Caveolin-1 promotes mitochondrial health and limits mitochondrial ROS through ROCK/AMPK regulation of basal mitophagic flux.

Caveolin-1 (CAV1), the main structural component of caveolae, is phosphorylated at tyrosine-14 (pCAV1), regulates signal transduction, mechanotransduction, and mitochondrial function, and plays contrasting roles in cancer progression. We report that CRISPR/Cas9 knockout (KO) of CAV1 increases mitochondrial oxidative phosphorylation, increases mitochondrial potential, and reduces ROS in MDA-MB-231 triple-negative breast cancer cells. Supporting a role for pCAV1, these effects are reversed upon expression of CAV1 phosphomimetic CAV1 Y14D but not non-phosphorylatable CAV1 Y14F. pCAV1 is a known effector of Rho-associated kinase (ROCK) signaling and ROCK1/2 signaling mediates CAV1 promotion of increased mitochondrial potential and decreased ROS production in MDA-MB-231 cells. CAV1/ROCK control of mitochondrial potential and ROS is caveolae-independent as similar results were observed in PC3 prostate cancer cells lacking caveolae. Increased mitochondrial health and reduced ROS in CAV1 KO MDA-MB-231 cells were reversed by knockdown of the autophagy protein ATG5, mitophagy regulator PINK1 or the mitochondrial fission protein Drp1 and therefore due to mitophagy. Use of the mitoKeima mitophagy probe confirmed that CAV1 signaling through ROCK inhibited basal mitophagic flux. Activation of AMPK, a major mitochondrial homeostasis protein inhibited by ROCK, is inhibited by CAV1-ROCK signaling and mediates the increased mitochondrial potential, decreased ROS, and decreased basal mitophagy flux observed in wild-type MDA-MB-231 cells. CAV1 regulation of mitochondrial health and ROS in cancer cells therefore occurs via ROCK-dependent inhibition of AMPK. This study therefore links pCAV1 signaling activity at the plasma membrane with its regulation of mitochondrial activity and cancer cell metabolism through control of mitophagy.

Exome sequencing in multiplex families with left-sided cardiac defects has high yield for disease gene discovery.

Congenital heart disease (CHD) is a common group of birth defects with a strong genetic contribution to their etiology, but historically the diagnostic yield from exome studies of isolated CHD has been low. Pleiotropy, variable expressivity, and the difficulty of accurately phenotyping newborns contribute to this problem. We hypothesized that performing exome sequencing on selected individuals in families with multiple members affected by left-sided CHD, then filtering variants by population frequency, in silico predictive algorithms, and phenotypic annotations from publicly available databases would increase this yield and generate a list of candidate disease-causing variants that would show a high validation rate. In eight of the nineteen families in our study (42%), we established a well-known gene/phenotype link for a candidate variant or performed confirmation of a candidate variant's effect on protein function, including variants in genes not previously described or firmly established as disease genes in the body of CHD literature: BMP10, CASZ1, ROCK1 and SMYD1. Two plausible variants in different genes were found to segregate in the same family in two instances suggesting oligogenic inheritance. These results highlight the need for functional validation and demonstrate that in the era of next-generation sequencing, multiplex families with isolated CHD can still bring high yield to the discovery of novel disease genes.

ROCK1 is a multifunctional factor maintaining the primordial follicle reserve and follicular development in mice.

The follicle is the basic structural and functional unit of the ovary in female mammals. The excessive depletion of follicles will lead to diminished ovarian reserve or even premature ovarian failure, resulting in diminished ovarian oogenesis and endocrine function. Excessive follicular depletion is mainly due to loss of primordial follicles. Our analysis of published human ovarian single-cell sequencing results by others revealed a significant increase in rho-associated protein kinase 1 (ROCK1) expression during primordial follicle development. However, the role of ROCK1 in primordial follicle development and maintenance is not clear. This study revealed a gradual increase in ROCK1 expression during primordial follicle activation. Inhibition of ROCK1 resulted in reduced primordial follicle activation, decreased follicular reserve, and delayed development of growing follicles. This effect may be achieved through the HIPPO pathway. The present study indicates that ROCK1 is a key molecule for primordial follicular reserve and follicular development.NEW & NOTEWORTHY ROCK1, one of the Rho GTPases, plays an important role in primordial follicle reserve and follicular development. ROCK1 was primarily expressed in the cytoplasm of oocytes and granulosa cell in mice. Inhibition of ROCK1 significantly reduced the primordial follicle reserve and delayed growing follicle development. ROCK1 regulates primordial follicular reserve and follicle development through the HIPPO signaling pathway. These findings shed new lights on the physiology of sustaining female reproduction.

RhoA/rho kinase pathway activation in age-associated endothelial cell dysfunction and thrombosis.

The small GTPase RhoA and the downstream Rho kinase (ROCK) regulate several cell functions and pathological processes in the vascular system that contribute to the age-dependent risk of cardiovascular disease, including endothelial dysfunction, excessive permeability, inflammation, impaired angiogenesis, abnormal vasoconstriction, decreased nitric oxide production and apoptosis. Frailty is a loss of physiological reserve and adaptive capacity with advanced age and is accompanied by a pro-inflammatory and pro-oxidative state that promotes vascular dysfunction and thrombosis. This review summarises the role of the RhoA/Rho kinase signalling pathway in endothelial dysfunction, the acquisition of the pro-thrombotic state and vascular ageing. We also discuss the possible role of RhoA/Rho kinase signalling as a promising therapeutic target for the prevention and treatment of age-related cardiovascular disease.

A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells.

Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.

CTNNAL1 promotes the structural integrity of bronchial epithelial cells through the RhoA/ROCK1 pathway.

Adhesion molecules play critical roles in maintaining the structural integrity of the airway epithelium in airways under stress. Previously, we reported that catenin alpha-like 1 (CTNNAL1) is downregulated in an asthma animal model and upregulated at the edge of human bronchial epithelial cells (HBECs) after ozone stress. In this work, we explore the potential role of CTNNAL1 in the structural adhesion of HBECs and its possible mechanism. We construct a CTNNAL1 ‒/‒ mouse model with CTNNAL1-RNAi recombinant adeno-associated virus (AAV) in the lung and a CTNNAL1-silencing cell line stably transfected with CTNNAL1-siRNA recombinant plasmids. Hematoxylin and eosin (HE) staining reveals that CTNNAL1 ‒/‒ mice have denuded epithelial cells and structural damage to the airway. Silencing of CTNNAL1 in HBECs inhibits cell proliferation and weakens extracellular matrix adhesion and intercellular adhesion, possibly through the action of the cytoskeleton. We also find that the expressions of the structural adhesion-related molecules E-cadherin, integrin ß1, and integrin ß4 are significantly decreased in ozone-treated cells than in vector control cells. In addition, our results show that the expression levels of RhoA/ROCK1 are decreased after CTNNAL1 silencing. Treatment with Y27632, a ROCK inhibitor, abolished the expressions of adhesion molecules induced by ozone in CTNNAL1-overexpressing HBECs. Overall, the findings of the present study suggest that CTNNAL1 plays a critical role in maintaining the structural integrity of the airway epithelium under ozone challenge, and is associated with epithelial cytoskeleton dynamics and the expressions of adhesion-related molecules via the RhoA/ROCK1 pathway.

[Trametenolic acid inhibits migration and invasion of hepatocellular carcinoma HepG2.2.15 cells via RhoC/ROCK1 pathway].

This study investigated the effect of trametenolic acid(TA) on the migration and invasion of human hepatocellular carcinoma HepG2.2.15 cells by using Ras homolog gene family member C(RhoC) as the target and probed into the mechanism, aiming to provide a basis for the utilization of TA. The methyl thiazolyl tetrazolium(MTT) assay was employed to examine the proliferation of HepG2.2.15 cells exposed to TA, and scratch and Transwell assays to examine the cell migration and invasion. The pull down assay was employed to determine the impact of TA on RhoC GTPase activity. Western blot was employed to measure the effect of TA on the transport of RhoC from cytoplasm to cell membrane and the expression of RhoC/Rho-associated kinase 1(ROCK1)/myosin light chain(MLC)/matrix metalloprotease 2(MMP2)/MMP9 pathway-related proteins. RhoC was over-expressed by transient transfection of pcDNA3.1-RhoC. The changes of F-actin in the cytoskeleton were detected by Laser confocal microscopy. In addition, the changes of cell migration and invasion, expression of proteins in the RhoC/ROCK1/MLC/MMP2/MMP9 pathway, and RhoC GTPase activity were detected. The subcutaneously transplanted tumor model of BALB/c nude mice and the low-, medium-, and high-dose(40, 80, and 120 mg·kg~(-1), respectively) TA groups were established and sorafenib(20 mg·kg~(-1)) was used as the positive control. The tumor volume and weight in each group were measured, and the expression of related proteins in the tumor tissue was determined by Western blot. The results showed that TA inhibited the proliferation of HepG2.2.15 cells in a concentration-dependent manner, with the IC_(50) of 66.65 and 23.09 µmol·L~(-1) at the time points of 24 and 48 h, respectively. The drug administration groups had small tumors with low mass. The tumor inhibition rates of sorafenib and low-, medium-and high-dose TA were 62.23%, 26.48%, 55.45%, and 62.36%, respectively. TA reduced migrating and invading cells and inhibited RhoC protein expression and RhoC GTPase activity in a concentration-dependent manner, dramatically reducing RhoC and membrane-bound RhoC GTPase. The expression of ROCK1, MLC, p-MLC, MMP2, and MMP9 downstream of RhoC can be significantly inhibited by TA, as confirmed in both in vitro and in vivo experiments. After HepG2.2.15 cells were transfected with pcDNA3.1-RhoC to overexpress RhoC, TA down-regulated the protein levels of RhoC, ROCK1, MLC, p-MLC, MMP2, and MMP9 and decreased the activity of RhoC GTPase, with the inhibition level comparable to that before overexpression. In summary, TA can inhibit the migration and invasion of HepG2.2.15 cells. It can inhibit the RhoC/ROCK1/MLC/MMP2/MMP9 signaling pathway by suppressing RhoC GTPase activity and down-regulating RhoC expression. This study provides a new idea for the development of autophagy modulators targeting HSP90α to block the proliferation and inhibit the invasion and migration of hepatocellular carcinoma cells via multiple targets of active components in traditional Chinese medicines.

LncRNA NEAT1 accelerates renal fibrosis progression via targeting miR-31 and modulating RhoA/ROCK signal pathway.

Renal fibrosis is the final pathway for chronic kidney disease to end-stage renal failure. Noncoding RNAs have been reported to play a crucial role in renal fibrosis. Here, the effects of long noncoding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) and miR-31 on renal fibrosis and their regulatory mechanism were evaluated. RT-qPCR was used to assess NEAT1, miR-31, and RhoA levels. Western blot was performed to analyze the expression of fibrosis markers, RhoA, rho-related kinase (ROCK1), and connective tissue growth factor (CTGF). RNA immunoprecipitation (RIP), fluorescence in situ hybridization (FISH), and luciferase reporter assays verified the interaction between miR-31 and NEAT1 or RhoA. Renal fibrosis and injury were observed by Masson and hematoxylin and eosin (H&E) staining. The expression level of inflammatory cytokines was detected by ELISA. Immunohistochemistry (IHC) was performed to examine the expression levels of α-smooth muscle actin (α-SMA) and RhoA in renal tissues. We showed that NEAT1 was highly expressed, whereas miR-31 was decreased in renal fibrosis. NEAT1 was found to directly bind miR-31 to positively regulate RhoA expression. Furthermore, NEAT1 silencing inhibited renal fibrosis and inflammation and suppressed the RhoA/ROCK1 signaling pathway. However, knockdown of miR-31 could reverse these effects. NEAT1 silencing or overexpression of miR-31 alleviated renal fibrosis in vivo. In conclusion, NEAT1 accelerates renal fibrosis progression via negative regulation of miR-31 and the activation of RhoA/ROCK1 pathway, thereby upregulating the expression level of CTGF, providing a theoretical basis for treatment and prognostic evaluation of renal fibrosis.

Legumain Is an Endogenous Modulator of Integrin αvß3 Triggering Vascular Degeneration, Dissection, and Rupture.

BACKGROUND: The development of thoracic aortic dissection (TAD) is closely related to extracellular matrix degradation and vascular smooth muscle cell (VSMC) transformation from contractile to synthetic type. LGMN (legumain) degrades extracellular matrix components directly or by activating downstream signals. The role of LGMN in VSMC differentiation and the occurrence of TAD remains elusive. METHODS: Microarray datasets concerning vascular dissection or aneurysm were downloaded from the Gene Expression Omnibus database to screen differentially expressed genes. Four-week-old male Lgmn knockout mice (Lgmn-/-), macrophage-specific Lgmn knockout mice (LgmnF/F;LysMCre), and RR-11a-treated C57BL/6 mice were given BAPN (ß-aminopropionitrile monofumarate; 1 g/kg/d) in drinking water for 4 weeks for TAD modeling. RNA sequencing analysis was performed to recapitulate transcriptome profile changes. Cell interaction was examined in macrophage and VSMC coculture system. The reciprocity of macrophage-derived LGMN with integrin αvß3 in VSMCs was tested by coimmunoprecipitation assay and colocalization analyses. RESULTS: Microarray datasets from the Gene Expression Omnibus database indicated upregulated LGMN in aorta from patients with TAD and mice with angiotensin II-induced AAA. Elevated LGMN was evidenced in aorta and sera from patients with TAD and mice with BAPN-induced TAD. BAPN-induced TAD progression was significantly ameliorated in Lgmn-deficient or inhibited mice. Macrophage-specific deletion of Lgmn alleviated BAPN-induced extracellular matrix degradation. Unbiased profiler polymerase chain reaction array and Gene Ontology analysis displayed that LGMN regulated VSMC phenotype transformation. Macrophage-specific deletion of Lgmn ameliorated VSMC phenotypic switch in BAPN-treated mice. Macrophage-derived LGMN inhibited VSMC differentiation in vitro as assessed by macrophages and the VSMC coculture system. Macrophage-derived LGMN bound to integrin αvß3 in VSMCs and blocked integrin αvß3, thereby attenuating Rho GTPase activation, downregulating VSMC differentiation markers and eventually exacerbating TAD development. ROCK (Rho kinase) inhibitor Y-27632 reversed the protective role of LGMN depletion in vascular dissection. CONCLUSIONS: LGMN signaling may be a novel target for the prevention and treatment of TAD.

IncRNA MALAT1 Regulates the Proliferation, Apoptosis, Migration, and Invasion of Osteosarcoma Cells by Targeting miR-873-5p/ROCK1.

The malignant bone tumor osteosarcoma (OS) was one of the most aggressive tumors. Despite breakthroughs in treatment options for OS recently, the survival rate of patients with metastasis or reoccurring disease has remained unchanged over the last 25 years, at around 20%. lncRNA expression dysregulation is linked to carcinogenesis, advancement, and metastasis. Additionally, the fundamental mechanism of lncRNAs in regulating OS cell biological activity and progression is still being investigated. The expression of miR-873-5p and MALAT1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in OS. The relationship between the expression level of MALAT1 and the survival rate of OS individuals was evaluated by the Kaplan-Meier plotter. The tumor cell's capability of proliferation was determined using the CCK-8. Transwell was used to test the migratory and invasive properties of tumor cells. ROCK1 protein expression was analyzed by western blot, while qRT-PCR was used to detect ROCK1 mRNA expression. Targeted genes of MALAT1 or miR-873-5p were predicted by StarBase2.0. The target association among miR-873-5p and MALAT1 or ROCK1 was confirmed using the luciferase assay. The relationship between ROCK1 and MALAT1 or miR-873-5p expression in OS was investigated using Spearman's correlation analysis. MALAT1 was up-regulated and was linked to a lower survival rate of patients in OS. The malignant behaviors of cells were inhibited by down-regulated MALAT1 in vitro. Dual-luciferase gene experiments confirmed the presence of MALAT1/miR-873-5p/ROCK1 axis. The up-regulated miR-873-5p blocked the promoted effects of MALAT1 on cell behaviors. Over-expressed MALAT1 promoted the malignant behaviors of cells by miR-873-5p/ROCK1 axis in OS.

ROCK2 inhibition triggers the collective invasion of colorectal adenocarcinomas.

The metastatic progression of cancer is a multi-step process initiated by the local invasion of the peritumoral stroma. To identify the mechanisms underlying colorectal carcinoma (CRC) invasion, we collected live human primary cancer specimens at the time of surgery and monitored them ex vivo. This revealed that conventional adenocarcinomas undergo collective invasion while retaining their epithelial glandular architecture with an inward apical pole delineating a luminal cavity. To identify the underlying mechanisms, we used microscopy-based assays on 3D organotypic cultures of Caco-2 cysts as a model system. We performed two siRNA screens targeting Rho-GTPases effectors and guanine nucleotide exchange factors. These screens revealed that ROCK2 inhibition triggers the initial leader/follower polarization of the CRC cell cohorts and induces collective invasion. We further identified FARP2 as the Rac1 GEF necessary for CRC collective invasion. However, FARP2 activation is not sufficient to trigger leader cell formation and the concomitant inhibition of Myosin-II is required to induce invasion downstream of ROCK2 inhibition. Our results contrast with ROCK pro-invasive function in other cancers, stressing that the molecular mechanism of metastatic spread likely depends on tumour types and invasion mode.

Tumor-suppressing effects of miR-381-3p in pediatric acute myeloid leukemia via ROCK1 downregulation.

MicroRNA (miR)-381-3p is the newly discovered tumor-associated miRNA, which is frequently associated with diverse human malignancies; but, it is still unknown about its effect on acute myeloid leukemia (AML) in children. This work focused on exploring miR-381-3p's effect on childhood AML and identifying the possible mechanisms facilitating new treatment development. Using qRT-PCR analysis, miR-381-3p expression remarkably reduced in pediatric AML patients and AML cell lines (HL-60 and U937). Following transfection of miR-381-3p mimic or inhibitor into HL-60 and U937 cells, we conducted MTT assay to evaluate cell proliferation, flow cytometry (FCM) to measured cell apoptosis and cell cycle, whereas Transwell assays to detect cell invasion and migration. Our results demonstrated that miR-381-3p overexpression remarkably repressed cell growth, invasion and migration; additionally, miR-381-3p overexpression resulted in arrest of cell cycle and enhanced cell apoptosis. In contrast, miR-381-3p knockdown led to an opposite effect. Moreover, we predicted miR-381's target gene and validated it by luciferase reporter assay and TargetScan, separately. We identified miR-381-3p's binding site in ROCK1 3'-UTR. As revealed by Western-blot (WB) assay, miR-381-3p overexpression notably suppressed ROCK1 level. Moreover, restoring ROCK1 expression abolished miR-381-3p's inhibition on cell proliferation, invasion and migration. Data in this work indicated the role of miR-381-3p as the tumor suppressor within pediatric AML by targeting ROCK1. Therefore, miR-381-3p might serve as a potential therapeutic target for the treatment of pediatric AML.

Dia- and Rok-dependent enrichment of capping proteins in a cortical region.

Rho signaling with its major targets the formin Dia, Rho kinase (Rok) and non-muscle myosin II (MyoII, encoded by zip in flies) control turnover, amount and contractility of actomyosin. Much less investigated has been a potential function for the distribution of F-actin plus and minus ends. In syncytial Drosophila embryos, Rho1 signaling is high between actin caps, i.e. the cortical intercap region. Capping protein binds to free plus ends of F-actin to prevent elongation of the filament. Capping protein has served as a marker to visualize the distribution of F-actin plus ends in cells and in vitro. In the present study, we probed the distribution of plus ends with capping protein in syncytial Drosophila embryos. We found that capping proteins are specifically enriched in the intercap region similar to Dia and MyoII but distinct from overall F-actin. The intercap enrichment of Capping protein was impaired in dia mutants and embryos, in which Rok and MyoII activation was inhibited. Our observations reveal that Dia and Rok-MyoII control Capping protein enrichment and support a model that Dia and Rok-MyoII control the organization of cortical actin cytoskeleton downstream of Rho1 signaling. This article has an associated First Person interview with the first authors of the paper.

Switching between blebbing and lamellipodia depends on the degree of non-muscle myosin II activity.

Cells can adopt both mesenchymal and amoeboid modes of migration through membrane protrusive activities, namely formation of lamellipodia and blebbing. How the molecular players control the transition between lamellipodia and blebs is yet to be explored. Here, we show that addition of the ROCK inhibitor Y27632 or low doses of blebbistatin, an inhibitor of non-muscle myosin II (NMII) ATPase activity and filament partitioning, induces blebbing to lamellipodia conversion (BLC), whereas addition of low doses of ML7, an inhibitor of myosin light chain kinase (MLCK), induces lamellipodia to blebbing conversion (LBC) in human MDA-MB-231 cells. Similarly, siRNA-mediated knockdown of ROCK and MLCK induces BLC and LBC, respectively. Interestingly, both blebs and lamellipodia membrane protrusions are able to maintain the ratio of phosphorylated to unphosphorylated regulatory light chain at cortices when MLCK and ROCK, respectively, are inhibited either pharmacologically or genetically, suggesting that MLCK and ROCK activities are interlinked in BLC and LBC. Such BLCs and LBCs are also inducible in other cell lines, including MCF7 and MCF10A. These studies reveal that the relative activity of ROCK and MLCK, which controls both the ATPase activity and filament-forming property of NMII, is a determining factor in whether a cell exhibits blebbing or lamellipodia.

MYC-activated LINC00607 promotes hepatocellular carcinoma progression by regulating the miR-584-3p/ROCK1 axis.

BACKGROUND: There have been many reports of long non-coding RNAs (lncRNAs) in tumors, and abnormally expressed lncRNA is closely related to hepatocellular carcinoma (HCC). The mechanism of LINC00607 in HCC has not been reported. METHODS: We utilized qPCR to evaluate the RNA expression level. The mechanism of MYC binding to the LINC00607 promoter was revealed through chromatin immunoprecipitation assay and dual luciferase reporter assay. The proliferation and invasive ability were evaluated by CCK-8 and transwell assays. The relation between LINC00607 and miR-584-3p was assessed by RNA immunoprecipitation assay and dual luciferase reporter assay. The level of ROCK1 was evaluated by qPCR and western blot. RESULTS: In this research, we found that the expression of LINC00607 was higher in HCC tissues when compared with that in the adjacent non-tumor tissues. Meanwhile, MYC was observed to interact with the LINC00607 promoter, leading to the upregulation of LINC00607 in HCC. We further revealed that LINC00607 functioned as a sponge for miR-584-3p. Cell proliferation and migration assays showed that miR-584-3p may inhibit the HCC progression. Moreover, we found that the miR-584-3p inhibitor could reverse the effects of LINC00607 downregulation in HCC through rescue experiments. Through verification, miR-584-3p bound to the 3' UTR of ROCK1 to downregulate its expression. CONCLUSION: LINC00607 regulated by MYC can promote the proliferation, migration and invasion of HCC cells through the miR-584-3p/ROCK1 axis.

Genomic variation across Chinook salmon populations reveals effects of a duplication on migration alleles and supports fine scale structure.

The distribution of ecotypic variation in natural populations is influenced by neutral and adaptive evolutionary forces that are challenging to disentangle. This study provides a high-resolution portrait of genomic variation in Chinook salmon (Oncorhynchus tshawytscha) with emphasis on a region of major effect for ecotypic variation in migration timing. With a filtered data set of ~13 million single nucleotide polymorphisms (SNPs) from low-coverage whole genome resequencing of 53 populations (3566 barcoded individuals), we contrasted patterns of genomic structure within and among major lineages and examined the extent of a selective sweep at a major effect region underlying migration timing (GREB1L/ROCK1). Neutral variation provided support for fine-scale structure of populations, while allele frequency variation in GREB1L/ROCK1 was highly correlated with mean return timing for early and late migrating populations within each of the lineages (r2  = .58-.95; p < .001). However, the extent of selection within the genomic region controlling migration timing was much narrower in one lineage (interior stream-type) compared to the other two major lineages, which corresponded to the breadth of phenotypic variation in migration timing observed among lineages. Evidence of a duplicated block within GREB1L/ROCK1 may be responsible for reduced recombination in this portion of the genome and contributes to phenotypic variation within and across lineages. Lastly, SNP positions across GREB1L/ROCK1 were assessed for their utility in discriminating migration timing among lineages, and we recommend multiple markers nearest the duplication to provide highest accuracy in conservation applications such as those that aim to protect early migrating Chinook salmon. These results highlight the need to investigate variation throughout the genome and the effects of structural variants on ecologically relevant phenotypic variation in natural species.

Regulation of oxidized LDL-induced proliferation and migration in human vascular smooth muscle cells by a novel circ_0007478/miR-638/ROCK2 ceRNA network.

BACKGROUND: Circular RNAs (circRNAs) have been implicated in the pathogenesis of atherosclerosis (AS) and the migration and proliferation of vascular smooth muscle cells (VSMCs) under oxidized low-density lipoprotein (ox-LDL). Here, we defined the exact action of human circ_0007478 in VSMC migration and proliferation induced by ox-LDL. METHODS: Human VSMCs (HVSMCs) were exposed to ox-LDL. Circ_0007478, microRNA (miR)-638, and rho-associated protein kinase 2 (ROCK2) levels were gauged by quantitative real-time PCR (qRT-PCR) and western blot. Cell viability and proliferation were assessed by MTT and EdU assays, respectively. Transwell assays were used to detect cell migration and invasion. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to evaluate the direct relationship between miR-638 and circ_0007478 or ROCK2. RESULTS: Our data indicated that circ_0007478 expression was augmented in AS serum samples and ox-LDL-treated HVSMCs. Depletion of circ_0007478 attenuated HVSMC proliferation, migration, and invasion induced by ox-LDL. Mechanistically, circ_0007478 targeted miR-638 by directly pairing to miR-638. Reduction of miR-638 reversed the effects of circ_0007478 depletion on ox-LDL-evoked proliferation, migration, and invasion in HVSMCs. ROCK2 was a direct miR-638 target and miR-638-mediated inhibition of ROCK2 relieved ox-LDL-evoked HVSMC proliferation, migration, and invasion. Furthermore, circ_0007478 was identified as a competing endogenous RNA (ceRNA) for miR-638 to modulate ROCK2 expression. CONCLUSION: Our present study establishes an undescribed ceRNA regulatory network, in which circ_0007478 targets miR-638 to upregulate ROCK2, thereby contributing to ox-LDL-induced proliferation and migration in HVSMCs.

MiR196a-5p in extracellular vesicles released from human nasopharyngeal carcinoma enhance the phagocytosis and secretion of microglia by targeting ROCK1.

The microenvironment of the brain has become increasingly recognized as an essential regulator in metastatic and primary brain tumors. Recent studies demonstrate that circulating tumor-derived exosomes are critical for the brain tumor microenvironment. Nasopharyngeal carcinoma (NPC), a malignant tumor of the head and neck, often invades the skull base but infrequently extends to brain parenchyma. Neurobiological communication between microglia and tumor-derived extracellular vesicles (EVs) has been extensively studied, but how NPC cells regulate the immune microenvironment in the brain remains unknown. Here, we report that NPC derived EVs lead to increased microglial phagocytosis and proliferation, and heightened levels of IL-6, IL-8, CXCL1 and TGF-ß1. Analysis of microRNAs in EVs reveal that miR196a-5p is the major effector microRNA. Moreover, we demonstrate an enrichment of miR196a-5p in the plasmatic EVs of NPC patients. Further investigation demonstrated that miR196a-5p was transferred to microglia and regulated microglial structure and functions by downregulating the expression of ROCK1. Therefore, these data indicate that NPC-derived EVs are potent modulators of microglial functions in brain microenvironment. Regardless of brain colonization, EVs-mediated functional changes in microglia may be a universal phenomenon that results in the alteration of the tumor host's microenvironment in the brain.

Deciphering Rho-associated, coiled-coil-containing protein kinase in diabetic nephropathy: small steps towards a giant leap.

Diabetic nephropathy is a public health problem worldwide. Our understanding of the molecular machinery, as well as the clinical therapies for diabetic nephropathy, has evolved dramatically in recent years. However, even with this progress, there are residual risks of kidney failure and cardiovascular events in patients with diabetes. Rho-associated, coiled-coil-containing protein kinase (ROCK) is activated in response to various pathologic stimuli in the context of diabetes. The contribution of ROCK has been investigated in vivo using gene deletion rodent models and specific inhibitors, which are providing key insights into the pathologic function of ROCK in diabetic nephropathy. ROCK has two isoforms, ROCK1 and ROCK2. Both isoforms are expressed in the kidney, including mesangial cells, podocytes, and endothelial cells. ROCK1 blunts AMP-activated protein kinase (AMPK), while ROCK2 negatively regulates peroxisome proliferator-activated receptor α (PPARα) to inhibit fatty acid oxidation, both of which lead to structural and functional impairment of glomeruli in diabetes. Of note, ROCK signaling is activated in the kidney of animal models and patients with diabetes. In addition, an observational study has shown that fasudil hydrochloride, an ATP-competitive selective ROCK inhibitor, significantly attenuated proteinuria among patients with diabetes. These findings highlight the promising prospects for the development of a ROCK-centered approach against the progression of diabetic nephropathy.

Semaphorin 3A inhibits tumor progression via the downregulation of Lin28B in ovarian cancer.

Semaphorin 3A (Sema3A) has recently been proven to play an essential role in tumorigenesis. Here, the role of Sema3A in ovarian cancer is explored. The prognostic value of Sema3A was evaluated using the Kaplan-Meier plotter database, and stable expression cells were established by the delivery of lentivirus harboring SEMA3A cDNA or shRNA into OVCA433 and SKOV3 cells, respectively. Then CCK-8 assay, colony-formation assay, wound-healing assay, and Transwell assay were utilized to verify the effect of Sema3A on tumorigenesis. Co-cultures of ovarian cancer cells (OVCA433 and SKOV3) with a conditional medium collected from the established cells were further utilized to confirm the function of Sema3A. Then, the RNA-seq assay was adopted to explore the underlying mechanism. The results demonstrated that low expression of Sema3A was predictive of poor overall survival in patients with ovarian cancer. Functional experiments revealed that Sema3A inhibited proliferation, migration, and invasion in ovarian cancer cells. Secreted Sema3A in a conditioned culture medium also exhibited an anti-tumor effect in ovarian cancer cells. RNA-seq assay suggested that focal adhesion and Lin28B were involved in regulating Sema3A. Rescue assays further verified that Lin28B/ROCK1 axis was vital in the regulation of Sema3A and Lin28B significantly upregulated ROCK1 through let-7g microRNA. The presented data indicate that Sema3A inhibits proliferation and metastasis via the downregulation of Lin28B/ROCK1 in ovarian cancer.