microRNA-135b expression silences Ppm1e to provoke AMPK activation and inhibit osteoblastoma cell proliferation

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.


INTRODUCTION
Osteoblastoma is one of the major causes of cancerrelated mortalities among children and young teenagers around the World [1][2][3][4]. Remarkable improvements have been achieved in the diagnosis and clinical treatment for osteoblastoma, and the five-year overall survival has been increased to over 70% [1][2][3][4]. However, for those with advanced, malignant and/or recurrent osteoblastoma, the prognosis is still very poor [1][2][3][4]. Therefore, our group has been dedicated to understanding the pathologic mechanisms of osteoblastoma tumorigenesis and progression, and developing novel anti-osteoblastoma agents [5,6].

U2OS tumor growth in SCID mice is inhibited after expressing Ppm1e shRNA or miR-135b-5p
The potential effect of miR-135b-5p on osteoblastoma cell growth in vivo was tested. U2OS cells, stably expressing Ppm1e shRNA ("-1", see Figure 4), Vec-miR-135b ("L1", see Figure 2) or empty vector ("Vec", see Figure 2), were inoculated into the severe combined immunodeficient (SCID) mice via s.c. injection. Tumor recordings were started when the tumors were around 100 mm 3 in volume for each group. Tumor growth curve results in Figure 6A demonstrated that growth of U2OS tumors was largely inhibited after expressing Ppm1e shRNA or Vec-miR-135b. Estimated daily tumor growth (in mm 3 per day) was also significantly lower in Ppm1e shRNA-or Vec-miR-135b-expressing tumors ( Figure 6B). At the end of the experiments (Week-7), the weight of tumors with Ppm1e shRNA or Vec-miR-135b was also much lower than "Vec" control tumors ( Figure 6C). The mice body weight was indifferent between the three groups ( Figure 6D). At Week-7, all xenografted tumors were isolated and lysed. Expressions of miR-135b-5p and Ppm1e were examined. As demonstrated, Ppm1e mRNA was indeed depleted in Ppm1e shRNA-or Vec-
Intriguingly, we here proposed that Ppm1e is the primary target of miR-135b in mediating its inhibition against osteoblastoma cells. shRNA-mediated knockdown of Ppm1e mimicked miR-135b-5p's actions, and similarly induced AMPK activation and inhibited osteoblastoma cell proliferation. Remarkably, forced-expression of miR-135b-5p failed to further inhibit the proliferation of Ppm1e-silneced cells. Therefore, miR-135b-5p expression was in-effective when Ppm1e was depleted, suggesting that Ppm1e is the direct target of this Anti-
oncomir. Notably, we showed that Ppm1e expression was significantly elevated in osteoblastoma tissues. On the other hand, shRNA-mediated Ppm1e knockdown potently inhibited osteoblastoma cell growth in vivo and in vivo. These results indicate that Ppm1e could be a novel oncotarget protein of osteoblastoma.

Chemicals and reagents
The Ppm1e antibody was provided by Dr. Cui's group at Nantong University [20]. All other antibodies were obtained from Cell Signaling Tech (Denver MA). The enhanced chemiluminescence (ECL) reagents were obtained from Pierce (Rockford, IL). The cell culture reagents were provided by Gibco Co. (Shanghai, China).

Human tissue specimens
As described previously [5,6], the surgery-isolated osteoblastoma tissues and surrounding normal bone tissues were separated carefully. Tissues were washed thoroughly and minced into small pieces. Tissues were then mechanically dissociated and lysed by the descried tissue lysis buffer [5,6]. Tissue lysates were stored in liquid nitrogen. This study was conducted according to the principles expressed in the Declaration of Helsinki. The protocol was approved by Internal Review Board and Ethic Review Board of Soochow University. Writteninformed consent was obtained from each participant.

CCK-8 cell proliferation assay
Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) assay was employed to test cell proliferation according to the attached instructions. The detailed protocol was described in our previous study [44].

Other cell proliferation assays
Clonogenicity assay of cell growth and BrdU ELISA assay of cell proliferation were described in detail in our previous studies [44,45].

Western blotting assay
Cell and tissue lysates (40 μg/sample) were separated by 10-12% SDS-PAGE gel, and were transferred onto PVDF membrane (Millipore, USA). Afterwards, the membrane was blocked, followed by incubation with specific primary and corresponding secondary antibodies. The detection was performed via ECL Supersingnal West Pico Chemiluminescent. Indicated band was quantified via ImageJ software.

Quantitative real-time PCR assay
The protocol of quantitative real-time reverse transcriptase polymerase chain reaction ("qRT-PCR") assay was described in detail in our previous studies [6,44,45]. We utilized the comparative Ct (2 −ΔΔCt ) method to quantify target mRNA expression [46]. GAPDH was tested as the reference gene [6]. mRNA primers for GAPDH and Ppm1e were provided by Dr. Cui [20]. The expression of mature miR-135b was tested by the TaqMan microRNA assay as described [47]. Ten ng of total RNA per sample was reverse-transcribed via the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystem, Shanghai, China) [47]. The miR-135b-5p primers were described early [48,49].

shRNA knock and stable cell selection
The two distinct lentiviral Ppm1e shRNAs were provided again by Dr. Cui [20]. The scramble control shRNA (sc-108065) and AMPKα1 shRNA (sc-45312-V) were purchased from Santa Cruz Biotech (Shanghai, China). Osteoblastoma cells were seeded onto six-well plates with 50-60% confluence. Ten μL/mL of lentiviral shRNA was added directly to cultured cells for 24 hours. Afterwards, puromycin (1 μg/mL, Sigma) was added to the culture medium for another 48 hours. The knockdown of targeted protein (Ppm1e or AMPKα1 ) was verified by Western blotting assay and/or qRT-PCR assay.

Mice U2OS xenograft assay
As described [5,50,51], CB.17 severe combined immuno-deficient (SCID) male mice (18-20 g) were purchased from the Animal Facility of Soochow University (Suzhou, China). Three million of U2OS cells (per mouse), with Ppm1e shRNA or "Vec-miR-135b", were inoculated subcutaneously (s.c.) into the flanks of the mice. When the xenografts were about 100 mm 3 in volume, recordings were starte as described [50][51][52]. The protocols were in accordance with the Institutional Animal Care and Use Committee (IACUC), and were approved by the Ethics Committee and Internal Review Board (IRB) of Soochow University.

Statistical analysis
The quantitative data presented in this study was mean ± standard deviation (SD). Statistical differences were analyzed by one-way ANOVA with post hoc Bonferroni test.

CONCLUSIONS
We conclude that miR-135b silences Ppm1e to provoke AMPK activation and inhibit osteoblastoma cell proliferation.