microRNA-29 mediates a novel negative feedback loop to regulate SCAP / SREBP-1 and lipid metabolism

The membrane-bound transcription factors, SREBPs (sterol regulatory element-binding proteins), play a central role in regulating lipid metabolism. The transcriptional activation of SREBPs requires the key protein SCAP (SREBP-cleavage activating protein) to translocate their precursors from the endoplasmic reticulum to the Golgi for subsequent proteolytic activation, a process tightly regulated by a cholesterol-mediated negative feedback loop. Our previous work showed that the SCAP/SREBP-1 pathway is significantly upregulated in human glioblastoma (GBM), the most deadly brain cancer, and that glucose-mediated N-glycosylation of SCAP is a prerequisite step for SCAP/SREBP trafficking. More recently, we demonstrated that microRNA-29 (miR-29) mediates a previously unrecognized negative feedback loop in SCAP/SREBP-1 signaling to control lipid metabolism. We found that SREBP-1, functioning as a transcription factor, promotes the expression of the miR-29 family members, miR-29a, -29b and -29c. In turn, the miR-29 isoforms reversely repress the expression of SCAP and SREBP-1. Moreover, treatment with miR-29 mimics effectively suppressed GBM tumor growth by inhibiting SCAP/SREBP-1 and de novo lipid synthesis. These findings, recently published in Cell Reports, strongly suggest that delivery of miR-29 in vivo may be a promising approach to treat cancer and metabolic diseases by suppressing SCAP/SREBP-1-regulated lipid metabolism.

Lipids are essential components of the cells, particularly phospholipids and cholesterol, constituting the basic structure of cell membrane system [1,2] .Furthermore, lipids also serve as important signaling molecules, regulating various cellular functions [3] .Dysregulation of lipid metabolism contributes to the pathogenesis of various metabolic syndromes, i.e., atherosclerosis, steatosis, obesity and diabetes [4] .Therefore, interfering with the dysregulated lipid metabolism in metabolic diseases has been a long-term focus of basic research and pharmacological development [4,5] .Nevertheless, the still incomplete understanding of the molecular mechanisms underlying the alteration of lipid metabolism significantly hinders progress.
The family of basic helix-loop-helix transcription factors, SREBPs (sterol regulatory element-binding proteins), plays a central role in lipid metabolism by controlling the de novo synthesis of fatty acids, phospholipids, cholesterol, and also
Brown & Goldstein put forth an elegant model of the regulation of SREBP activation through a cholesterol-mediated negative feedback loop (Fig. 1) [4,6] .
After translation, SREBPs immediately bind to the key protein, SCAP (SREBP-cleavage activating protein), to form a complex.SCAP further binds to Insig (insulin-induced gene protein), an endoplasmic reticulum (ER)-anchored protein, resulting in the formation of the Insig/SCAP/SREBP complex, which is retained in the ER by high levels of cholesterol [15][16][17] .When cholesterol levels decrease, SCAP dissociates from Insig, resulting in the degradation of Insig.SCAP then interacts with COPII proteins that translocate the SCAP/SREBP complex from the ER to the Golgi, where SREBPs are sequentially cleaved by site-1 and site-2 proteinases to release their transcriptionally active N-terminal fragments that enter into the nucleus to promote the transcription of lipogenic genes including Insig-1 [4,6,10,11,17,18] .Consequently, the levels of cholesterol and Insig are restored to bind again to the SCAP/SREBP complex, which Our previous study showed that glucose-mediated SCAP N-glycosylation enables SCAP/SREBP-1 trafficking from the ER to the Golgi for subsequent proteolytic activation.Furthermore, EGFR signaling enhances glucose uptake, thereby increasing SCAP N-glycosylation and SREBP-1 activation to promote tumor growth [23][24][25] .High levels of cholesterol increase the association of Insig and SCAP, resulting in the retention of the complex in the ER [6] .Our newly discovered negative feedback loop shows that SREBP-1 transcriptionally activates the expression of pri-miR-29a/b1 and pri-miR-29b2/c, which generate the mature miR-29a, -29b and 29c.In turn, miR-29 reversely inhibits the expression of SCAP and SREBP-1 by binding to their 3'-UTRs, resulting in the downregulation of lipogenesis genes [55] .SRE, sterol regulatory element (SREBP-binding motif present in the promoters of SREBP target genes).S1P, site 1 protease.S2P, site 2 protease.
To explore whether other factors are critical for SCAP/SREBP trafficking, we investigated the role of glucose-mediated N-glycosylation modification of the SCAP protein, and showed that it was a prerequisite step for SCAP/SREBP trafficking and activation upon cholesterol reduction [23][24][25] .We found that N-glycosylation stabilizes SCAP and reduces its association with Insig-1, allowing SCAP/SREBP movement from the ER to the Golgi (Fig. 1).Our study demonstrated that glucose is an essential activator of SCAP/SREBP trafficking, while cholesterol functions as a key inhibitor of this process [23][24][25] .
We identified miRNA-29 as a critical mediator of a novel negative feedback loop in the regulation of SCAP/SREBP-1 signaling [54] , providing a promising new approach to target GBM.The miRNA-29 family includes 3 members, miR-29a, -29b and -29c, which share the same seed sequence.miR-29b is encoded by pri-miR-29b1 and pri-miR-29b2, which are located on different chromosomes but generate the same mature miR-29b.Interestingly, pri-miR-29a and pri-miR-29b1 are both located on chromosome 7 and share the same promoter.Similarly, pri-miR-29b2 and pri-miR-29c are located on chromosome 1 and are co-transcribed by the same promoter [55,56] .
In our study, we found that expression of all 3 mature miR-29s was positively correlated with SREBP-1 gene expression in samples from a large cohort of GBM patients with altered EGFR (amplification or mutation) [55] .Furthermore, activating EGFR/PI3K/Akt signaling via EGF stimulation significantly enhanced the expression of all 3 miR-29s in GBM cell lines.Interestingly, both SREBP-1a and -1c directly bind to the promoter region of miR-29a/b1 and miR-29b2/c, activating their expression and generating mature miRNA-29a, -29b and -29c.We also showed that the 3'-untranslated region (3'-UTR) of SREBP-1 has binding sites for miR-29, and demonstrated that miR-29a, -29b and -29c inhibited the mRNA and protein levels of SREBP-1 by directly binding to these complementary sites.Importantly, our intracranial GBM xenograft studies show that miR-29 treatment significantly suppressed tumor growth via inhibition of SCAP/SREBP-1 and lipid synthesis [54] .miR-29 has been shown to be transcriptionally inhibited by transcription factors such as c-Myc, TGF-β and NF-κB in cancer cells [57][58][59] .Our study was the first to show that miR-29 expression is controlled by SREBP-1, and that miR-29 is directly involved in the regulation of lipid metabolism.This newly discovered negative feedback loop regulation of SCAP/SREBP-1 by miR-29 further demonstrates that lipid homeostasis is elegantly regulated by multi-layer of mechanisms in addition to cholesterol and glucose regulation [23][24][25]54] . Consiering the simple synthesis and easy delivery of mature microRNAs, miR-29 treatment may be a feasible and promising approach to treat cancers and other metabolic diseases.

Figure 1 .
Figure1.miR-29 mediates a novel negative feedback loop in SCAP/SREBP-1 signaling and regulates lipid metabolism.Our previous study showed that glucose-mediated SCAP N-glycosylation enables SCAP/SREBP-1 trafficking from the ER to the Golgi for subsequent proteolytic activation.Furthermore, EGFR signaling enhances glucose uptake, thereby increasing SCAP N-glycosylation and SREBP-1 activation to promote tumor growth[23][24][25] .High levels of cholesterol increase the association of Insig and SCAP, resulting in the retention of the complex in the ER[6] .Our newly discovered negative feedback loop shows that SREBP-1 transcriptionally activates the expression of pri-miR-29a/b1 and pri-miR-29b2/c, which generate the mature miR-29a, -29b and 29c.In turn, miR-29 reversely inhibits the expression of SCAP and SREBP-1 by binding to their 3'-UTRs, resulting in the downregulation of lipogenesis genes[55] .SRE, sterol regulatory element (SREBP-binding motif present in the promoters of SREBP target genes).S1P, site 1 protease.S2P, site 2 protease.