Abstract
Long non-coding RNA THOR (Lnc-THOR) binds to IGF2BP1, essential for its function. We here show that Lnc-THOR is expressed in human glioma tissues and cells. Its expression is extremely low or even undetected in normal brain tissues, as well as in human neuronal cells and astrocytes. We show that Lnc-THOR directly binds to IGF2BP1 in established and primary human glioma cells. shRNA-mediated Lnc-THOR knockdown or CRISPR/Cas9-induced Lnc-THOR knockout potently inhibited cell survival and proliferation, while provoking glioma cell apoptosis. Contrarily, forced overexpression of Lnc-THOR promoted glioma cell growth and migration. Importantly, Lnc-THOR shRNA or knockout activated MAGEA6-AMPK signaling in glioma cells. AMPK inactivation, by AMPKα1 shRNA, knockout, or dominant-negative mutation (T172A), attenuated Lnc-THOR shRNA-induced A172 glioma cell apoptosis. Moreover, CRISPR/Cas9-induced IGF2BP1 knockout activated MAGEA6-AMPK signaling as well, causing A172 glioma cell apoptosis. Significantly, Lnc-THOR shRNA was ineffective in IGF2BP1 KO A172 cells. In vivo, Lnc-THOR silencing or knockout potently inhibited subcutaneous A172 xenograft tumor growth in mice. MAGEA6 downregulation and AMPK activation were detected in Lnc-THOR-silenced/-KO A172 tumor tissues. Taken together, Lnc-THOR depletion inhibits human glioma cell survival possibly by activating MAGEA6-AMPK signaling.
Highlights
Glioma is among the most aggressive human malignancies, causing significant human mortalities each year[1,2,3]
We have previously shown that MAGEA6 knockdown by targeted short hairpin RNA restored AMPKα1 expression, causing glioma cell death and apoptosis[21]
As described in our previous studies[21], a total of five pairs of human glioma tissues (“T”) and paired surrounding normal brain tissues (“N”) were analyzed, and Quantitative real-time reverse transcriptase polymerase chain reaction (qPCR) assay results in Fig. 1a show that Lnc-THOR levels are high in human glioma tissues, whereas its levels in normal brain tissues are, extremely low (Fig. 1a)
Summary
Lnc-THOR knockdown was verified by qPCR assay. The lentiviral GV248-Lnc-THOR construct (“LV-Lnc-THOR”) was transfected to glioma cells (plated at a density of 1 × 105 cells/well into 6-well plates), followed by selection using puromycin (2.5 μg/ mL) for 10–12 days. Lnc-THOR overexpression was verified by qPCR assay. Puromycin (2.5 μg/mL)containing complete medium was added to select stable cells for 5–6 days. IGF2BP1 or AMPKα1 KO in the stable cells was confirmed by western blotting and/or qPCR assays. The biotin-labeled fulllength Lnc-THOR (provided by Dr Wang39) was folded in RNA structure buffer and incubated with cleared nuclei lysates of the glioma cells together with Dynabeads MyOne Streptavidin C1 magnetic beads (“Beads,” again provided by Dr Wang[39]). Two-tailed unpaired T test (Excel 2013) was applied to test significance between the two treatment groups. p < 0.05 was considered significant
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