caspase-3; miR-125a-5p; migration; proliferation.

Figure 1

Expression of miR-125a-5p in prostate cancer cell lines and clinical specimens. Notes: (A) qRT-PCR was used to evaluate the expression levels of miR-125a-5p in AR-negative prostate cancer cell lines, DU145, PC-3, NCI-H660 cells and AR-positive prostate cancer cell lines, 22RV-1, LNCaP, VCaP cells. The control cell line is a nontumorigenic human prostate epithelial cell line RWPE-1. (*P<0.05 vs RWPE-1). (B) qRT-PCR was used to compare the expression levels of miR-125a-5p between paired T and ANT specimens in nine patients with prostate cancer (*P<0.05). Abbreviations: qRT-PCR, quantitative reverse transcription-polymerase chain reaction; AR, androgen receptor; T, carcinoma tissues; ANT, adjacent noncarcinoma tissues.

Figure 2

Effect of miR-125a-5p downregulation on prostate cancer cell proliferation and migration. Notes: DU145 and VCaP cells were transduced with miR-125a-5p inhibitor lentivirus, 125a-5p-inhibitor, or C-miRNA. After lentiviral transduction, qRT-PCR was used to evaluate the knockdown efficiency of lentiviral transduction in DU145 (A) and VCaP (B) cells (*P<0.05). Cancer cell proliferation in DU145 (C) and VCaP (D) cells was evaluated by MTT assay (*P<0.05). Cancer migration in DU145 (E) and VCaP (F) cells was evaluated by a transwell assay and H&E staining (*P<0.05). Abbreviations: C-miRNA, control-microRNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; H&E, hematoxylin and eosin.

Figure 3

Effect of miR-125a-5p on apoptotic pathway in prostate cancer. Notes: (A) Diagram shows the binding of miR-125a-5p on WT NAIF1 3′-UTR. An MT NAIF1 3′-UTR with modified miR-125a-5p binding site was also demonstrated. (B) HEK293T cells were cotransfected with miR-125a-5p and firefly-WT-NAIF1, or firefly-MT-NAIF1, or Renilla luciferase vector (Renilla) for 48 hours. A dual-luciferase reporter assay was then used to evaluate the binding between miR-125a-5p and NAIF1 (*P<0.05, ^∆P>0.05). DU145 and VCaP cells were transduced with 125a-5p-inhibitor or C-miRNA. qRT-PCR was used to evaluate the gene expression levels of NAIF1 and caspase-3 in DU145 (C) and VCaP (D) cells (*P<0.05 vs C-miRNA). Western blot was used to evaluate the protein expressions of NAIF1 and caspase-3 in DU145 (E) and VCaP (F) cells. Abbreviations: WT, wild type; 3′-UTR, 3′-untranslated regions; MT, mutant; firefly-WT-NAIF1, WT NAIF1 firefly luciferase vector; firefly-MT-NAIF1, MT NAIF1 firefly luciferase vector; C-miRNA, control-microRNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

Figure 4

Effect of NAIF1 upregulation on prostate cancer cell proliferation and migration. Notes: (A, B) DU145 and VCaP cells were transfected with NAIF1 overexpression vector, pcDNA3.1-NAIF1 or an empty overexpression vector, pcDNA3.1-C. After transfection, qRT-PCR was used to evaluate the gene expression levels of NAIF1 and caspase-3 in DU145 (A) and VCaP (B) cells (*P<0.05). Cancer cell proliferation in DU145 (C) and VCaP (D) cells was evaluated by MTT assay (*P<0.05). Cancer migration in DU145 (E) and VCaP (F) cells was evaluated by a transwell assay and H&E staining (*P<0.05). Abbreviations: qRT-PCR, quantitative reverse transcription-polymerase chain reaction; H&E, hematoxylin and eosin.

Figure 5

Effect of miR-125a-5p downregulation or NAIF1 overexpression on prostate cancer in vivo xenograft. Notes: (A) DU145 cells were transduced with miR-125a-5p inhibitor lentivirus, 125a-5p-inhibitor or C-miRNA lentivirus. One million healthy DU145 cells were then inoculated into 6-week-old female null mice. The growth curves were plotted to monitor tumor volumes for 5 weeks (*P<0.05). (B) After 5-week in vivo growth, prostate cancer xenografts were extracted. Ki-67 immunostaining was then performed. (C, D) DU145 cells were transfected with NAIF1 overexpression vector, pcDNA3.1-NAIF1 or an empty overexpression vector, pcDNA3.1-C, followed by in vivo xenograft assay. The growth curves were also monitored for 5 weeks (C, *P<0.05); and Ki-67 staining was performed after that (D). Abbreviation: C-miRNA, control-microRNA.