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mTOR signaling pathway in glioblastoma under the regulation of miR-137
Author’s Accepted Manuscript PTP4A3 is a target for inhibition of cell proliferatin, migration and invasion through Akt/mTOR signaling pathway in glioblastoma under the regulation of miR-137 Liling Wang, Jianxun Liu, Zhiqiang Zhong, Xuhai Gong, Wei Liu, Lei Shi, Xuesong Li www.elsevier.com/locate/brainres
PII: DOI: Reference:
S0006-8993(16)30450-4 http://dx.doi.org/10.1016/j.brainres.2016.06.026 BRES44977
To appear in: Brain Research Received date: 7 April 2016 Revised date: 14 June 2016 Accepted date: 16 June 2016 Cite this article as: Liling Wang, Jianxun Liu, Zhiqiang Zhong, Xuhai Gong, Wei Liu, Lei Shi and Xuesong Li, PTP4A3 is a target for inhibition of cell proliferatin, migration and invasion through Akt/mTOR signaling pathway in glioblastoma under the regulation of miR-137, Brain Research, http://dx.doi.org/10.1016/j.brainres.2016.06.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PTP4A3 is a target for inhibition of cell proliferatin, migration and invasion through Akt/mTOR signaling pathway in glioblastoma under the regulation of miR137 Liling Wanga, Jianxun Liua, Zhiqiang Zhonga, Xuhai Gonga, Wei Liub, Lei Shic, Xuesong Lia* a
Department of Neurology, Daqing Oilfield General Hospital, 9 Zhongkang Road,
Daqing, Heilongjiang 163000, China b
Department of General Surgery, Daqing Oilfield General Hospital, 9 Zhongkang Road,
Daqing, Heilongjiang 163000, China c
Department of Obstetrics and Gynecology, Daqing Oilfield General Hospital, 9
Zhongkang Road, Daqing, Heilongjiang 163000, China *
Corresponding author. Tel.: +86 13329391666; fax: +86 0459 5994114.
[email protected]
Abstract Glioblastoma multiforme (GBM) is one of the most common primary malignant adult brain tumors. It is characterized by aggressive progression and poor prognosis. There is significant need to understand the mechanism of GBM malignancy and develop improved therapeutic options for GBM patients. We systematically studied the function of PTP4A3 in the malignancy of GBM. We found that PTP4A3 was upregulated in GBM tissues and cells. Knockdown of PTP4A3 expression in GBM cells inhibited cell proliferation, migration, and invasion. PTP4A3 knockdown modulated the activity of the
Akt/mTOR signaling pathway by inducing de-phosphorylation of Akt and mTOR. We identified PTP4A3 as a direct target of miR-137. MiR-137 has been reported as a tumor suppressor in GBM development. In this study, overexpression of miR-137 in GBM cells also inhibited cell proliferation, migration, and invasion. Finally, restoration of PTP4A3 expression in miR-137 overexpressing cells partially reversed the inhibition of GBM cell malignancy, and the de-phosphorylation of Akt and mTOR. We identified that PTP4A3 regulated GBM via miR-137-mediated Akt/mTOR signaling pathway.
Keywords miR-137, PTP4A3, Akt, mTOR, glioblastoma multiforme
1. Introduction Glioblastoma multiforme (GBM) is one of the most common primary malignant adult brain tumors (Louis et al., 2007). GBM is characterized by its aggressiveness and poor prognosis, with a median survival of only about 15 months (Stupp et al., 2005). Currently, the standard treatment regimen for GBM includes surgery removal, chemotherapy and radiotherapy (Stupp et al., 2005). Despite recent advancement in the therapeutic regimen, the successful rate for GBM treatment remains low. This situation is mainly due to the development of drug resistance by cancer cells, and the intrinsic ability of the cancer cells to migrate and invade through normal brain tissue, making complete surgical removal of cancer tissue nearly impossible. Therefore, there is a significant need
to understand the mechanism of GBM malignancy and develop improved therapeutic options for GBM patients. Protein tyrosine phosphatases (PTP) play important roles in modulating the levels of tyrosine phosphorylation. Increasing recent evidence has revealed that deregulation of protein tyrosine phosphatase activity is involved in a variety of diseases, including cancer (Ostman et al., 2006). One of the PTPs, PTP4A3, is characterized by the unique prenylation motif in its C-terminal end. Non-prenylated PTP4A3 is inactive and is usually located in the nucleus, and prenylated PTP4A3 is active and can be found in the membranes and intracellular structures (Zeng et al., 2000). PTP4A3 is known for its role in promoting cancer metastasis (Al-Aidaroos and Zeng, 2010). PTP4A3 upregulation was found in metastatic specimens from multiples cancer types (Polato et al., 2005; Radke et al., 2006; Saha et al., 2001; Zhou et al., 2009) including colorectal cancer (Saha et al., 2001), bladder cancer (Yeh et al., 2015), breast cancer (den Hollander et al., 2016) and glioma (Kong et al., 2007), and is associated with poor prognosis. Research has shown that the oncogenic function of PTP4A3 is accomplished via different cellular mechanisms. For example, PTP4A3 promotes cell proliferation, angiogenesis, and cell cycle progression (Basak et al., 2008; Guo et al., 2006; Polato et al., 2005). PTP4A3 also induces cell migration and invasion, and promotes metastasis through activation of the Rho GTPase and the PI3K/Akt pathway (Fiordalisi et al., 2006; Wang et al., 2007; Zeng et al., 2003). Recent studies demonstrated that autophagy could also play a critical role in PTP4A3-driven cancer progression (Huang et al., 2014). MicroRNAs (miRNAs) are a group of short, non-coding RNAs that modulate protein expression by complementing with the 3’-UTR of mRNAs (Bartel, 2004). MiRNAs have
been shown to induce the translational silence or cleavage of target genes and thereby participate in a variety of key cellular progresses that are associated with the onset and progression of cancer development (Gurtan and Sharp, 2013). Increasing recent evidences demonstrated aberrant regulation of miRNAs in a number of cancers such as gastric cancer, breast cancer, and gliomas (Zhu et al., 2014). Of particular interest to this study is miR-137. MiR-137 is located on chromosome 1p22, and has been found to be downregulated in many cancers including lung cancer (Zhu et al., 2013), colorectal cancer (Balaguer et al., 2010), gastric cancer (Chen et al., 2011), melanoma (Deng et al., 2011), oral cancer (Wiklund et al., 2011), and breast cancer (Zhao et al., 2012). In this study, we for the first time systematically studied the function of PTP4A3 and miR-137 in the malignancy of GBM. We found that PTP4A3 was upregulated in GBM tissues and cells. Knockdown of PTP4A3 expression in GBM cells inhibited cell proliferation, migration, and invasion. Mechanistically, we found that PTP4A3 was able to modulate of activity of the Akt/mTOR signaling pathway. We also identified PTP4A3 as a direct target of miR-137. Finally, we demonstrated that miR-137 regulated GBM cell malignancy, and this regulation was realized partially through PTP4A3-mediated Akt/mTOR signaling pathway.
2. Results 2.1. PTP4A3 is upregulated in GBM PTP4A3 has been shown to function as an oncogene in several types of cancers (Sharlow et al., 2014). Overexpression of PTP4A3 was reported in glioma (Kong et al., 2007).
However, the roles of PTP4A3 in glioblastoma multiforme (GBM) development have not been studied before. In order to reveal the possible regulatory mechanism of PTP4A3 in GBM, we first examined the expression levels of PTP4A3 in 20 GBM samples by qRTPCR. We found that the endogenous PTP4A3 levels were significantly higher in GBM tissues compared to those in non-tumor tissues (P < 0.001) (Fig. 1A). This finding was corroborated by immunohistochemistry results, which revealed that PTP4A3 staining was much stronger in GBM tissues than that in non-tumor brain tissues (Fig. 1B). We further confirmed elevated PTP4A3 protein levels in GBM by performing Western blot analysis on four randomly selected GBM samples (Fig. 1C). In addition, upregulated levels of PTP4A3 were also observed in U87, U251 and LN229 GBM cell lines (Fig. 1D). These results collectively indicated that PTP4A3 was upregulated in GBM tissues and cells.
2.2. Knockdown of PTP4A3 inhibits GBM cell proliferation, migration and invasion To study the biological roles of PTP4A3 in GBM, we transfected siRNA against PTP4A3 into LN229 and U87 cells to knock down endogenous PTP4A3 expression. Both mRNA (Fig. 2A) and protein (Fig. 2B) levels of PTP4A3 were decreased by this treatment. With these cell lines, we then evaluated the effects of PTP4A3 downregulation on a series of behaviors of GBM cells. We first performed MTT cell proliferation and colony formation assays. We found that knockdown of PTP4A3 significantly inhibited cell proliferation (Fig. 2C) and colony formation (Fig. 2D) of both LN229 and U87 cells. We also examined the effects of PTP4A3 knockdown in GBM cell migration and invasion. With wound healing assay, we were able to determine that PTP4A3 knockdown substantially suppressed the migration of both LN229 and U87 cells. The migration distances of
PTP4A3 knockdown LN229 and U87 cells were reduced by approximately 52.3% and 58.4%, respectively (Fig. 3A). Consistently, in transwell invasion assay, we found that the invasive abilities of LN229 and U87 cells were decreased by approximately 68.1% and 63.4% by PTP4A3 knockdown, respectively (Fig. 3B). In summary, knockdown of PTP4A3 expression inhibited the proliferation, migration, and invasion of GBM cells.
2.3. PTP4A3 regulates the Akt/mTOR signaling pathway PTP4A3 has been reported to affect cancer development by regulating the Akt signaling pathway (Lee et al., 2012; Wang et al., 2007). To confirm whether PTP4A3 regulates Akt signaling pathway in GBM, we first examined the phosphorylation status of Akt in LN229 and U87 cells. We found that knockdown of PTP4A3 significantly reduced the levels of phosphorylated Akt in both cell lines, while the total Akt levels remained unchanged (Fig. 4A). Akt signaling has been shown to modulate the activity of the mTOR pathway and to play important roles of GBM development (Li et al., 2016). In this context, we sought to examine mTOR phosphorylation in LN229 and U87 cells, which was an indicator of the activity of mTOR signaling. We found that while total mTOR levels remained constant, the levels of phosphorylated mTOR were significantly decreased in PTP4A3 knockdown cells (Fig. 4A). Furthermore, we examined the levels of a series of downstream target genes of the Akt/mTOR signaling pathway, including cyclin D1, p21, p27 and MMP-9. As expected, PTP4A3 knockdown led to altered protein levels of all these genes. Specifically, the levels of cyclin D1 and MMP-9 were reduced, and those of p21 and p27 were increased (Fig. 4B). Together, these results strongly
supported the participation of PTP4A3 in the regulation of the Akt/mTOR signaling pathway.
2.4. PTP4A3 is a direct target of miR-137 Recent studies have highlighted the pivotal roles of miRNAs in regulating GBM development, and miRNAs could serve as potential targets for cancer therapy (Costa et al., 2015). Therefore, we sought to determine whether PTP4A3 could be regulated by miRNAs. Using miRNA target prediction algorithm TargetScan (www.targetscan.org), we were able to identify that PTP4A3 was a predicted target of miR-137 (Fig. 5A). It is interesting to note that miR-137 has been demonstrated to be a tumor suppressor during GBM development (ref Liang et al., 2016; Sun et al., 2013; Sun et al., 2015). In order to confirm the prediction, we performed luciferase assay by co-transfecting miR-137 with either wild type or mutant 3’-UTR of PTP4A3 into HEK293 cells. The mutant 3’-UTR of PTP4A3 abolished the binding ability of miR-137. We found that the luciferase activity driven by the wild type 3’-UTR of PTP4A3 was significantly decreased in the presence of miR-137. Importantly, miR-137 had no effect on luciferase activity driven by the mutant 3’-UTR of PTP4A3. In addition, in both LN229 and U87 cells, both mRNA (Fig. 5C) and protein (Fig. 5D) levels of PTP4A3 were significantly reduced by the transfection of miR-137. Taken together, these data suggested that PTP4A3 was a direct target of miR-137.
2.5. MiR-137 regulates GBM cell malignancy partially through PTP4A3-mediated Akt/mTOR signaling pathway In this study, we have demonstrated that PTP4A3 regulates some of the key aspects of GBM cell malignancy, including cell proliferation, migration and invasion, likely via a pathway involving Akt/mTOR signaling. In this context, since PTP4A3 is a target of miR-137, we hypothesized that miR-137 could regulate the proliferation, migration and invasion of GBM cells through PTP4A3 and PTP4A3-mediated Akt/mTOR signaling pathway. To test this hypothesis, in miR-137 transfected LN229 and U87 cells, we reintroduced PTP4A3 by co-transfection to restore the decreased levels of PTP4A3 induced by miR-137 back to normal (Fig. 6A). We then assessed cell proliferation, migration and invasion in these cells. Consistent with our hypothesis, ectopic expression of miR-137 significantly inhibited the proliferation (Fig. 6B), migration (Fig. 6C) and invasion (Fig. 6D) of both LN229 and U87 cells. Importantly, restoration of PTP4A3 expression was able to partially relieve the inhibition of cell proliferation (Fig. 6B), migration (Fig. 6C) and invasion (Fig. 6D) in both GBM cell lines. In addition, we also examined the effects of miR-137 on the activities of the Akt/mTOR signaling pathway. We found that ectopic overexpression of miR-137 significantly reduced the phosphorylation of both Akt and mTOR (Fig. 7), and that re-introduction of PTP4A3 partially restored the levels of phosphorylation of Akt and mTOR (Fig. 7). Collectively, these results were consistent with our hypothesis that miR-137 regulated GBM cell malignancy, and this regulation was realized partially through PTP4A3-mediated Akt/mTOR signaling pathway.
3. Discussion Previous studies on PTP4A3 and miR-137 have demonstrated that they respectively play important roles in the development of multiple types of cancers. However, their regulatory relationship and their involvement in GBM progression have not been systematically investigated. In this study, we performed in-depth analysis of the function of PTP4A3 and miR-137 in the malignancy of GBM. We first confirmed that PTP4A3 is upregulated in GBM. This conclusion was supported by data from both clinical samples and cell lines, and is also consistent with the roles of PTP4A3 in other cancer types. PTP4A3 has been shown by previous studies to promote cancer malignancy and is associated with poor prognosis (Al-Aidaroos and Zeng, 2010). PTP4A3 upregulation was observed in metastatic specimens from multiples cancer types (Polato et al., 2005; Radke et al., 2006; Saha et al., 2001; Zhou et al., 2009). The mechanisms by which PTP4A3 regulates malignancy-associated cellular processes have been proposed and investigated before. PTP4A3 was demonstrated to promote cell proliferation, angiogenesis, and cell cycle progression (Basak et al., 2008; Guo et al., 2006; Polato et al., 2005). PTP4A3 was also shown to induce cell migration and invasion, and promote metastasis through activation of the Rho GTPase family and the PI3K/Akt pathway (Fiordalisi et al., 2006; Wang et al., 2007; Zeng et al., 2003). Recent studies indicated that autophagy also played a critical role in PTP4A3 regulated cancer development (ref). In our study, we focused our investigation on the Akt/mTOR signaling pathway, as this is a well-documented pathway that participates in the regulation of a series of key cellular functions, and this pathway has been implicated in PTP4A3 mechanism of action in other cancer types (Lee et al., 2012; Wang et al., 2007). Our data
indicated that in GBM cells, down-regulation of PTP4A3 led to de-phosphorylation and inactivation of the Akt/mTOR pathway. Since the Akt/mTOR pathway is pro-survival and anti-apoptosis (Downward, 1998), this mode of regulation is consistent with PTP4A3 upregulation in GBM tissue and cells. It is important to note that all our experiments were performed in two independent GBM cell lines, therefore the conclusions of our study are not restricted to a specific cell line. We identified miR-137 as a regulator of PTP4A3 by bioinformatics prediction. This prediction was then confirmed by evidence from different aspects. First, miR-137 was able to inhibit the 3’-UTR activity of PTP4A3 gene in a luciferase assay, but could not inhibit the 3’-UTR harboring a mutation that abolished miR-137 binding. Second, miR137 overexpression reduced the expression of endogenous PTP4A3. Our functional study on miR-137 further supported its role as a regulator of PTP4A3, as overexpression of miR-137 almost perfectly mimicked the phenotype on cell proliferation, cell migration and cell invasion induced by PTP4A3 knockdown. Collectively, these lines of evidence consistently supported that PTP4A3 is a bona fide target for miR-137. In our study, we found that restoration of PTP4A3 expression could only partially rescue the inhibition of cell proliferation, migration and invasion induced by miR-137 overexpression. We postulate that this phenomenon could be caused by the possibility that miR-137 has other targets than PTP4A3, and those other targets may also contribute to the inhibitory regulation of cell malignancy. It is actually not uncommon that a miRNA has many targets (Thomson et al., 2011). In addition, although in our study we focused our investigation on the activity of the Akt/mTOR pathway, we could not exclude the possibility that PTP4A3 is also involved in the regulation of other pathways
to affect GBM cell malignancy. These factors may have contributed to the incomplete rescue by PTP4A3, and we will seek to further our investigation on these factors in our future study.
4. Experimental procedure 4.1. Human samples This study was approved by the Institutional Review Board of Daqing Oilfield General Hospital. Following informed consent, 20 samples with World Health Organization (WHO) grade IV glioblastoma (GBM) and eight normal brain tissues were obtained from patients undergoing surgery at Daqing Oilfield General Hospital. Collected tissues were immediately snap-frozen and stored at -80°C.
4.2. Immunohistochemistry (IHC) IHC was performed as previously described (Guo et al., 2014). Anti-PTP4A3 primary antibody was purchased from Abcam (Cambridge, MA, USA).
4.3. Cell lines and cell culture GBM cell lines (LN229, U87 and U251), and HEK293 cells were maintained in Dulbecco's modified Eagle medium (DMEM) (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and penicillin/streptomycin (Invitrogen).
4.4. Western blot Total protein from GBM tissues and cell lines was extracted using RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 5.0 mM EDTA pH 8.0, 0.5 mM dithiothreitol, and 1 mM phenylmethylsulfonylfluoride). Protein concentrations were determined using the BCA method (Thermo Scientific, Rockford, IL, USA). Equal quantities of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked and then incubated with anti-PTP4A3 (Abcam), anti-p-Akt, anti-Akt, anti-p-mTOR, anti-TOR and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, membranes were washed and then incubated with secondary antibodies (Santa Cruz) for 1 h. Bound antibodies were detected using Enhanced Chemiluminescence (ECL) Super Signal West Pico (Pierce Biotech,Rockford, IL, USA).
4.5. Quantity Real-Time PCR (qRT-PCR) qRT-PCR was performed as previously described (Li et al., 2015). The following primers were used: PTP4A3 forward, 5’-CGGCAAGGTAGTGGAAGAC-3’; reverse, 5’GGCGGATGAACTGGATGG-3’.
4.6. 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay MTT assay was performed to examine cell proliferation. 5 × 103 cells per well of LN229 or U87 cells were seeded into a 96-well plate and incubated at 37°C with 5% CO2. MTT reagent (20 µl) was added and incubated for 3 - 4 h at 37°C. Then the MTT reagent was removed and 150 µl of dimethyl sulfoxide (DMSO) was added to each well to solubilize the formazan. The absorbance was measured at 590 nm using a microplate reader.
4.7. Colony formation assay The plate colony formation assay was performed to assess GBM cell growth. LN229 or U87 cells were seeded into 6-well plate (500 cell/well) and cultured in incubator. After ten days, the cells were washed two times with PBS, fixed with 4% paraformaldehyde for 10 min and stained with crystal violet at room temperature. The number of colonies was counted.
4.8. Wound healing assay Ability of GBM cell migration was assessed by wound healing assay. Cells were seeded in 6-well plates. When grown to 80-90% confluence, the cells in each well were scratched with wound lines vertically to the bottom of the well with a 200µL pipette tip. After being washed with PBS two times, cells were incubated in growing medium. The wound width was determined 48 h later under a microscope (Olympus, Tokyo, Japan).
4.9. Transwell assay Transwell assay was used to analyze GBM cell invasive ability using Matrigel covered 8µl pore membrane transwell inserts (BD Biosciences, San Jose, CA, USA). LN229 or U87 cells (1×105 cells per chamber) were seeded in the upper chambers in serum-free DMEM medium, and complete DMEM medium with 10% FBS was placed in the lower chambers. After 24 h, cells on the upper surface of the chamber were removed with a cotton swab and the inserts were fixed with 4% paraformaldehyde and stained with crystal violet. Invaded cells attached to the lower surface of the filter were counted in six randomly selected areas under a microscope (Olympus).
4.10. Luciferase assay Wild-type full-length PTP4A3 3’UTR were amplified from the genomic DNA and ligated into the psi-CHECKTM luciferase reporter vector (Promega, Madison, WI, USA). Mutant 3’UTR luciferase reporter vectors containing four mutated nucleotides on the predicted miR-137 binding sites were constructed using the site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). These plasmids were sequenced and then cotransfected with miR-NC or miR-137 into HEK293 using X-tremeGENE transfection reagent (Roche, Mannheim, Germany). Renilla luciferase plasmid (Promega) was co-transfected to normalize the relative luciferase values. Forty-eight hours post-transfection, cells were collected, lysed and their luciferase activity measured 48 h post-transfection using a dualluciferase reporter assay system (Promega).
4.11. Statistics Data is expressed as mean ± SD of three independent experiments. Differences among groups were analyzed using one-way ANOVA or student’s t test. A p-value < 0.05 was considered significant. All statistical analyses were conducted using SPSS 20.0.
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Figure legends Fig. 1. PTP4A3 is upregulated in GBM. (A) PTP4A3 mRNA expression levels in GBM samples (n = 20) as determined by qRTPCR compared to those in non-tumor brain samples (n = 8) . P < 0.001. (B)
Representative PTP4A3 immunohistochemistry staining in GBM tissue and in non-tumor tissue. (C) PTP4A3 protein expression levels in 4 randomly selected GBM patients as determined by Western blot. β-actin was used as a loading control. (D) PTP4A3 protein expression levels in U87, U251 and LN229 GBM cells as determined by Western blot.
Fig. 2. Knockdown of PTP4A3 reduces GBM cell proliferation. (A) mRNA levels as determined by qRT-PCR and (B) protein levels as determined by Western blot of PTP4A3 in LN229 and U87 cells transfected with PTP4A3 siRNA (PTP4A3-si) or Control siRNA (Control-si). (C) MTT assay and (D) colony formation assay performed on LN229 and U87 cells transfected with PTP4A3 siRNA (PTP4A3-si) or Control siRNA (Control-si). *** indicates P < 0.001 compared to Control-si transfected cells.
Fig. 3. Knockdown of PTP4A3 inhibits GBM cell migration and invasion. (A) Wound healing assay and (B) Matrigel transwell assay performed on LN229 and U87 cells transfected with PTP4A3 siRNA (PTP4A3-si) or Control siRNA (Control-si). *** indicates P < 0.001 compared to Control-si transfected cells.
Fig. 4. PTP4A3 regulates Akt/mTOR signaling pathway. (A) Protein levels of total Akt, phosphorylated Akt, total mTOR and phosphorylated mTOR as determined by Western blot in LN229 and U87 cells transfected with PTP4A3 siRNA (PTP4A3-si) or Control siRNA (Control-si). β-actin was used as a loading
control. (B) Protein levels of the downstream target genes of Akt/mTOR signaling including Cyclin D1, p21, p27 and MMP9, as determined by Western blot performed on LN229 and U87 cells transfected with PTP4A3 siRNA (PTP4A3-si) or Control siRNA (Control-si). Quantification of each bind was shown. *** indicates P < 0.001 compared to Control-si transfected cells.
Fig. 5. PTP4A3 is a direct target of miR-137. (A) Schematic diagram of the computational predicted seed region in the 3’-UTR of PTP4A3 with miR-137 shown. Mutations on the “seed” sequence were designed as shown. (B) Luciferase activities in HEK293T cells co-transfected with either miR-137 or a control miRNA (miR-NC), and plasmids with either wild type or mutant 3’-UTR of PTP4A3. (C) mRNA levels as determined by qRT-PCR and (D) protein levels as determined by Western blot of PTP4A3 in LN229 and U87 cells transfected with either miR-137 or a control miRNA (miR-NC). β-actin was used as a loading control. * indicates P < 0.05, ** indicates P < 0.01 compared to miR-NC transfected cells.
Fig. 6. Restoration of PTP4A3 expression partially rescued miR-137 induced cell malignancy. (A) Protein levels of PTP4A3 as determined by western blot analysis in LN229 and U87 cells transfected with a control miRNA (miR-NC), miR-137, or co-transfected with miR137 and PTP4A3. Quantification of each bind was shown. (B) cell proliferation as determined by MTT assay, (C) cell migration as determined by would healing assay, and
(D) cell invasion as determined by transwell assay in LN229 and U87 cells transfected with a control miRNA (miR-NC), miR-137, or co-transfected with miR-137 and PTP4A3. ** indicates P < 0.01, *** indicates P < 0.001 compared to miR-NC transfected cells; & indicates P < 0.05, && indicates P < 0.01, &&& indicates P < 0.001 compared to miR-137 transfected cells.
Fig. 7. Restoration of PTP4A3 partially rescued miR-137 induced repression of Akt/mTOR signaling. Protein levels of total Akt, phosphorylated Akt, total mTOR and phosphorylated mTOR as determined by Western blot in LN229 and U87 cells transfected with a control miRNA (miR-NC), miR-137, or co-transfected with miR-137 and PTP4A3. * indicates P < 0.01, ** indicates P < 0.01, *** indicates P < 0.001 compared to miR-NC transfected cells; &&& indicates P < 0.001 compared to miR-137 transfected cells.
Figures
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Highlights
PTP4A3 is upregulated in GBM
Knockdown of PTP4A3 inhibits GBM cell proliferation, migration and invasion
PTP4A3 regulates the Akt/mTOR signaling pathway
PTP4A3 is a direct target of miR-137
MiR-137 regulates GBM cell malignancy partially through PTP4A3-mediated Akt/mTOR signaling pathway
PII: DOI: Reference:
S0006-8993(16)30450-4 http://dx.doi.org/10.1016/j.brainres.2016.06.026 BRES44977
To appear in: Brain Research Received date: 7 April 2016 Revised date: 14 June 2016 Accepted date: 16 June 2016 Cite this article as: Liling Wang, Jianxun Liu, Zhiqiang Zhong, Xuhai Gong, Wei Liu, Lei Shi and Xuesong Li, PTP4A3 is a target for inhibition of cell proliferatin, migration and invasion through Akt/mTOR signaling pathway in glioblastoma under the regulation of miR-137, Brain Research, http://dx.doi.org/10.1016/j.brainres.2016.06.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PTP4A3 is a target for inhibition of cell proliferatin, migration and invasion through Akt/mTOR signaling pathway in glioblastoma under the regulation of miR137 Liling Wanga, Jianxun Liua, Zhiqiang Zhonga, Xuhai Gonga, Wei Liub, Lei Shic, Xuesong Lia* a
Department of Neurology, Daqing Oilfield General Hospital, 9 Zhongkang Road,
Daqing, Heilongjiang 163000, China b
Department of General Surgery, Daqing Oilfield General Hospital, 9 Zhongkang Road,
Daqing, Heilongjiang 163000, China c
Department of Obstetrics and Gynecology, Daqing Oilfield General Hospital, 9
Zhongkang Road, Daqing, Heilongjiang 163000, China *
Corresponding author. Tel.: +86 13329391666; fax: +86 0459 5994114.
[email protected]
Abstract Glioblastoma multiforme (GBM) is one of the most common primary malignant adult brain tumors. It is characterized by aggressive progression and poor prognosis. There is significant need to understand the mechanism of GBM malignancy and develop improved therapeutic options for GBM patients. We systematically studied the function of PTP4A3 in the malignancy of GBM. We found that PTP4A3 was upregulated in GBM tissues and cells. Knockdown of PTP4A3 expression in GBM cells inhibited cell proliferation, migration, and invasion. PTP4A3 knockdown modulated the activity of the
Akt/mTOR signaling pathway by inducing de-phosphorylation of Akt and mTOR. We identified PTP4A3 as a direct target of miR-137. MiR-137 has been reported as a tumor suppressor in GBM development. In this study, overexpression of miR-137 in GBM cells also inhibited cell proliferation, migration, and invasion. Finally, restoration of PTP4A3 expression in miR-137 overexpressing cells partially reversed the inhibition of GBM cell malignancy, and the de-phosphorylation of Akt and mTOR. We identified that PTP4A3 regulated GBM via miR-137-mediated Akt/mTOR signaling pathway.
Keywords miR-137, PTP4A3, Akt, mTOR, glioblastoma multiforme
1. Introduction Glioblastoma multiforme (GBM) is one of the most common primary malignant adult brain tumors (Louis et al., 2007). GBM is characterized by its aggressiveness and poor prognosis, with a median survival of only about 15 months (Stupp et al., 2005). Currently, the standard treatment regimen for GBM includes surgery removal, chemotherapy and radiotherapy (Stupp et al., 2005). Despite recent advancement in the therapeutic regimen, the successful rate for GBM treatment remains low. This situation is mainly due to the development of drug resistance by cancer cells, and the intrinsic ability of the cancer cells to migrate and invade through normal brain tissue, making complete surgical removal of cancer tissue nearly impossible. Therefore, there is a significant need
to understand the mechanism of GBM malignancy and develop improved therapeutic options for GBM patients. Protein tyrosine phosphatases (PTP) play important roles in modulating the levels of tyrosine phosphorylation. Increasing recent evidence has revealed that deregulation of protein tyrosine phosphatase activity is involved in a variety of diseases, including cancer (Ostman et al., 2006). One of the PTPs, PTP4A3, is characterized by the unique prenylation motif in its C-terminal end. Non-prenylated PTP4A3 is inactive and is usually located in the nucleus, and prenylated PTP4A3 is active and can be found in the membranes and intracellular structures (Zeng et al., 2000). PTP4A3 is known for its role in promoting cancer metastasis (Al-Aidaroos and Zeng, 2010). PTP4A3 upregulation was found in metastatic specimens from multiples cancer types (Polato et al., 2005; Radke et al., 2006; Saha et al., 2001; Zhou et al., 2009) including colorectal cancer (Saha et al., 2001), bladder cancer (Yeh et al., 2015), breast cancer (den Hollander et al., 2016) and glioma (Kong et al., 2007), and is associated with poor prognosis. Research has shown that the oncogenic function of PTP4A3 is accomplished via different cellular mechanisms. For example, PTP4A3 promotes cell proliferation, angiogenesis, and cell cycle progression (Basak et al., 2008; Guo et al., 2006; Polato et al., 2005). PTP4A3 also induces cell migration and invasion, and promotes metastasis through activation of the Rho GTPase and the PI3K/Akt pathway (Fiordalisi et al., 2006; Wang et al., 2007; Zeng et al., 2003). Recent studies demonstrated that autophagy could also play a critical role in PTP4A3-driven cancer progression (Huang et al., 2014). MicroRNAs (miRNAs) are a group of short, non-coding RNAs that modulate protein expression by complementing with the 3’-UTR of mRNAs (Bartel, 2004). MiRNAs have
been shown to induce the translational silence or cleavage of target genes and thereby participate in a variety of key cellular progresses that are associated with the onset and progression of cancer development (Gurtan and Sharp, 2013). Increasing recent evidences demonstrated aberrant regulation of miRNAs in a number of cancers such as gastric cancer, breast cancer, and gliomas (Zhu et al., 2014). Of particular interest to this study is miR-137. MiR-137 is located on chromosome 1p22, and has been found to be downregulated in many cancers including lung cancer (Zhu et al., 2013), colorectal cancer (Balaguer et al., 2010), gastric cancer (Chen et al., 2011), melanoma (Deng et al., 2011), oral cancer (Wiklund et al., 2011), and breast cancer (Zhao et al., 2012). In this study, we for the first time systematically studied the function of PTP4A3 and miR-137 in the malignancy of GBM. We found that PTP4A3 was upregulated in GBM tissues and cells. Knockdown of PTP4A3 expression in GBM cells inhibited cell proliferation, migration, and invasion. Mechanistically, we found that PTP4A3 was able to modulate of activity of the Akt/mTOR signaling pathway. We also identified PTP4A3 as a direct target of miR-137. Finally, we demonstrated that miR-137 regulated GBM cell malignancy, and this regulation was realized partially through PTP4A3-mediated Akt/mTOR signaling pathway.
2. Results 2.1. PTP4A3 is upregulated in GBM PTP4A3 has been shown to function as an oncogene in several types of cancers (Sharlow et al., 2014). Overexpression of PTP4A3 was reported in glioma (Kong et al., 2007).
However, the roles of PTP4A3 in glioblastoma multiforme (GBM) development have not been studied before. In order to reveal the possible regulatory mechanism of PTP4A3 in GBM, we first examined the expression levels of PTP4A3 in 20 GBM samples by qRTPCR. We found that the endogenous PTP4A3 levels were significantly higher in GBM tissues compared to those in non-tumor tissues (P < 0.001) (Fig. 1A). This finding was corroborated by immunohistochemistry results, which revealed that PTP4A3 staining was much stronger in GBM tissues than that in non-tumor brain tissues (Fig. 1B). We further confirmed elevated PTP4A3 protein levels in GBM by performing Western blot analysis on four randomly selected GBM samples (Fig. 1C). In addition, upregulated levels of PTP4A3 were also observed in U87, U251 and LN229 GBM cell lines (Fig. 1D). These results collectively indicated that PTP4A3 was upregulated in GBM tissues and cells.
2.2. Knockdown of PTP4A3 inhibits GBM cell proliferation, migration and invasion To study the biological roles of PTP4A3 in GBM, we transfected siRNA against PTP4A3 into LN229 and U87 cells to knock down endogenous PTP4A3 expression. Both mRNA (Fig. 2A) and protein (Fig. 2B) levels of PTP4A3 were decreased by this treatment. With these cell lines, we then evaluated the effects of PTP4A3 downregulation on a series of behaviors of GBM cells. We first performed MTT cell proliferation and colony formation assays. We found that knockdown of PTP4A3 significantly inhibited cell proliferation (Fig. 2C) and colony formation (Fig. 2D) of both LN229 and U87 cells. We also examined the effects of PTP4A3 knockdown in GBM cell migration and invasion. With wound healing assay, we were able to determine that PTP4A3 knockdown substantially suppressed the migration of both LN229 and U87 cells. The migration distances of
PTP4A3 knockdown LN229 and U87 cells were reduced by approximately 52.3% and 58.4%, respectively (Fig. 3A). Consistently, in transwell invasion assay, we found that the invasive abilities of LN229 and U87 cells were decreased by approximately 68.1% and 63.4% by PTP4A3 knockdown, respectively (Fig. 3B). In summary, knockdown of PTP4A3 expression inhibited the proliferation, migration, and invasion of GBM cells.
2.3. PTP4A3 regulates the Akt/mTOR signaling pathway PTP4A3 has been reported to affect cancer development by regulating the Akt signaling pathway (Lee et al., 2012; Wang et al., 2007). To confirm whether PTP4A3 regulates Akt signaling pathway in GBM, we first examined the phosphorylation status of Akt in LN229 and U87 cells. We found that knockdown of PTP4A3 significantly reduced the levels of phosphorylated Akt in both cell lines, while the total Akt levels remained unchanged (Fig. 4A). Akt signaling has been shown to modulate the activity of the mTOR pathway and to play important roles of GBM development (Li et al., 2016). In this context, we sought to examine mTOR phosphorylation in LN229 and U87 cells, which was an indicator of the activity of mTOR signaling. We found that while total mTOR levels remained constant, the levels of phosphorylated mTOR were significantly decreased in PTP4A3 knockdown cells (Fig. 4A). Furthermore, we examined the levels of a series of downstream target genes of the Akt/mTOR signaling pathway, including cyclin D1, p21, p27 and MMP-9. As expected, PTP4A3 knockdown led to altered protein levels of all these genes. Specifically, the levels of cyclin D1 and MMP-9 were reduced, and those of p21 and p27 were increased (Fig. 4B). Together, these results strongly
supported the participation of PTP4A3 in the regulation of the Akt/mTOR signaling pathway.
2.4. PTP4A3 is a direct target of miR-137 Recent studies have highlighted the pivotal roles of miRNAs in regulating GBM development, and miRNAs could serve as potential targets for cancer therapy (Costa et al., 2015). Therefore, we sought to determine whether PTP4A3 could be regulated by miRNAs. Using miRNA target prediction algorithm TargetScan (www.targetscan.org), we were able to identify that PTP4A3 was a predicted target of miR-137 (Fig. 5A). It is interesting to note that miR-137 has been demonstrated to be a tumor suppressor during GBM development (ref Liang et al., 2016; Sun et al., 2013; Sun et al., 2015). In order to confirm the prediction, we performed luciferase assay by co-transfecting miR-137 with either wild type or mutant 3’-UTR of PTP4A3 into HEK293 cells. The mutant 3’-UTR of PTP4A3 abolished the binding ability of miR-137. We found that the luciferase activity driven by the wild type 3’-UTR of PTP4A3 was significantly decreased in the presence of miR-137. Importantly, miR-137 had no effect on luciferase activity driven by the mutant 3’-UTR of PTP4A3. In addition, in both LN229 and U87 cells, both mRNA (Fig. 5C) and protein (Fig. 5D) levels of PTP4A3 were significantly reduced by the transfection of miR-137. Taken together, these data suggested that PTP4A3 was a direct target of miR-137.
2.5. MiR-137 regulates GBM cell malignancy partially through PTP4A3-mediated Akt/mTOR signaling pathway In this study, we have demonstrated that PTP4A3 regulates some of the key aspects of GBM cell malignancy, including cell proliferation, migration and invasion, likely via a pathway involving Akt/mTOR signaling. In this context, since PTP4A3 is a target of miR-137, we hypothesized that miR-137 could regulate the proliferation, migration and invasion of GBM cells through PTP4A3 and PTP4A3-mediated Akt/mTOR signaling pathway. To test this hypothesis, in miR-137 transfected LN229 and U87 cells, we reintroduced PTP4A3 by co-transfection to restore the decreased levels of PTP4A3 induced by miR-137 back to normal (Fig. 6A). We then assessed cell proliferation, migration and invasion in these cells. Consistent with our hypothesis, ectopic expression of miR-137 significantly inhibited the proliferation (Fig. 6B), migration (Fig. 6C) and invasion (Fig. 6D) of both LN229 and U87 cells. Importantly, restoration of PTP4A3 expression was able to partially relieve the inhibition of cell proliferation (Fig. 6B), migration (Fig. 6C) and invasion (Fig. 6D) in both GBM cell lines. In addition, we also examined the effects of miR-137 on the activities of the Akt/mTOR signaling pathway. We found that ectopic overexpression of miR-137 significantly reduced the phosphorylation of both Akt and mTOR (Fig. 7), and that re-introduction of PTP4A3 partially restored the levels of phosphorylation of Akt and mTOR (Fig. 7). Collectively, these results were consistent with our hypothesis that miR-137 regulated GBM cell malignancy, and this regulation was realized partially through PTP4A3-mediated Akt/mTOR signaling pathway.
3. Discussion Previous studies on PTP4A3 and miR-137 have demonstrated that they respectively play important roles in the development of multiple types of cancers. However, their regulatory relationship and their involvement in GBM progression have not been systematically investigated. In this study, we performed in-depth analysis of the function of PTP4A3 and miR-137 in the malignancy of GBM. We first confirmed that PTP4A3 is upregulated in GBM. This conclusion was supported by data from both clinical samples and cell lines, and is also consistent with the roles of PTP4A3 in other cancer types. PTP4A3 has been shown by previous studies to promote cancer malignancy and is associated with poor prognosis (Al-Aidaroos and Zeng, 2010). PTP4A3 upregulation was observed in metastatic specimens from multiples cancer types (Polato et al., 2005; Radke et al., 2006; Saha et al., 2001; Zhou et al., 2009). The mechanisms by which PTP4A3 regulates malignancy-associated cellular processes have been proposed and investigated before. PTP4A3 was demonstrated to promote cell proliferation, angiogenesis, and cell cycle progression (Basak et al., 2008; Guo et al., 2006; Polato et al., 2005). PTP4A3 was also shown to induce cell migration and invasion, and promote metastasis through activation of the Rho GTPase family and the PI3K/Akt pathway (Fiordalisi et al., 2006; Wang et al., 2007; Zeng et al., 2003). Recent studies indicated that autophagy also played a critical role in PTP4A3 regulated cancer development (ref). In our study, we focused our investigation on the Akt/mTOR signaling pathway, as this is a well-documented pathway that participates in the regulation of a series of key cellular functions, and this pathway has been implicated in PTP4A3 mechanism of action in other cancer types (Lee et al., 2012; Wang et al., 2007). Our data
indicated that in GBM cells, down-regulation of PTP4A3 led to de-phosphorylation and inactivation of the Akt/mTOR pathway. Since the Akt/mTOR pathway is pro-survival and anti-apoptosis (Downward, 1998), this mode of regulation is consistent with PTP4A3 upregulation in GBM tissue and cells. It is important to note that all our experiments were performed in two independent GBM cell lines, therefore the conclusions of our study are not restricted to a specific cell line. We identified miR-137 as a regulator of PTP4A3 by bioinformatics prediction. This prediction was then confirmed by evidence from different aspects. First, miR-137 was able to inhibit the 3’-UTR activity of PTP4A3 gene in a luciferase assay, but could not inhibit the 3’-UTR harboring a mutation that abolished miR-137 binding. Second, miR137 overexpression reduced the expression of endogenous PTP4A3. Our functional study on miR-137 further supported its role as a regulator of PTP4A3, as overexpression of miR-137 almost perfectly mimicked the phenotype on cell proliferation, cell migration and cell invasion induced by PTP4A3 knockdown. Collectively, these lines of evidence consistently supported that PTP4A3 is a bona fide target for miR-137. In our study, we found that restoration of PTP4A3 expression could only partially rescue the inhibition of cell proliferation, migration and invasion induced by miR-137 overexpression. We postulate that this phenomenon could be caused by the possibility that miR-137 has other targets than PTP4A3, and those other targets may also contribute to the inhibitory regulation of cell malignancy. It is actually not uncommon that a miRNA has many targets (Thomson et al., 2011). In addition, although in our study we focused our investigation on the activity of the Akt/mTOR pathway, we could not exclude the possibility that PTP4A3 is also involved in the regulation of other pathways
to affect GBM cell malignancy. These factors may have contributed to the incomplete rescue by PTP4A3, and we will seek to further our investigation on these factors in our future study.
4. Experimental procedure 4.1. Human samples This study was approved by the Institutional Review Board of Daqing Oilfield General Hospital. Following informed consent, 20 samples with World Health Organization (WHO) grade IV glioblastoma (GBM) and eight normal brain tissues were obtained from patients undergoing surgery at Daqing Oilfield General Hospital. Collected tissues were immediately snap-frozen and stored at -80°C.
4.2. Immunohistochemistry (IHC) IHC was performed as previously described (Guo et al., 2014). Anti-PTP4A3 primary antibody was purchased from Abcam (Cambridge, MA, USA).
4.3. Cell lines and cell culture GBM cell lines (LN229, U87 and U251), and HEK293 cells were maintained in Dulbecco's modified Eagle medium (DMEM) (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and penicillin/streptomycin (Invitrogen).
4.4. Western blot Total protein from GBM tissues and cell lines was extracted using RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 5.0 mM EDTA pH 8.0, 0.5 mM dithiothreitol, and 1 mM phenylmethylsulfonylfluoride). Protein concentrations were determined using the BCA method (Thermo Scientific, Rockford, IL, USA). Equal quantities of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were blocked and then incubated with anti-PTP4A3 (Abcam), anti-p-Akt, anti-Akt, anti-p-mTOR, anti-TOR and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, membranes were washed and then incubated with secondary antibodies (Santa Cruz) for 1 h. Bound antibodies were detected using Enhanced Chemiluminescence (ECL) Super Signal West Pico (Pierce Biotech,Rockford, IL, USA).
4.5. Quantity Real-Time PCR (qRT-PCR) qRT-PCR was performed as previously described (Li et al., 2015). The following primers were used: PTP4A3 forward, 5’-CGGCAAGGTAGTGGAAGAC-3’; reverse, 5’GGCGGATGAACTGGATGG-3’.
4.6. 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay MTT assay was performed to examine cell proliferation. 5 × 103 cells per well of LN229 or U87 cells were seeded into a 96-well plate and incubated at 37°C with 5% CO2. MTT reagent (20 µl) was added and incubated for 3 - 4 h at 37°C. Then the MTT reagent was removed and 150 µl of dimethyl sulfoxide (DMSO) was added to each well to solubilize the formazan. The absorbance was measured at 590 nm using a microplate reader.
4.7. Colony formation assay The plate colony formation assay was performed to assess GBM cell growth. LN229 or U87 cells were seeded into 6-well plate (500 cell/well) and cultured in incubator. After ten days, the cells were washed two times with PBS, fixed with 4% paraformaldehyde for 10 min and stained with crystal violet at room temperature. The number of colonies was counted.
4.8. Wound healing assay Ability of GBM cell migration was assessed by wound healing assay. Cells were seeded in 6-well plates. When grown to 80-90% confluence, the cells in each well were scratched with wound lines vertically to the bottom of the well with a 200µL pipette tip. After being washed with PBS two times, cells were incubated in growing medium. The wound width was determined 48 h later under a microscope (Olympus, Tokyo, Japan).
4.9. Transwell assay Transwell assay was used to analyze GBM cell invasive ability using Matrigel covered 8µl pore membrane transwell inserts (BD Biosciences, San Jose, CA, USA). LN229 or U87 cells (1×105 cells per chamber) were seeded in the upper chambers in serum-free DMEM medium, and complete DMEM medium with 10% FBS was placed in the lower chambers. After 24 h, cells on the upper surface of the chamber were removed with a cotton swab and the inserts were fixed with 4% paraformaldehyde and stained with crystal violet. Invaded cells attached to the lower surface of the filter were counted in six randomly selected areas under a microscope (Olympus).
4.10. Luciferase assay Wild-type full-length PTP4A3 3’UTR were amplified from the genomic DNA and ligated into the psi-CHECKTM luciferase reporter vector (Promega, Madison, WI, USA). Mutant 3’UTR luciferase reporter vectors containing four mutated nucleotides on the predicted miR-137 binding sites were constructed using the site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). These plasmids were sequenced and then cotransfected with miR-NC or miR-137 into HEK293 using X-tremeGENE transfection reagent (Roche, Mannheim, Germany). Renilla luciferase plasmid (Promega) was co-transfected to normalize the relative luciferase values. Forty-eight hours post-transfection, cells were collected, lysed and their luciferase activity measured 48 h post-transfection using a dualluciferase reporter assay system (Promega).
4.11. Statistics Data is expressed as mean ± SD of three independent experiments. Differences among groups were analyzed using one-way ANOVA or student’s t test. A p-value < 0.05 was considered significant. All statistical analyses were conducted using SPSS 20.0.
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Figure legends Fig. 1. PTP4A3 is upregulated in GBM. (A) PTP4A3 mRNA expression levels in GBM samples (n = 20) as determined by qRTPCR compared to those in non-tumor brain samples (n = 8) . P < 0.001. (B)
Representative PTP4A3 immunohistochemistry staining in GBM tissue and in non-tumor tissue. (C) PTP4A3 protein expression levels in 4 randomly selected GBM patients as determined by Western blot. β-actin was used as a loading control. (D) PTP4A3 protein expression levels in U87, U251 and LN229 GBM cells as determined by Western blot.
Fig. 2. Knockdown of PTP4A3 reduces GBM cell proliferation. (A) mRNA levels as determined by qRT-PCR and (B) protein levels as determined by Western blot of PTP4A3 in LN229 and U87 cells transfected with PTP4A3 siRNA (PTP4A3-si) or Control siRNA (Control-si). (C) MTT assay and (D) colony formation assay performed on LN229 and U87 cells transfected with PTP4A3 siRNA (PTP4A3-si) or Control siRNA (Control-si). *** indicates P < 0.001 compared to Control-si transfected cells.
Fig. 3. Knockdown of PTP4A3 inhibits GBM cell migration and invasion. (A) Wound healing assay and (B) Matrigel transwell assay performed on LN229 and U87 cells transfected with PTP4A3 siRNA (PTP4A3-si) or Control siRNA (Control-si). *** indicates P < 0.001 compared to Control-si transfected cells.
Fig. 4. PTP4A3 regulates Akt/mTOR signaling pathway. (A) Protein levels of total Akt, phosphorylated Akt, total mTOR and phosphorylated mTOR as determined by Western blot in LN229 and U87 cells transfected with PTP4A3 siRNA (PTP4A3-si) or Control siRNA (Control-si). β-actin was used as a loading
control. (B) Protein levels of the downstream target genes of Akt/mTOR signaling including Cyclin D1, p21, p27 and MMP9, as determined by Western blot performed on LN229 and U87 cells transfected with PTP4A3 siRNA (PTP4A3-si) or Control siRNA (Control-si). Quantification of each bind was shown. *** indicates P < 0.001 compared to Control-si transfected cells.
Fig. 5. PTP4A3 is a direct target of miR-137. (A) Schematic diagram of the computational predicted seed region in the 3’-UTR of PTP4A3 with miR-137 shown. Mutations on the “seed” sequence were designed as shown. (B) Luciferase activities in HEK293T cells co-transfected with either miR-137 or a control miRNA (miR-NC), and plasmids with either wild type or mutant 3’-UTR of PTP4A3. (C) mRNA levels as determined by qRT-PCR and (D) protein levels as determined by Western blot of PTP4A3 in LN229 and U87 cells transfected with either miR-137 or a control miRNA (miR-NC). β-actin was used as a loading control. * indicates P < 0.05, ** indicates P < 0.01 compared to miR-NC transfected cells.
Fig. 6. Restoration of PTP4A3 expression partially rescued miR-137 induced cell malignancy. (A) Protein levels of PTP4A3 as determined by western blot analysis in LN229 and U87 cells transfected with a control miRNA (miR-NC), miR-137, or co-transfected with miR137 and PTP4A3. Quantification of each bind was shown. (B) cell proliferation as determined by MTT assay, (C) cell migration as determined by would healing assay, and
(D) cell invasion as determined by transwell assay in LN229 and U87 cells transfected with a control miRNA (miR-NC), miR-137, or co-transfected with miR-137 and PTP4A3. ** indicates P < 0.01, *** indicates P < 0.001 compared to miR-NC transfected cells; & indicates P < 0.05, && indicates P < 0.01, &&& indicates P < 0.001 compared to miR-137 transfected cells.
Fig. 7. Restoration of PTP4A3 partially rescued miR-137 induced repression of Akt/mTOR signaling. Protein levels of total Akt, phosphorylated Akt, total mTOR and phosphorylated mTOR as determined by Western blot in LN229 and U87 cells transfected with a control miRNA (miR-NC), miR-137, or co-transfected with miR-137 and PTP4A3. * indicates P < 0.01, ** indicates P < 0.01, *** indicates P < 0.001 compared to miR-NC transfected cells; &&& indicates P < 0.001 compared to miR-137 transfected cells.
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Highlights
PTP4A3 is upregulated in GBM
Knockdown of PTP4A3 inhibits GBM cell proliferation, migration and invasion
PTP4A3 regulates the Akt/mTOR signaling pathway
PTP4A3 is a direct target of miR-137
MiR-137 regulates GBM cell malignancy partially through PTP4A3-mediated Akt/mTOR signaling pathway