MicroRNA-301b promotes the proliferation and invasion of glioma cells through enhancing activation of Wnt/ß-catenin signaling via targeting Glypican-5.

Accumulating evidence has suggested that Glypican-5 (GPC5) is a tumor suppressor gene in many types of cancers. However, whether GPC5 is involved in glioma remains unknown. This study was designed to explore the expression, biological function and regulatory mechanism of GPC5 in glioma. Our results demonstrated that GPC5 expression was significantly decreased in multiple glioma cell lines. Gain-of-function experiments showed that the ectopic expression of GPC5 markedly inhibited the proliferation, invasion and Wnt/ß-catenin signaling of glioma cell lines. GPC5 was identified as a target gene of microRNA-301b (miR-301b). Further data showed that miR-301b expression was significantly up-regulated in glioma tissues and cell lines. In addition, miR-301b expression was inversely correlated with GPC5 expression in clinical glioma tissues. The overexpression of miR-301b promoted the proliferation, invasion and Wnt/ß-catenin signaling of glioma cell lines, whereas the inhibition of miR-301b showed the opposite effect. However, the silencing of GPC5 significantly reversed the antitumor effect of miR-301b inhibition. Overall, our results revealed a tumor suppressive role of GPC5 in glioma and suggested that GPC5 expression was regulated by miR-301b. Our study indicates that the inhibition of miR-301b represses the proliferation and invasion of glioma cells by up-regulating GPC5 expression.

mTOR enhances foam cell formation by suppressing the autophagy pathway.

Recently, autophagy has drawn more attention in cardiovascular disease as it has important roles in lipid metabolism. Mammalian target of rapamycin (mTOR) is a key regulator of autophagy; however, its effect on atherosclerosis and the underlying mechanism remains undefined. In this study, an obvious upregulation of mTOR and p-mTOR protein was observed in macrophage-derived foam cells. Blocking mTOR expression with specific small interference RNA (siRNA) dramatically suppressed foam cell formation, accompanied by a decrease of lipid deposition. Further mechanistic analysis indicated that suppressing mTOR expression significantly upregulated autophagic marker LC3 expression and downregulated autophagy substrate p62 levels, indicating that mTOR silencing triggered autophagosome formation. Moreover, blocking mTOR expression obviously accelerated neutral lipid delivery to lysosome and cholesterol efflux from foam cells, implying that mTOR could induce macrophage foam cell formation by suppressing autophagic pathway. Further, mTOR silencing significantly upregulated ULK1 expression, which was accounted for mTOR-induced foam cell formation via autophagic pathway as treatment with ULK1 siRNA dampened LC3-II levels and increased p62 expression, concomitant with lipid accumulation and decreased cholesterol efflux from foam cells. Together, our data provide an insight into how mTOR accelerates the pathological process of atherosclerosis. Accordingly, blocking mTOR levels may be a promising therapeutic agent against atherosclerotic complications.

Knockdown of mTOR by lentivirus­mediated RNA interference suppresses atherosclerosis and stabilizes plaques via a decrease of macrophages by autophagy in apolipoprotein E­deficient mice.

Atherosclerotic plaque destabilization and rupture leads to acute coronary syndromes which cause serious damage to human health worldwide. However, there is currently a lack of efficient therapeutic methods. Mammalian target of rapamycin (mTOR) has been suggested to be involved in the development of atherosclerotic plaques and serves as a therapeutic target. The present study was performed to determine whether RNA interference (RNAi) of mTOR in vivo by LV­mediated small hairpin RNA (shRNA) was capable of inhibiting the progression of atherosclerotic plaques. LV­mediated shRNA against mTOR (LV­shmTOR) was designed and obtained. Male apolipoprotein E­deficient mice were fed a high­fat diet and a constrictive collar was placed around the right carotid arteries of these mice to induce plaque formation. Eight weeks after surgery, mice were randomly divided into the mTOR RNA interference (LV­shmTOR) group, receiving treatment with LV­mTOR­shRNA; the LV­shCON group, receiving treatment with LV­non­specific­shRNA; and the control group, receiving treatment with phosphate­buffered saline. Following transfection, the mice were sacrificed to evaluate the effects of mTOR expression silencing on atherosclerosis. Transfection of LV­mTOR­shRNA markedly inhibited the mRNA and protein expression levels. Knockdown of mTOR ameliorated dysregulated blood lipid metabolism and stabilized aortic atherosclerotic plaques by decreasing the plaque area and increasing the fibrous cap and cap­to­core ratio. Furthermore, macrophages were decreased by silencing mTOR in atherosclerotic plaques. In addition, western blot analysis revealed that the knockdown of mTOR increased autophagy­related protein 13 (Atg13) dephosphorylation and light chain 3­I/light chain 3­II (LC3­I/LC3­II) ratios, both of which were associated with a high activity of autophagy, suggesting an increase of autophagy in atherosclerotic plaques. Moreover, genes including matrix metalloproteinase 2, monocyte chemoattractant protein 1 and tissue factor, which promote plaque instability, were downregulated by silencing mTOR. These results demonstrate that LV­mediated mTOR silencing by RNAi treatment induces macrophage autophagy and is a potential strategy for the treatment of atherosclerotic plaques.