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. 2017 Apr 18;8(16):26424-26433.
doi: 10.18632/oncotarget.15477.

microRNA-135b Expression Silences Ppm1e to Provoke AMPK Activation and Inhibit Osteoblastoma Cell Proliferation

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Free PMC article

microRNA-135b Expression Silences Ppm1e to Provoke AMPK Activation and Inhibit Osteoblastoma Cell Proliferation

Zheng-Wei Li et al. Oncotarget. .
Free PMC article

Abstract

Forced-activation of AMP-activated protein kinase (AMPK) can possibly inhibit osteoblastoma cells. Here, we aim to provoke AMPK activation via microRNA silencing its phosphatase Ppm1e (protein phosphatase Mg2+/Mn2+-dependent 1e). We showed that microRNA-135b-5p ("miR-135b-5p"), the anti-Ppm1e microRNA, was significantly downregulated in human osteoblastoma tissues. It was correlated with Ppm1e upregulation and AMPKα1 de-phosphorylation. Forced-expression of miR-135b-5p in human osteoblastoma cells (MG-63 and U2OS lines) silenced Ppm1e, and induced a profound AMPKα1 phosphorylation (at Thr-172). Osteoblastoma cell proliferation was inhibited after miR-135b-5p expression. Intriguingly, Ppm1e shRNA knockdown similarly induced AMPKα1 phosphorylation, causing osteoblastoma cell proliferation. Reversely, AMPKα1 shRNA knockdown or dominant negative mutation almost abolished miR-135b-5p's actions in osteoblastoma cells. Further in vivo studies demonstrated that U2OS tumor growth in mice was dramatically inhibited after expressing miR-135b-5p or Ppm1e shRNA. Together, our results suggest that miR-135b-induced Ppm1e silence induces AMPK activation to inhibit osteoblastoma cell proliferation.

Keywords: AMPK; Ppm1e; cell proliferation; microRNA-135b; osteoblastoma.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflicts of interests.

Figures

Figure 1
Figure 1. miR-135b-5p upregulation correlates with Ppm1e upregulation and AMPKα1 de-phosphorylation in human osteoblastoma tissues
Expressions of miR-135b-5p (A, qRT-PCR assay), Ppm1e mRNA (B, qRT-PCR assay) and listed proteins (C–D, Western blotting assay) in ten (10) different human osteoblastoma tissues (“Tumor”) and surrounding normal bone tissues (“Normal”) were tested. Ppm1e protein expression (vs. Erk1/2) and AMPKα1 phosphorylation (vs. total AMPKα1) were quantified (CD). *p < 0.05 vs. “Normal” group.
Figure 2
Figure 2. Forced-expression of miR-135b-5p silences Ppm1e, causing AMPK activation in human osteoblastoma cells
Stable MG-63 cells (AC) or U2OS cells (DF), expressing miR-135b vector (“Vec-miR-135b”, three lines each, “L1/L2/L3”), microRNA control (“mi-C”) vector or the empty vector (“Vec”, pSuper-puro), were subjected to qRT-PCR assay to test expression of miR-135b-5p (A and D) and Ppm1e mRNA (B and E); Listed proteins in above cells were tested by Western blot assay, Ppm1e protein expression (vs. Tubulin) and AMPKα1 phosphorylation (vs. total AMPKα1) were quantified (C and F). For each assay, n = 5. *p < 0.05 vs. group “mi-C”. Experiments in this figure were repeated three times, and similar results were obtained.
Figure 3
Figure 3. Forced-expression of miR-135b-5p inhibits osteoblastoma cell proliferation
Stable MG-63 cells (AC) or U2OS cells (DF), expressing miR-135b vector (“Vec-miR-135b”, three lines each, “L1/L2/L3”), microRNA control (“mi-C”) vector or the empty vector (“Vec”, pSuper-puro), were subjected to following cell proliferation assays: CCK-8 assay (A and D), colony formation assay (B and E) and BrdU incorporation assay (C and F). Notably, exact same number of viable cells of different background were plated initially for these proliferation assays (Same for all Figures). For each assay, n = 5. *p < 0.05 vs. group “mi-C”. Experiments in this figure were repeated three times, and similar results were obtained.
Figure 4
Figure 4. Ppm1e shRNA knockdown activates AMPK and inhibits osteoblastoma cell proliferation
Stable MG-63 cells with listed Ppm1e shRNA (“shPpm1e-1” or “shPpm1e-1”) or scramble shRNA control (“sh-C”), as well as the parental control MG-63 cells (“Ctrl”) were subjected to qRT-PCR assay to test expression of Ppm1e mRNA (A) and miR-135b-5p (C); Listed proteins in above cells were also tested, and blot data were quantified (B). Proliferation of above cells was tested by the CCK-8 assay (D) and colony formation assay (E). MG-63 cells with listed Ppm1e shRNA (“shPpm1e-1” or “shPpm1e-1”) were also transfected with miR-135b vector (“Vec-miR-135b”), expression of miR-135b-5p was tested afterwards (F); Cell proliferation was examined by CCK-8 assay (G). Ppm1e protein expression (vs. Tubulin) and AMPKα1 phosphorylation (vs. total AMPKα1) were quantified (B). Notably, exact same number of viable cells of different background were plated initially for these proliferation assays. For each assay, n = 5. *p < 0.05 vs. group “sh-C”/“mi-C”. Experiments in this figure were repeated three times, and similar results were obtained.
Figure 5
Figure 5. AMPKα1 shRNA knockdown or mutation abolishes miR-135b-5p's actions against osteoblastoma cells
Stable MG-63 cells with miR-135b vector (“Vec-miR-135b”, “L1”) or the empty vector (“Vec”, pSuper-puro) were further constructed with AMPKα1 shRNA (“shAMPKα1”), scramble control shRNA (“sh-C”) or a dominant negative mutation of AMPKα1 (T172A, Flag-tagged, ““dnAMPKα1”), expressions of listed proteins were shown (A); miR-135b-5p mRNA expression was tested by qRT-PCR assay (B); Cell proliferation was tested by the CCK-8 assay (C) and BrdU ELISA assay (D). Ppm1e protein expression (vs. Erk1/2) and AMPKα1 phosphorylation (vs. Tubulin) were quantified (A). For each assay, n = 5. *p < 0.05 vs. group “Vec” only. #p < 0.05 vs. group “sh-C”. Experiments in this figure were repeated three times, and similar results were obtained.
Figure 6
Figure 6. U2OS tumor growth in SCID mice is inhibited after expressing Ppm1e shRNA or miR-135b-5p
Stable U2OS cells, with Ppm1e shRNA (“-1”), Vec-miR-135b (“L1”) or empty vector (“Vec”), were inoculated into the SCID mice via s.c. injection. When the tumors were around 100 mm3 in volume, the recording were started. Tumor volume (A) and mice body weight (D), which subtracting estimated tumor weight) were recorded every week for a total of 6 weeks (Week1 to Week7); Estimated daily tumor growth, in mm3 per day, was also calculated (B); At week-7, tumors of each group were isolated and weighted (C); Expressions of Ppm1e mRNA (E), miR-135b-5 (F) in tumor tissue lysates were also tested. For each group, n = 10. *p < 0.05 vs. “Vec” control tumors.

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