| Research BackgroundGliomas are primary brain tumors that originates from the brain and spinal glial cells. Gliomas are the most common primary brain tumors in adults and represent 70%-80% of all human primary brain malignancies [1,2]. There are three types of gliomas, including astrocytomas, oligodendrogliomas, ependymomas, and glioblastoma multiforme (GBM). GBM, the most common and deadliest type of malignant primary brain tumor, has a very poor prognosis, which seriously affects the survival and quality of life of the patients and it also results in a heavy economic burden for the family and society [3-5]. The surgery also called "maximum safe resection", aims to remove as much tumor as is safely possible to preserve neurological function. Since surgery can't completely remove all the small lesions, it is not a cure and this is also the reason why post-surgical recurrence of GBM is nearly universal [6]. The ultimate cause of death in most GBM patients was due to continuous extension of local invasion of tumor cells into surrounding brain tissues, resulting in further loss of brain function and leading to respiratory failure or multiple organ dysfunction [7]. Therefore, it remains a main challenge to get an advanced scientific understanding of the molecular aspects of tumor cell invasion and provide a strategy to prevent tumor cells from invading surrounding healthy brain tissues. Although post-surgical radiotherapy and combined chemotherapy with temozolomide can improve survival, about 72% of patients will still expect to experience tumor recurrence within 17 months after the surgery [8], and the 5-year survival rate is only 9.8%[9]. Due to the poor prognosis of GBM, researchers have spent huge efforts over the past decades on identification of molecular markers that can help classify GBM and predict their occurrence and prognosis [10,11,12].MicroRNAs (miRNAs) are a group of small (20-25 nucleotides) non-coding RNA fragments that can bind to target messenger RNAs (mRNAs) and result in translational repression or silencing of their target mRNAs. miRNAs have been found to be involved in the pathogenesis and progression of many types of human cancers. Therefore, they have great potential to transform cancer diagnosis and treatment. So far, more than 1,000 miRNAs have been identified and they were found to be either up-regulated or down-regulated in many different types of human cancers. Researchers have demonstrated that multiple miRNAs might have been involved in the pathogenesis of GBM and have the potential to be used as prognostic biomarkers [13-15]. In 2013, Dr. Hermansen, Dr. Kristensen and their colleagues described in details the procedure for the synthesis of miRNAs and introduced the methods for assessing miRNA expression. These preliminary studies and subsequent in-depth experiments have significantly advanced our understanding of the pathogenesis of GBM. The meta-analysis performed by other research groups have systematically explored the differential expression of miRNAs in multiple studies and provided us a unique miRNA expression profile that can help to improve the diagnosis and prognosis of GBM patients [16-19]. Researchers have evaluated the possibility of using the differential expression profile of some specific miRNA to predict the prognosis of GBM patients. Compared to healthy brain tissues, it was found that the expression of miR-7 [21], miR-34a [22], miR-128 [23], miR-218 [24], and miR-873 [25] were down-regulated in GBM patients while the expression of miR-10 was up-regulated. However, whether these biomarkers, such as the miRNA signature found in the above mentioned two studies, can be used as a molecular approach to better classify GBM and even replace the currently most widely used histopathological scoring system is yet unknown.MicroRNA-522 (miR-522) is a member of one of the most recently studied microRNAs. It has been demonstrated that miR-522 is closely related to multiple biological processes that involved in tumorigenesis. Wang and colleagues have shown that the small molecule miR-522 was significantly up-regulated in HBV-related HCC and its regulation of tumorigenesis was mainly mediated by modulation of CCND3 and CHC gene activities [26]. Zhang et al. have also demonstrated that miR-522 can promote the proliferation of liver tumor cells through modulation of DKK1 and SFRP2 gene activities [27]. The team also shown that miR-522 can act as a tumor suppressor gene in inhibiting the proliferation and metastasis of non-small cell lung cancers, by up-regulation of the expression of DENND 2D gene [27]. However, the molecular mechanisms of miR-522 in glioblastoma pathogenesis and progression are still largely unknown. Here, we present a study to assess the expression of miR-522 in GBM patients, the association of miR-522 expression with tumor cell proliferation and further investigate the mechanisms of miR-522 in regulation of GBM pathogenesis and progression.Researchers at the University of California in the United States first discovered pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatases 1 (PHLPP1) [28]. PHLPP1 has been shown to act as a tumor suppressor gene in the cytoplasm and nucleus as well as the cell membrane. The levels of PHLPP1 expression varied greatly in most of human tissues. However, it is often expressed at fairly low levels or not expressed in some tumor tissues, in the meanwhile, the expression of phosphorylated serine/threonine protein kinase B (AKT, also known as PKB) was significantly increased in these tissues. It is well known that PI3K/AKT is a key molecular signaling pathway that plays a critical role in the development and metastasis of many human tumors. The tumor suppression role of PHLPP1 was mainly mediated through dephosphorylating of AKT at Ser473, resulting negative regulation ofAKT expression. Therefore, through negative regulation of the PI3K/ AKT signaling pathway, PHLPP 1 can inhibit tumor formation [29].It has been found that PHLPP1 gene belongs to the PHLPP family and it includes two subunits:PHLPP1 a and PHLPP1 ?, which are located at the short arm of chromosome 18,18q21.33. The total length of its coding region is 6,401 base pairs (bp), including 17 exons and four transcript regions. The PHLPP1 a protein contains 1,205 amino acids and it molecular weight is very small, only 140 kDa. While the coding region of PHLPP1 ? gene is longer than that of PHLPP1 a. PHLPP1 0 is 6,156 bp in length; its protein contains 1,717 amino acids and molecular weight is 170 kDa. Although both of them are parts of the PHLPP1 gene, their coding regions were located in two different loci. PHLPP1 contains leucine-rich repeats (LRR) domains, including a PH domain (PH) and a PDZ ligand at the C terminal. They vary in size with PHLPP1 ? is larger and PHLPP1 ? is smaller, the former has a 56 kDa extension protein at the N terminus [30].As a tumor suppressor gene, PHLPP1 was down-regulated in most of the tumor cells. For example, in colorectal cancer, it was found that increased expression of PHLPP1 can significantly inhibit the proliferation of colorectal cancer cells and sensitize tumor cells to the PI3K inhibitors. Because PHLPP1 acts as a suppressor gene and suppresses the entire molecular processes of tumorigenesis, tumors with low expression of PHLPP1 proliferated rapidly while tumor with high expression of PHLPP1 showed slow progress [31]. This is exactly what was found in chronic lymphocytic leukemia (CLL). The expression of both PHLPPI and PHLPP2 were significantly reduced or undetectable in patients with 13q14 deletions and the lack of PHLPPI and PHLPP2 expression was observed in about 50% of the cases [32]. A variety of human tumors showed low or no expression of PHLPPI and PHLPP2, especially the malignant tumors such as breast cancer and ovarian cancer in female, renal nephroblastoma, prostate cancer in male, hepatocellular carcinoma, and pancreatic cancer occur in the body of pancreas [33-35]. Therefore, this study aims to elucidate the relationship of PHLPP1, as a tumor suppressor gene, with miR-522 in glioblastoma pathogenesis.The purpose of this study is to investigate the role and mechanisms of miR-522 in the development and progression of human GBM, and to explore whether its regulatory is through the tumor suppressor gene PHLPP1. In this study, we firstly examined the expression of miR-522 in human GBM and whether or not it has a stimulatory or inhibitory role in the proliferation of GBM. We further investigated the mechanisms of miR-522 in GBM pathogenesis and its relationship with the tumor suppressor gene PHLPP1.The mechanisms of miR-522 in modulating proliferation of human glioblastoma cellsObjectivesTo examine the expression of miR-522 in human glioblastoma multiforme (GBM), investigate the clinical significance of miR-522 dysregulation, and further explore the role and mechanisms of miR-522 in modulating GBM cell proliferation.MethodsTo investigate whether GBM cell proliferation can be modulated by miR-522, we transfected U87MG, the GBM cell line, with miR-522 mimics and miR-522 inhibitor respectively. To further verify the regulation of miR-522 in PHLPP1 activity, we constructed the PHLPP1 plasmid with wildtype and mutated 3'UTR and co-transfected it into U87MG cells with the miR-522 mimics or miR-522 inhibitor. The Real-time PCR was performed to assess the transfection efficiency. MTT assay and colony formation assay were used to determine the changes in the proliferation ability of GBM cells. Quantitative RT-PCR (QPCR) was used to examine the expression of miR-522 and western blot was used to examine the protein expression of PHLPP1. Targetscan prediction and dual luciferase reporter assay were used to evaluate the interaction between miR-522 and PHLPP1.Results1. MiR-522 was significantly upregulated in human GBM tissues and GBM cell lines. Compared to adjacent non-cancerous brain tissue, the QPCR assay clearly demonstrated that miR-522 expression was significantly upregulated in GBM tissues. We further verified this finding in GBM cell lines. The level of MiR-522 expression was significantly higher in GBM cell lines than that of the normal human astrocytes (NHAs).2. MiR-522 promotes GBM cell proliferation. We found that miR-522 expression was significantly increased in cells transfected with miR-522 mimics compared to the control group. The MTT and colony formation assays revealed that miR-522 significantly promoted U87MG cell proliferation, and this was further indicated by the anchorage-independent growth assay.3. miR-522 depletion suppresses U87MG cell proliferation. Consistent with the above findings, the proliferation of U87MG cells was significantly inhibited by the miR-522 inhibitor, anti-miR-522. miR-522 expression was dramatically decreased in cells transfected with anti-miR-522 and depletion of miR-522 suppressed U87MG proliferation. The suppression was clearly indicated by MTT assay, colony formation assay, and the anchorage-independent growth assay (Fig.3B-D).4. MiR-522 directly regulates PHLPP1 activity by binding to its 3'UTR and modulates cell proliferation. Using a bioinformatic approach, TargetScan Huaman v6.2, we found that miR-522 contains a seed sequence that is complementary to the sequence at the 3'UTR of the tumor suppressor gene PHLPP1, i.e. PHLPP1 has a binding site for miR-522 and thus it was selected as a target of miR-522 for further analysis. Western blotting assay showed that ectopic expression of miR-522 in U87MG cells induced by miR-522 mimics dramatically down-regulated PHLPP1 protein expression. Consistently, suppression of miR-522 with miR-522 inhibitor led to significant upregulation of PHLPP1 protein expression. With the luciferase reporter assay, we further verified the role of miR-522 in modulating PHLPP1 activity. Compared to the empty vector control, miR-522 overexpression induced by miR-522 mimics resulted in a significant decrease in the luciferase activity of the wildtype PHLPP, while miR-522 downregulation by miR-522 inhibitor dramatically increased the luciferase activity of the wildtype PHLPP. However, the luciferase activity of the PHLPP with mutant 3'UTR was unaffected. Our data strongly suggested that miR-522 is a negative regulator of PHLPP1 and its regulation was mediated by direct binding to its 3'UTR region of the gene.We also examined the expression levels of several other key mediators in cell proliferation. As expected, p27 was strikingly downregulated at both the mRNA and protein levels in miR-522-overexpressing cells. Interestingly, we also found that the cyclin D1, a CDK regulator and also a key player in cell proliferation, was dramatically upregulated at both the mRNA and protein levels in miR-522 overexpressing cells. Consistently, the mRNA and protein expression of cyclin D1 was significantly decreased when miR-522 expression was suppressed. Our results indicated that miR-522 modulates cell proliferation through regulation of some key cell proliferation regulators, such as p27 and cyclin D1.5. PHLPP1 downregulation contributed to miR-522-induced proliferation of GBM cells. As expected, western blot assay verified that PHLPP1-siRNA effectively decreased the expression of PHLPP1 in miR-522-transfected U87MG cells (Fig.5A). The colony formation assay and anchorage-independent growth assay revealed that PHLPP1 silencing in miR-522-transfected U87MG cells promoted cell proliferation (Fig.5B and C). Taken together, these results demonstrated that PHLPP1 downregulation contributed to miR-522-induced GBM cell proliferation.ConclusionIn summary, our preliminary results suggested that miR-522 plays an important role in modulating GBM cell proliferation and it may represent a promising novel therapeutic target for the treatment of GBM patients. |
PDF Full Text Request