Silent information regulator 3 modulates lysosome function to inhibit apoptosis of U87 cell by mTOR pathway

Abstract

Objective To investigate the role of silent information regulator 3 (Sirt3) in glioma U87 cells, and provide new targets for early diagnosis and treatment of glioma. Methods Based on the China Brain Glioma Database (CGGA) and GEPIA2 database, the expression of Sirt3 in different grades of glioma was analyzed by bioinformatics, and the protein level of Sirt3 in the adjacent and tumor samples of 6 glioma patients was further detected by Western blotting. Then, shRNA lentivirus and CRISPR/Cas9 system were used to interfere with the expression of Sirt3 in U87 cells, and TUNEL staining, PI-Annexin V double fluorescent labeling and Western blotting were used to detect the expression of apoptosis-related proteins(Bax, Cleaved-caspase3) and lysosome membrane proteins (LAMP1, LAMP2, LAMP2A), TFEB, mTOR, and other proteins. Then mTOR small molecule inhibitor, rapamycin was employed to treat Sirt3 knockdown cells, the changes in related proteins and cell apoptosis were detected. In addition, nuclear and cytoplasmic separation experiments were carried out on U87 cells before and after Sirt3 knockout, and protein change of p-TFEB was detected. Finally, the change of lysosome membrane protein after overexpression of TFEB and Sirt3 was detected in U87 cells with knocking down and out of Sirt3. Results Sirt3 was significantly highly expressed in glioma tissue, and the level was closely associated with poor survival of the glioma patients. Sirt3 knockdown significantly enhanced cell apoptosis (P < 0.01), down-regulated the expression of LAMP1, LAMP2 and LAMP2A (P < 0.01), and improved the expression of p-mTOR (P < 0.01). Additionally, Sirt3 knockout also up-regulated p-TFEB protein level in the cytoplasm fraction (P < 0.01) while, in the Sirt3 knockout U87 cells, its overexpression reserved the protein levels of LAMP1 and LAMP2 and increased the amount of lysosome. In the Sirt3 knockdown cells, overexpression of TFEB resulted in up-regulation of LAMPs proteins(P < 0.01). Conclusion Sirt3 may affect the nuclear-cytoplasmic distribution of TFEB by regulating the mTOR signaling pathway, up-regulate the expression of LAMPs, enhance the function of lysosome, and then promote the survival and malignant proliferation of glioma cells. Our study suggests that Sirt3 may be a molecular marker for clinical diagnosis and progression of glioma.

Keywords

• silent information regulator 3
• glioma
• apoptosis
• lysosome
• mtor
• tfeb