Knockdown of PHLDA2 promotes apoptosis and autophagy of glioma cells through the AKT/mTOR pathway.

Pleckstrin homology like domain family A member 2 (PHLDA2) is an imprinted gene expressed in placenta and has been shown to be associated with tumor progression. However, the effect of PHLDA2 on glioma cell growth has not been reported yet. Data based on TCGA database showed that PHLDA2 was up-regulated in glioma tissues. Moreover, PHLDA2 was also elevated in glioma cells. Functional assays showed that siRNA-mediated knockdown of PHLDA2 reduced cell viability of glioma cells and suppressed the cell proliferation. Cell apoptosis of glioma cells was promoted by silencing of PHLDA2 with increased Bax and decreased Bcl-2. Silencing of PHLDA2 reduced protein expression of p62, enhanced LC3 and Beclin1 to promote autophagy. Phosphorylated AKT and mTOR were down-regulated in glioma cells by interference of PHLDA2. In conclusion, downregulation of PHLDA2 inhibited glioma cell proliferation, and promoted cell apoptosis and autophagy through inactivation of AKT/mTOR signaling.

Downregulation of ZMYND11 induced by miR-196a-5p promotes the progression and growth of GBM.

ZMYND11 (zinc finger MYND-type containing 11) has been widely regarded to be involved in a variety of cancers as a potential suppressor. However, the biological role and mechanism of ZMYND11 in glioblastoma multiform (GBM) remain unknown. In this study, we found that ZMYND11 expression was remarkably decreased in GBM tissues from 20 cases and cell line (U87) compared to normal brain tissue from 10 cases (P < 0.001). Furthermore, we explored that ZMYND11 upregulation significantly suppressed U87 cells proliferation and invasion, induced cell cycle arrest and apoptosis in vitro. Subsequently, we identified increased ZMYND11 inhibited the tumor growth using tumor cells xenograft experiment on rude mice. Moreover, we explored that ZMYND11 was a new direct and functional target of miR-196a-5p in U87 via luciferase reporter assay. In addition, we confirmed the negative correlation between miR-196a-5p and ZMYND11 in GBM tissue and U87 cells by changing the expression level of miR-196a-5p with lentivirus and plasmid vector. Furthermore, we demonstrated that decreased ZMYND11 could reverse suppressive effect of downregulated miR-196a-5p on U87 by rescue experiment. Taken together, ZMYND11 was demonstrated to be a potential and extremely promising suppressor of GBM, while miRNA-196a-5p was quite an important target of treatment of GBM.

LATS2 inhibits cell proliferation and metastasis through the Hippo signaling pathway in glioma.

As a core kinase in the Hippo pathway, large tumor suppressor kinase 2 (LATS2) regulates cell proliferation, migration and invasion through numerous signaling pathways. However, its functions on cell proliferation, migration and invasion in glioma have yet to be elucidated. The present study revealed that LATS2 was downregulated in glioma tissues and cells, as determined by reverse transcription­quantitative polymerase chain reaction and immunohistochemistry. In addition, Cell Counting Kit­8, scratch wound healing and Transwell assays revealed that overexpression of LATS2 in U­372 MG cells inhibited cell proliferation, migration and invasion. Furthermore, western blot analysis indicated that the expression levels of phosphorylated (p)­yes­associated protein and p­tafazzin were increased in cells with LATS2 overexpression. These results indicated that LATS2 is a potential tumor suppressor, and downregulation of LATS2 in glioma may contribute to cancer progression.

Cytoplasmic polyadenylation element-binding protein 4 is highly expressed in human glioma.

Cytoplasmic polyadenylation element-binding protein 4 (CPEB4) is a highly conserved, sequence-specific RNA-binding protein that recruits translational repression or cytoplasmic polyadenylation machinery to target mRNAs. Recent studies have shown that CPEBs are expressed in somatic tissues and have essential functions supporting tumor growth, vascularization, and invasion. Overexpression of CPEB4 has been reported in pancreatic ductal adenocarcinoma and is associated with poor prognoses. However, whether CPEB4 plays a role in the tumorigenesis of gliomas is unknown. Here, we analyzed the expression of CPEB4 in gliomas. The expression profiles of CPEB4 mRNA and protein in nine normal brain tissues and 63 gliomas were detected using immunohistochemistry, real-time PCR, and western blotting. CPEB4-positive expression was significantly correlated with the pathological grade of glioma; abundant expression was observed in high-grade gliomas, whereas little or no expression was observed in normal astrocytes. Immunohistochemistry staining indicated that CPEB4 was mainly localized in the cytoplasm. In addition, CPEB4 was more highly expressed in U87 glioma cells than in U251 cells. CPEB4 expression significantly correlated with the grade in clinical gliomas. This study suggested that CPEB4 might play a role in the pathogenesis of glioma.

Downregulation of miR-95-3p inhibits proliferation, and invasion promoting apoptosis of glioma cells by targeting CELF2.

Gliomas are the most common and aggressive types of tumors in human brain, of which the prognosis remains dismal because of their biological behavior. The involvement of miRNAs in tumorigenesis of various kinds of cancers drives us to explore new miRNAs related to gliomas. We measured expression level of miR­95­3p by qRT-PCR in human glioma and non-neoplasm brain tissues and found that higher level of miR­95­3p in glioma tissues of higher grade. Biological functions of miR­95­3p on glioma cells were investigated by MTT assay, flow cytometry and transwell assay. We discovered the cell lines transfected with miR­95­3p ASO (antisense oligonucleotide) had retarded proliferation and invasion but enhanced apoptosis ability. We searched on-line tool Targetscan and selected CELF (CUGBP- and ETR-3-like family 2) as a putative target. Luciferase reporter was employed to confirm the binding sites in 3'UTR region of CELF2 for miR­95­3p. The correlation between expression of CELF2 and miR­95­3p was determined by western blotting and qRT-PCR both in cell lines and human samples. Results showed CELF2 was a direct target of miR­95­3p and expression levels of CELF2 and miR­95­3p were negatively correlated. Finally, CELF2 largely abrogated the effects of miR­95­3p on proliferation, invasion and apoptosis of glioma cells in rescue experiments, which verified the role of CELF2 in miR­95­3p regulating glioma biological behavior. In conclusion, our data suggest the expression level of miR­95­3p is positively related to glioma grade and downregulation of miR­95­3p affects proliferation, invasion and apoptosis of glioma cells by targeting CELF2. We identified miR­95­3p as a putative therapeutic target and CELF2 as a potential tumor suppressor.