STEM-23. LncRNA HOTAIR SPONGES miR301a-3p TO PROMOTE GLIOMA’S PROLIFERATION AND INVASION THROUGH THE ACTIVATION OF FosL1

BACKGROUND Non-coding RNAs, including LncRNAs, function in regulating glioblastomas’ numerous oncogenic phenotypes, such as invasiveness and stemness of glioblastoma stem cells. These LncRNAs contain long non-encoding RNA transcripts >200 nucleotides in length, many of which show cell type-specific expression. In this project, we investigated how TRPM7, a promoter for glioma’s proliferation and invasion, regulates LncRNA and contributes to glioma tumorigenesis. METHODS 1) Total RNA, from either with A172 glioma cells or A172 glioma cells with TRPM7 knocked out (A172 KO), were extracted and then parallelly subjected to Human Cancer Pathway Finder, RT2 LncRNA PCR Array. 2) We then analyzed the prognostic role of LncRNAs using a publicly available bioinformatics data set (www.Oncolanc.org). 3) We examined the effects of TRPM7-regulated LncRNA, and its downstream molecules miR301a-3p and FosL1 oncogene, on the proliferation and invasion of glioma cells in A172 and PDX-L12 cells by either inhibition or activation of the above molecules. RESULTS 1) Data analysis from RT2 data resulted in a list of 10 downregulated and 7 upregulated LncRNAs whose transcripts are statistically significant with fold changes greater than 2.0 by TRPM7 knockout. 2) The results showed that TRPM7 functions as a positive regulator of LncRNA, HOTAIR, which is an unfavorable prognostic factor of 7-year overall survival in glioma patients. 3) In addition, we found that LncRNA HOTAIR sponges miR-301a-3p to promote proliferation, invasion, and stemness of glioma through upregulating the oncogene FosL1. 4) Furthermore, we identified that FosL1 is a novel prognostic marker of glioma patients. CONCLUSION Our results demonstrated the role of TRPM7-regulated LncRNA HOTAIR as a miRNA sponge in glioma, which shed new light on LncRNA-directed therapeutics in glioma.

KRIBB11 Induces Apoptosis in A172 Glioblastoma Cells via MULE-Dependent Degradation of MCL-1

KRIBB11, an HSF1 inhibitor, was shown to sensitize various types of cancer cells to treatment with several anticancer drugs. However, the exclusive effects of KRIBB11 in preventing the growth of glioblastoma cells and the related mechanisms have not been elucidated yet. Herein, we aimed to examine the potential of KRIBB11 as an anticancer agent for glioblastoma. Using MTT and colony formation assays and Western blotting for c-PARP, we demonstrated that KRIBB11 substantially inhibits the growth of A172 glioma cells by inducing apoptosis. At the molecular level, KRIBB11 decreased anti-apoptotic protein MCL-1 levels, which was attributable to the increase in MULE ubiquitin ligase levels. However, the constitutive activity of HSF1 in A172 cells was not influenced by the exclusive treatment with KRIBB11. Additionally, based on cycloheximide chase assay, we found that KRIBB11 markedly retarded the degradation of MULE. In conclusion, stabilization of MULE upon KRIBB11 treatment is apparently an essential step for degradation of MCL-1 and the subsequent induction of apoptosis in A172 cells. Our results have expanded the knowledge on molecular pathways controlled by KRIBB11 and could be potentially effective for developing an inhibitory therapeutic strategy for glioblastoma.

Clock-Controlled Mitochondrial Dynamics Correlates with Cyclic Pregnenolone Synthesis

Neurosteroids are steroids synthetized in the nervous system, with the first step of steroidogenesis taking place within mitochondria with the synthesis of pregnenolone. They exert important brain-specific functions by playing a role in neurotransmission, learning and memory processes, and neuroprotection. Here, we show for the first time that mitochondrial neurosteroidogenesis follows a circadian rhythm and correlates with the rhythmic changes in mitochondrial morphology. We used synchronized human A172 glioma cells, which are steroidogenic cells with a functional core molecular clock, to show that pregnenolone levels and translocator protein (TSPO) are controlled by the clock, probably via circadian regulation of mitochondrial fusion/fission. Key findings were recapitulated in mouse brains. We also showed that genetic or pharmacological abrogation of fusion/fission activity, as well as disturbing the core molecular clock, abolished circadian rhythms of pregnenolone and TSPO. Our findings provide new insights into the crosstalk between mitochondrial function (here, neurosteroidogenesis) and circadian cycles.

Mambalgin-2 Induces Cell Cycle Arrest and Apoptosis in Glioma Cells via Interaction with ASIC1a

Gliomas are fast growing and highly invasive brain tumors, characterized by tumor microenvironment acidification that drives glioma cell growth and migration. Channels containing Acid-sensing Ion Channel 1a subunit (ASIC1a) mediate amiloride-sensitive cation influx in late stage glioma cells, but not in normal astrocytes. Thus, selective targeting of ASIC1a can be a perspective strategy for glioma treatment. Here, ASIC1a expression in U251 MG and A172 glioma cells, but not in normal astrocytes, was demonstrated. Recombinant analog of mambalgin-2 from black mamba Dendroaspis polylepis inhibited amiloride-sensitive currents at ASIC1a both in Xenopus laevis oocytes and in U251 MG cells, while its mutants with impaired activity towards this channel did not. Mambalgin-2 inhibited U251 MG and A172 glioma cells growth with EC50 in the nanomolar range without affecting the proliferation of normal astrocytes. Notably, mambalgin-2 mutants did not affect glioma cell proliferation, pointing on ASIC1a as the main molecular target of mambalgin-2 in U251 MG and A172 cells. Mambalgin-2 induced a cell cycle arrest, inhibited Cyclin D1 and cyclin-dependent kinases (CDK) phosphorylation and caused apoptosis in U251 MG and A172 cells. Moreover, mambalgin-2 inhibited the growth of low-passage primary cells from a patient with glioblastoma. Altogether, our data point to mambalgin-2 as a useful hit for the development of new drugs for glioma treatment.

MicroRNA-29a-3p Downregulation Causes Gab1 Upregulation to Promote Glioma Cell Proliferation

Background/Aims: Glioma causes significant human mortalities annually. Molecularly-targeted therapy is a focus of glioma research. Methods: Grb2-associated binding 1 (Gab1) expression and microRNA-29a-3p (“miR-29a-3p”) expression in human glioma cells and tissues were tested by Western blotting assay and qRT-PCR assay. shRNA/siRNA strategy was applied to silence Gab1 in human glioma cells. miR-29a or anti-sense miR-29a construct was transfected to human glioma cells. Cell proliferation was tested by BrdU ELISA assay and cell counting assay. Results: We show that expression of Gab1 was significantly elevated in human glioma tissues and cells, which correlated with downregulation of its putative microRNA: miR-29a-3p. In A172 glioma cells and primary human glioma cells, Gab1 shRNA/siRNA inhibited Akt-Erk activation and cell proliferation. Forced-expression of miR-29a-3p downregulated Gab1, inhibiting glioma cell proliferation, whereas miR-29a-3p was in-effective on cell proliferation in Gab1-silenced A172 cells. Furthermore, introduction of a 3’-untranslated region (3’-UTR) mutant Gab1 (UTR-G160A) blocked miR-29a-3p-induced inhibition on Akt signaling and A172 cell proliferation. Conclusions: miR-29a-3p downregulation leads to Gab1 upregulation to promote glioma cell proliferation.