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MiR-125b inhibits stromal cell proliferation in giant cell tumor of bone by targeting parathyroid hormone 1 receptor.

Wu PF, Liang JY, Yu F, Zhou ZB, Tang JY, Li KH - Iran J Basic Med Sci (2015)

Bottom Line: We found that miR-125b was significantly down-regulated in GCT tissues.Our results suggest that miR-125b acts as a tumor suppressor through suppression of the PTH1R/RANKL signaling pathway.These findings contribute to our understanding of the functions of miR-125b in GCT.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedics, Xiang Ya Hospital, Central South University, Changsha 410008, China.

ABSTRACT

Objectives: miR-125b has been identified as a tumor suppressor in many tumors, but its role in giant cell tumor (GCT) of bone remains poorly understood. The current study aimed to investigate the potential role and mechanism of miR-125b in GCT.

Materials and methods: Expression levels of miR-125b in GCT tissues were determined using RT-PCR. The cell proliferation was surveyed by direct cell counting, MTS and CCK-8, and the apoptotic cells were evaluated by Annexin V-FITC and propidium iodine staining assay. The target gene expression was determined using RT-PCR and western blot. Parathyroid hormone 1 receptor (PTH1R) 3'-UTR was cloned into luciferase reporter plasmid to confirm direct targeting.

Results: We found that miR-125b was significantly down-regulated in GCT tissues. Using both gain- and loss-of-function analyses, we further revealed that miR-125b suppressed GCT stromal cell proliferation and induced cell apoptosis. Furthermore, we revealed that PTH/PTHrP type 1 receptor is a direct and functional target of miR-125b.

Conclusion: Our results suggest that miR-125b acts as a tumor suppressor through suppression of the PTH1R/RANKL signaling pathway. These findings contribute to our understanding of the functions of miR-125b in GCT.

No MeSH data available.


Related in: MedlinePlus

PTH1R is a direct target of miR-125b. (A) PTH1R 3’UTR contains one predicted miR-125b binding site. The mutagenic nucleotides are indicated in grey. (B) Dual luciferase reporter assay. HEK293 cells were transfected with wild type 3’UTR-reporter or mutant (Mut) constructs together with miR-125b mimics or negative controls (NC). Relative firefly luciferase expression was normalized to Renilla luciferase. (C, D) The expression levels of PTH1R in GCTSCs transfected with NC, miR-125b or anti-miR-125b lentivirus by qRT-PCR and western blot. (E) qRT-PCR to measure RANKL and IL-8 expression in GCTSCs transfected with NC, miR-125b or anti-miR-125b lentivirus. (F, G and H) PTH1R rescues the suppressive roles of miR-125b in GCTSC proliferation. GCTSCs miR-125b or NC were transfect with or without PTH1R plasmids. Cell proliferation analysis was performed by cell counting (F), MTS assay (G) and CCK-8 assay (H). Data are presented as mean±sd. *P<0.05; ** P<0.01
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Figure 3: PTH1R is a direct target of miR-125b. (A) PTH1R 3’UTR contains one predicted miR-125b binding site. The mutagenic nucleotides are indicated in grey. (B) Dual luciferase reporter assay. HEK293 cells were transfected with wild type 3’UTR-reporter or mutant (Mut) constructs together with miR-125b mimics or negative controls (NC). Relative firefly luciferase expression was normalized to Renilla luciferase. (C, D) The expression levels of PTH1R in GCTSCs transfected with NC, miR-125b or anti-miR-125b lentivirus by qRT-PCR and western blot. (E) qRT-PCR to measure RANKL and IL-8 expression in GCTSCs transfected with NC, miR-125b or anti-miR-125b lentivirus. (F, G and H) PTH1R rescues the suppressive roles of miR-125b in GCTSC proliferation. GCTSCs miR-125b or NC were transfect with or without PTH1R plasmids. Cell proliferation analysis was performed by cell counting (F), MTS assay (G) and CCK-8 assay (H). Data are presented as mean±sd. *P<0.05; ** P<0.01

Mentions: Using in silicon prediction programs, PTH1R was identified as a potential target for miR-125b. PTH1R was selected out for its role in osteoclastogenesis and pathogenesis of bone tumor (17, 18). To verify whether PTH1R is a direct target of miR-125b, we cloned the wild-type 3’UTR or the mutant (lacking the 7-bp seed sequence) into a luciferase reporter vector. When we cotransfected HEK293 cells with the cloned UTR and miR-125b mimics, we observed that miR-125b overexprssion caused a consistent reduction in luciferase activity for wild type 3’UTR-reporter. Conversely, cotransfection of miR-125b mimics with the mutated form of 3’UTR-reporter resulted in no significant change in luciferase activity (Figure 3A, B), suggesting that miR-125b directly targeted the PTH1R 3’-UTR. In agreement, miR-125b overexpression significantly reduced both mRNA and protein expression for PTH1R in GCTSC cells. Conversely, miR-125b inhibitors transfection increased its mRNA and protein levels (Figure 3C, D). Overexpression of miR-125b also inhibited PTH1R downstream targets such as RANKL and IL-8 expression, whereas knockdown of this miRNA increased RANKL and IL-8 expression (Figure 3E), further indicating that PTH1R is a target of miR-125b in GCT cells.


MiR-125b inhibits stromal cell proliferation in giant cell tumor of bone by targeting parathyroid hormone 1 receptor.

Wu PF, Liang JY, Yu F, Zhou ZB, Tang JY, Li KH - Iran J Basic Med Sci (2015)

PTH1R is a direct target of miR-125b. (A) PTH1R 3’UTR contains one predicted miR-125b binding site. The mutagenic nucleotides are indicated in grey. (B) Dual luciferase reporter assay. HEK293 cells were transfected with wild type 3’UTR-reporter or mutant (Mut) constructs together with miR-125b mimics or negative controls (NC). Relative firefly luciferase expression was normalized to Renilla luciferase. (C, D) The expression levels of PTH1R in GCTSCs transfected with NC, miR-125b or anti-miR-125b lentivirus by qRT-PCR and western blot. (E) qRT-PCR to measure RANKL and IL-8 expression in GCTSCs transfected with NC, miR-125b or anti-miR-125b lentivirus. (F, G and H) PTH1R rescues the suppressive roles of miR-125b in GCTSC proliferation. GCTSCs miR-125b or NC were transfect with or without PTH1R plasmids. Cell proliferation analysis was performed by cell counting (F), MTS assay (G) and CCK-8 assay (H). Data are presented as mean±sd. *P<0.05; ** P<0.01
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Figure 3: PTH1R is a direct target of miR-125b. (A) PTH1R 3’UTR contains one predicted miR-125b binding site. The mutagenic nucleotides are indicated in grey. (B) Dual luciferase reporter assay. HEK293 cells were transfected with wild type 3’UTR-reporter or mutant (Mut) constructs together with miR-125b mimics or negative controls (NC). Relative firefly luciferase expression was normalized to Renilla luciferase. (C, D) The expression levels of PTH1R in GCTSCs transfected with NC, miR-125b or anti-miR-125b lentivirus by qRT-PCR and western blot. (E) qRT-PCR to measure RANKL and IL-8 expression in GCTSCs transfected with NC, miR-125b or anti-miR-125b lentivirus. (F, G and H) PTH1R rescues the suppressive roles of miR-125b in GCTSC proliferation. GCTSCs miR-125b or NC were transfect with or without PTH1R plasmids. Cell proliferation analysis was performed by cell counting (F), MTS assay (G) and CCK-8 assay (H). Data are presented as mean±sd. *P<0.05; ** P<0.01
Mentions: Using in silicon prediction programs, PTH1R was identified as a potential target for miR-125b. PTH1R was selected out for its role in osteoclastogenesis and pathogenesis of bone tumor (17, 18). To verify whether PTH1R is a direct target of miR-125b, we cloned the wild-type 3’UTR or the mutant (lacking the 7-bp seed sequence) into a luciferase reporter vector. When we cotransfected HEK293 cells with the cloned UTR and miR-125b mimics, we observed that miR-125b overexprssion caused a consistent reduction in luciferase activity for wild type 3’UTR-reporter. Conversely, cotransfection of miR-125b mimics with the mutated form of 3’UTR-reporter resulted in no significant change in luciferase activity (Figure 3A, B), suggesting that miR-125b directly targeted the PTH1R 3’-UTR. In agreement, miR-125b overexpression significantly reduced both mRNA and protein expression for PTH1R in GCTSC cells. Conversely, miR-125b inhibitors transfection increased its mRNA and protein levels (Figure 3C, D). Overexpression of miR-125b also inhibited PTH1R downstream targets such as RANKL and IL-8 expression, whereas knockdown of this miRNA increased RANKL and IL-8 expression (Figure 3E), further indicating that PTH1R is a target of miR-125b in GCT cells.

Bottom Line: We found that miR-125b was significantly down-regulated in GCT tissues.Our results suggest that miR-125b acts as a tumor suppressor through suppression of the PTH1R/RANKL signaling pathway.These findings contribute to our understanding of the functions of miR-125b in GCT.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedics, Xiang Ya Hospital, Central South University, Changsha 410008, China.

ABSTRACT

Objectives: miR-125b has been identified as a tumor suppressor in many tumors, but its role in giant cell tumor (GCT) of bone remains poorly understood. The current study aimed to investigate the potential role and mechanism of miR-125b in GCT.

Materials and methods: Expression levels of miR-125b in GCT tissues were determined using RT-PCR. The cell proliferation was surveyed by direct cell counting, MTS and CCK-8, and the apoptotic cells were evaluated by Annexin V-FITC and propidium iodine staining assay. The target gene expression was determined using RT-PCR and western blot. Parathyroid hormone 1 receptor (PTH1R) 3'-UTR was cloned into luciferase reporter plasmid to confirm direct targeting.

Results: We found that miR-125b was significantly down-regulated in GCT tissues. Using both gain- and loss-of-function analyses, we further revealed that miR-125b suppressed GCT stromal cell proliferation and induced cell apoptosis. Furthermore, we revealed that PTH/PTHrP type 1 receptor is a direct and functional target of miR-125b.

Conclusion: Our results suggest that miR-125b acts as a tumor suppressor through suppression of the PTH1R/RANKL signaling pathway. These findings contribute to our understanding of the functions of miR-125b in GCT.

No MeSH data available.


Related in: MedlinePlus