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Hypoxia-Responsive Mir-301a and Mir-301b Promote Radioresistance of Prostate Cancer Cells via Downregulating NDRG2.

Wang W, Liu M, Guan Y, Wu Q - Med. Sci. Monit. (2016)

Bottom Line: Higher level of miR-301a and miR-301b expression results in elevated autophagy and increased radioresistance in LNCaP cells.MiR-301a and miR-301b simultaneously target NDRG2 and decrease its expression.Knockdown of NDRG2 leads to increased autophagy and radioresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Henan Provincial People's Hospital, Zhengzhou, Henan, China (mainland).

ABSTRACT
BACKGROUND MiR-301a and miR-301b are 2 oncomiRs involved in multiple types of cancer. In this study, we explored the expression change of miR-301a and miR-301b in prostate cancer cells in hypoxia and studied their regulation of autophagy and radiosensitivity of prostate cancer cells. MATERIAL AND METHODS QRT-PCR was performed to quantify the expression change of miR-301a and miR-301b in hypoxia. Their effects on autophagy were measured by Western blot analysis, and their effects on radiosensitivity were measured by clonogenic assay and flow cytometry. In addition, the regulation of miR-301a and miR-301b on NDRG2, a tumor-suppressor gene in prostate cancer, was also studied. The effect of miR-301a/b-NDRG2 axis on autophagy and radiosensitivity of prostate cancer cells was further investigated. RESULTS MiR-301a and miR-301b are 2 hypoxia responsive miRNAs that are significantly upregulated in hypoxia in prostate cancer cells. Higher level of miR-301a and miR-301b expression results in elevated autophagy and increased radioresistance in LNCaP cells. MiR-301a and miR-301b simultaneously target NDRG2 and decrease its expression. Knockdown of NDRG2 leads to increased autophagy and radioresistance. CONCLUSIONS MiR-301a and miR-301b are 2 hypoxia-responsive miRNAs that decrease autophagy of prostate cancer cells in hypoxia by targeting NDRG2. Through downregulating NDRG2, miR-301a and miR-301b can promote radioresistance of prostate cancer cells.

No MeSH data available.


Related in: MedlinePlus

MiR-301a and miR-301b simultaneously targets NDRG2 and decrease its expression. (A) The predicted binding sequence between miR-301a, miR301b and 3′UTR of NDRG2. (B–D) LNCaP cells were transfected with 100 nM miR-301a mimics, miR-301b mimics, or NDRG2 siRNA. QRT-PCR analysis was performed to detect miR-301a, miR-301b (B), and NDRG2 mRNA (C) expression. NDRG2 protein expression (D) was measured by Western blot. (D, E) LNCaP cells were co-transfected with 100-nM miR-301a mimics (D) or miR-301b mimics (E) and pmirGLO-NDRG2-WT or pmirGLO-NDRG2-MT. The relative luciferase activity was measured 24 h after transfection. * p<0.05, ** p<0.01, *** p<0.001.
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f2-medscimonit-22-2126: MiR-301a and miR-301b simultaneously targets NDRG2 and decrease its expression. (A) The predicted binding sequence between miR-301a, miR301b and 3′UTR of NDRG2. (B–D) LNCaP cells were transfected with 100 nM miR-301a mimics, miR-301b mimics, or NDRG2 siRNA. QRT-PCR analysis was performed to detect miR-301a, miR-301b (B), and NDRG2 mRNA (C) expression. NDRG2 protein expression (D) was measured by Western blot. (D, E) LNCaP cells were co-transfected with 100-nM miR-301a mimics (D) or miR-301b mimics (E) and pmirGLO-NDRG2-WT or pmirGLO-NDRG2-MT. The relative luciferase activity was measured 24 h after transfection. * p<0.05, ** p<0.01, *** p<0.001.

Mentions: By performing target prediction using online databases, we observed that the 3′UTR of NDRG2 has a sequence targeted by both miR-301a and miR-301b (Figure 2A). Previous studies confirmed that NDRG2 is a tumor-suppressor gene in prostate cancer [13,14]. LNCaP cells were first transfected with miR-301a or miR-301b mimics for overexpression (Figure 2B). Both miR-301a and miR-301b overexpression could substantially inhibit NDRG2 expression, and the effect was similar to that of NDRG2 siRNA (Figure 2C). To further verify the direct binding between miR-301a/miR-301b and 3′UTR of NDRG2, we produced dual luciferase reporter plasmids inserted with wild-type or mutant predicted binding site. Both miR-301a and miR-301b substantially suppressed the relative luciferase activity of wild-type reporter plasmids, but had little effect on the mutant reporter plasmids (Figure 2D, 2E).


Hypoxia-Responsive Mir-301a and Mir-301b Promote Radioresistance of Prostate Cancer Cells via Downregulating NDRG2.

Wang W, Liu M, Guan Y, Wu Q - Med. Sci. Monit. (2016)

MiR-301a and miR-301b simultaneously targets NDRG2 and decrease its expression. (A) The predicted binding sequence between miR-301a, miR301b and 3′UTR of NDRG2. (B–D) LNCaP cells were transfected with 100 nM miR-301a mimics, miR-301b mimics, or NDRG2 siRNA. QRT-PCR analysis was performed to detect miR-301a, miR-301b (B), and NDRG2 mRNA (C) expression. NDRG2 protein expression (D) was measured by Western blot. (D, E) LNCaP cells were co-transfected with 100-nM miR-301a mimics (D) or miR-301b mimics (E) and pmirGLO-NDRG2-WT or pmirGLO-NDRG2-MT. The relative luciferase activity was measured 24 h after transfection. * p<0.05, ** p<0.01, *** p<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920099&req=5

f2-medscimonit-22-2126: MiR-301a and miR-301b simultaneously targets NDRG2 and decrease its expression. (A) The predicted binding sequence between miR-301a, miR301b and 3′UTR of NDRG2. (B–D) LNCaP cells were transfected with 100 nM miR-301a mimics, miR-301b mimics, or NDRG2 siRNA. QRT-PCR analysis was performed to detect miR-301a, miR-301b (B), and NDRG2 mRNA (C) expression. NDRG2 protein expression (D) was measured by Western blot. (D, E) LNCaP cells were co-transfected with 100-nM miR-301a mimics (D) or miR-301b mimics (E) and pmirGLO-NDRG2-WT or pmirGLO-NDRG2-MT. The relative luciferase activity was measured 24 h after transfection. * p<0.05, ** p<0.01, *** p<0.001.
Mentions: By performing target prediction using online databases, we observed that the 3′UTR of NDRG2 has a sequence targeted by both miR-301a and miR-301b (Figure 2A). Previous studies confirmed that NDRG2 is a tumor-suppressor gene in prostate cancer [13,14]. LNCaP cells were first transfected with miR-301a or miR-301b mimics for overexpression (Figure 2B). Both miR-301a and miR-301b overexpression could substantially inhibit NDRG2 expression, and the effect was similar to that of NDRG2 siRNA (Figure 2C). To further verify the direct binding between miR-301a/miR-301b and 3′UTR of NDRG2, we produced dual luciferase reporter plasmids inserted with wild-type or mutant predicted binding site. Both miR-301a and miR-301b substantially suppressed the relative luciferase activity of wild-type reporter plasmids, but had little effect on the mutant reporter plasmids (Figure 2D, 2E).

Bottom Line: Higher level of miR-301a and miR-301b expression results in elevated autophagy and increased radioresistance in LNCaP cells.MiR-301a and miR-301b simultaneously target NDRG2 and decrease its expression.Knockdown of NDRG2 leads to increased autophagy and radioresistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Henan Provincial People's Hospital, Zhengzhou, Henan, China (mainland).

ABSTRACT
BACKGROUND MiR-301a and miR-301b are 2 oncomiRs involved in multiple types of cancer. In this study, we explored the expression change of miR-301a and miR-301b in prostate cancer cells in hypoxia and studied their regulation of autophagy and radiosensitivity of prostate cancer cells. MATERIAL AND METHODS QRT-PCR was performed to quantify the expression change of miR-301a and miR-301b in hypoxia. Their effects on autophagy were measured by Western blot analysis, and their effects on radiosensitivity were measured by clonogenic assay and flow cytometry. In addition, the regulation of miR-301a and miR-301b on NDRG2, a tumor-suppressor gene in prostate cancer, was also studied. The effect of miR-301a/b-NDRG2 axis on autophagy and radiosensitivity of prostate cancer cells was further investigated. RESULTS MiR-301a and miR-301b are 2 hypoxia responsive miRNAs that are significantly upregulated in hypoxia in prostate cancer cells. Higher level of miR-301a and miR-301b expression results in elevated autophagy and increased radioresistance in LNCaP cells. MiR-301a and miR-301b simultaneously target NDRG2 and decrease its expression. Knockdown of NDRG2 leads to increased autophagy and radioresistance. CONCLUSIONS MiR-301a and miR-301b are 2 hypoxia-responsive miRNAs that decrease autophagy of prostate cancer cells in hypoxia by targeting NDRG2. Through downregulating NDRG2, miR-301a and miR-301b can promote radioresistance of prostate cancer cells.

No MeSH data available.


Related in: MedlinePlus