SCYL3, as a novel binding partner and regulator of ROCK2, promotes hepatocellular carcinoma progression.

Background & Aims: SCY1-like pseudokinase 3 (SCYL3) was identified as a binding partner of ezrin, implicating it in metastasis. However, the clinical relevance and functional role of SCYL3 in cancer remain uncharacterized. In this study, we aimed to elucidate the role of SCYL3 in the progression of hepatocellular carcinoma (HCC). Methods: The clinical significance of SCYL3 in HCC was evaluated in publicly available datasets and by qPCR analysis of an in-house HCC cohort. The functional significance and mechanistic consequences of SCYL3 were examined in SCYL3-knockdown/overexpressing HCC cells. In vivo tumor progression was evaluated in Tp53 KO/c-Myc OE mice using the sleeping beauty transposon system. Potential downstream pathways were investigated by co-immunoprecipitation, western blotting analysis and immunofluorescence staining. Results: SCYL3 is often overexpressed in HCC; it is preferentially expressed in metastatic human HCC tumors and is associated with worse patient survival. Suppression of SCYL3 in HCC cells attenuated cell proliferation and migration as well as in vivo metastasis. Intriguingly, endogenous SCYL3 overexpression increased tumor development and metastasis in Tp53 KO/c-Myc OE mice. Mechanistic investigations revealed that SCYL3 physically binds and regulates the stability and transactivating activity of ROCK2 (Rho kinase 2) via its C-terminal domain, leading to the increased formation of actin stress fibers and focal adhesions. Conclusions: These findings reveal that SCYL3 plays a critical role in promoting the progression of HCC and have implications for developing new therapeutic strategies to tackle metastatic HCC. Impact and implications: SCYL3 was first reported to be a binding partner of a metastasis-related gene, ezrin. To date, the clinical relevance and functional role of SCYL3 in cancer remain uncharacterized. Herein, we uncover its crucial role in liver cancer progression. We show that it physically binds and regulates the stability and transactivating activity of ROCK2 leading to HCC tumor progression. Our data provide mechanistic insight that SCYL3-mediated ROCK2 protein stability plays a pivotal role in growth and metastasis of HCC cells. Targeting SCYL3/ROCK2 signaling cascade may be a novel therapeutic strategy for treatment of HCC patients.

LncRNA SNHG16 promotes epithelial-mesenchymal transition by upregulating ITGA6 through miR-488 inhibition in osteosarcoma.

BACKGROUND: Osteosarcoma is a primary cause of cancer-associated death in children and adolescents worldwide. Long non-coding RNAs SNHG16 (lncRNA SNHG16) and integrin subunit-a 6 (ITGA6) are recently reported to be involved in the tumorigenesis of osteosarcoma by multiple mechanisms. However, the correlation between SNHG16 and ITGA6 in osteosarcoma remains undetermined. METHODS: Expression of miR-488, SNHG16 and ITGA6, as well as epithelial-mesenchymal transition (EMT) associated markers in osteosarcoma tissues and cell lines were examined by qRT-PCR or Western blotting. Effects of miR-488, SNHG16 and ITGA6 on cell migration, invasion were evaluated by wound-healing assay and transwell assay. Bioinformatics analysis and dual-luciferase reported assays were applied to assess the interaction among miR-488, SNHG16 and ITGA6. RNA immunoprecipitation (RIP) was also used to verify SNHG16 and miR-488 interaction. Finally, animal study was used to detect the effect of SNHG16 on osteosarcoma in vivo. RESULTS: SNHG16 and ITGA6 were significantly increased while miR-488 was decreased in osteosarcoma. ITGA6 was screened as a target gene of miR-488, and SNHG16 was sponged by miR-488 in osteosarcoma cells. MiR-488 overexpression and SNHG16 knockdown suppressed migration, invasion and EMT of osteosarcoma cells. Moreover, rescue assays proved that the influences of SNHG16 on osteosarcoma cells migration, invasion and EMT were dependent on miR-488 and ITGA6. In addition, the promotive effects of SNHG16 on osteosarcoma tumor growth and metastasis were further supported by xenograft tumor growth assay. CONCLUSION: SNHG16 promoted migration, invasion and EMT of osteosarcoma by sponging miR-488 to release ITGA6.

Inhibition of long noncoding RNA HIF1A-AS2 confers protection against atherosclerosis via ATF2 downregulation.

INTRODUCTION: In atherosclerotic lesions, extensive inflammation of the vessel wall contributes to plaque instability. Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes in atherosclerosis. OBJECTIVES: Here, we aim to identify the functional role and regulatory mechanisms of lncRNA hypoxia-inducible factor 1 alpha-antisense RNA 2 (HIF1A-AS2) in atherosclerotic inflammation. METHODS: An atherosclerotic mouse model was induced in ApoE-/- mice by high fat diet (HFD). Endothelial cells (ECs), human aortic smooth muscle cells (SMCs) or human coronary artery endothelial cells (HCAECs) were exposed to ox-LDL to develop the in vitro model. The effects of lncRNA HIF1A-AS2 on inflammation were evaluated by determining levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) and levels of adhesion molecules vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and macrophage cationic peptide 1 (MCP-1). RESULTS: It was established that lncRNA HIF1A-AS2 and ATF2 were highly expressed in atherosclerotic ApoE-/- mice. Downregulating lncRNA HIF1A-AS2 in ox-LDL-exposed ECs, SMCs and HCAECs inhibited inflammation by reducing levels of pro-inflammatory factors and adhesion molecules. LncRNA HIF1A-AS2 bound to the transcription factor USF1 to elevate ATF2 expression. USF1 overexpression counteracted the suppressive effect of lncRNA HIF1A-AS2 silencing on ox-LDL-induced inflammation. Knockdown of lncRNA HIF1A-AS2 or ATF2 could also attenuate inflammation in atherosclerotic mice. Collectively, the present study demonstrates that downregulation of lncRNA HIF1A-AS2 represses the binding of USF1 to the ATF2 promoter region and then inhibits ATF2 expression, thereby suppressing atherosclerotic inflammation. CONCLUSION: This study suggests lncRNA HIF1A-AS2 as an promising therapeutic target for atherosclerosis.

Mechanical strain-mediated reduction in RANKL expression is associated with RUNX2 and BRD2.

Mechanical loading-related strains trigger bone formation by osteoblasts while suppressing resorption by osteoclasts, uncoupling the processes of formation and resorption. Osteocytes may orchestrate this process in part by secreting sclerostin (SOST), which inhibits osteoblasts, and expressing receptor activator of nuclear factor-κB ligand (RANKL/TNFSF11) which recruits osteoclasts. Both SOST and RANKL are targets of the master osteoblastic transcription factor RUNX2. Subjecting human osteoblastic Saos-2 cells to strain by four point bending down-regulates their expression of SOST and RANKL without altering RUNX2 expression. RUNX2 knockdown increases basal SOST expression, but does not alter SOST down-regulation following strain. Conversely, RUNX2 knockdown does not alter basal RANKL expression, but prevents its down-regulation by strain. Chromatin immunoprecipitation revealed RUNX2 occupies a region of the RANKL promoter containing a consensus RUNX2 binding site and its occupancy of this site decreases following strain. The expression of epigenetic acetyl and methyl writers and readers was quantified by RT-qPCR to investigate potential epigenetic bases for this change. Strain and RUNX2 knockdown both down-regulate expression of the bromodomain acetyl reader BRD2. BRD2 and RUNX2 co-immunoprecipitate, suggesting interaction within regulatory complexes, and BRD2 was confirmed to interact with the RUNX2 promoter. BRD2 also occupies the RANKL promoter and its occupancy was reduced following exposure to strain. Thus, RUNX2 may contribute to bone remodeling by suppressing basal SOST expression, while facilitating the acute strain-induced down-regulation of RANKL through a mechanosensitive epigenetic loop involving BRD2.

Role of Blood Oxygen Saturation During Post-Natal Human Cardiomyocyte Cell Cycle Activities.

Blood oxygen saturation (SaO2) is one of the most important environmental factors in clinical heart protection. This study used human heart samples and human induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) to assess how SaO2 affects human CM cell cycle activities. The results showed that there were significantly more cell cycle markers in the moderate hypoxia group (SaO2: 75% to 85%) than in the other 2 groups (SaO2 <75% or >85%). In iPSC-CMs 15% and 10% oxygen (O2) treatment increased cell cycle markers, whereas 5% and rapid change of O2 decreased the markers. Moderate hypoxia is beneficial to the cell cycle activities of post-natal human CMs.

Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: evidence for involvement of splicing regulatory proteins.

Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations.