ROCK1 Gene Research Articles

Investigation of RhoA, ROCK1, and ROCK2 Gene Expressions in Autism Spectrum Disorders

Investigation of RhoA, ROCK1, and ROCK2 Gene Expressions in Autism Spectrum Disorders

Proteomic identification of potential biomarkers for heat tolerance in Caracu beef cattle using high and low thermotolerant groups

BackgroundHeat stress has deleterious effects on physiological and performance traits in livestock. Within this context, using tropically adapted cattle breeds in pure herds or terminal crossbreeding schemes to explore heterosis is attractive for increasing animal production in warmer climate regions. This study aimed to identify biological processes, pathways, and potential biomarkers related to thermotolerance in Caracu, a tropically adapted beef cattle breed, by proteomic analysis of blood plasma. To achieve this goal, 61 bulls had their thermotolerance evaluated through a heat tolerance index. A subset of 14 extreme animals, including the seven most thermotolerant (HIGH group) and the seven least thermotolerant (LOW group), had their blood plasma samples used for proteomic analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The differentially regulated proteins detected between HIGH and LOW groups were used to perform functional enrichment analysis and a protein-protein interaction network analysis.ResultsA total of 217 proteins were detected only in the HIGH thermotolerant group and 51 only in the LOW thermotolerant group. In addition, 81 and 87 proteins had significantly higher and lower abundancies in the HIGH group, respectively. Regarding proteins with the highest absolute log-fold change values, we highlighted those encoded by DUSP5, IGFALS, ROCK2, RTN4, IRAG1, and NNT genes based on their functions. The functional enrichment analysis detected several biological processes, molecular functions, and pathways related to cellular responses to stress, immune system, complement system, and hemostasis in both HIGH and LOW groups, in addition to terms and pathways related to lipids and calcium only in the HIGH group. Protein-protein interaction (PPI) network revealed as important nodes many proteins with roles in response to stress, hemostasis, immune system, inflammation, and homeostasis. Additionally, proteins with high absolute log-fold change values and proteins detected as essential nodes by PPI analysis highlighted herein are potential biomarkers for thermotolerance, such as ADRA1A, APOA1, APOB, APOC3, C4BPA, CAT, CFB, CFH, CLU, CXADR, DNAJB1, DNAJC13, DUSP5, FGA, FGB, FGG, HBA, HBB, HP, HSPD1, IGFALS, IRAG1, KNG1, NNT, OSGIN1, PROC, PROS1, ROCK2, RTN4, RYR1, TGFB2, VLDLR, VTN, and VWF.ConclusionsIdentifying potential biomarkers, molecular mechanisms and pathways that act in response to heat stress in tropically adapted beef cattle contributes to developing strategies to improve performance and welfare traits in livestock under tropical climates.

Evaluation of the Rock1 and microRNA-148a expression in biopsies collected from patients with Helicobacter pylori induced gastritis

BackgroundHelicobacter pylori infection is one of the most common chronic bacterial infections, especially in developing countries. MicroRNA-148a is involved in the regulation of various genes, including Rock1, which is altered in gastric cancer. Decreased expression of mir-148a leads to tumor metastasis and increased Rock1 gene expression in gastric cancer. This study aimed to investigate the expression of these genes in biopsies collected from patients with H. pylori induced gastritis.MethodsInformed consent forms were gotten from the studied patients with gastritis who needed endoscopy. Gastric biopsies were taken by a gastroenterologist from patients with inflammation. Rapid urease test, stool antigen detection, and histopathological staining were used to determine the H. pylori infected patients. Real time PCR was used to evaluate the miRNA and Rock1 expression levels.ResultsThe Rock1 expression level in biopsies that were positive for H. pylori was significantly increased compared to our control gastritis group that were H. pylori-negative, but the results were not statistically significant. Moreover, the mir-148a expression level in H. pylori-positive patients with gastritis was increased compared to our control group. However, the results were not statistically significant. We did not find a significant relation between the expression levels of Rock1 and mir-148a in samples with gastritis infected or uninfected by H. pylori. This result may be due to the small sample size.ConclusionWe suggest that this test should be carried out with more samples, and the comparison should be done between biopsies with inflammation and no inflammation in a patient.

CRISPR-CasRx-mediated disruption of Aqp1/Adrb2/Rock1/Rock2 genes reduces intraocular pressure and retinal ganglion cell damage in mice

Glaucoma affects approximately 80 million individuals worldwide, a condition for which current treatment options are inadequate. The primary risk factor for glaucoma is elevated intraocular pressure. Intraocular pressure is determined by the balance between the secretion and outflow of aqueous humor. Here we show that using the RNA interference tool CasRx based on shH10 adenovirus-associated virus can reduce the expression of the aqueous humor circulation related genes Rock1 and Rock2, as well as aquaporin 1 and β2 adrenergic receptor in female mice. This significantly reduced intraocular pressure in female mice and provided protection to the retina ganglion cells, ultimately delaying disease progression. In addition, we elucidated the mechanisms by which the knockdown of Rock1 and Rock2, or aquaporin 1 and β2 adrenergic receptor in female mice, reduces the intraocular pressure and secures the retina ganglion cells by single-cell sequencing.

Rock2 heterozygosity improves recognition memory and endothelial function in a mouse model of 16p11.2 deletion autism syndrome

Rho-associated protein kinase-2 (ROCK2) is a critical player in many cellular processes and was incriminated in cardiovascular and neurological disorders. Recent evidence has shown that non-selective pharmacological blockage of ROCKs ameliorates behavioral alterations in a mouse model of 16p11.2 haploinsufficiency. We had revealed that 16p11.2-deficient mice also display cerebrovascular abnormalities, including endothelial dysfunction. To investigate whether genetic blockage of ROCK2 also exerts beneficial effects on cognition and angiogenesis, we generated mice with both 16p11.2 and Rock2 haploinsufficiency (16p11.2df/+;Rock2+/−). We find that Rock2 heterozygosity on a 16p11.2df/+ background significantly improved recognition memory. Furthermore, brain endothelial cells from 16p11.2df/+;Rock2+/− mice display improved angiogenic capacity compared to cells from 16p11.2df/+ littermates. Overall, this study implicates Rock2 gene as a modulator of 16p11.2-associated alterations, highlighting its potential as a target for treatment of autism spectrum disorders.

Nigella sativa, Anthemis hyaline and Citrus sinensis extracts reduce SARS-CoV-2 replication by fluctuating Rho GTPase, PI3K-AKT, and MAPK/ERK pathways in HeLa-CEACAM1a cells

Traditional remedies have long utilized Anthemis hyaline, Nigella sativa, and Citrus sinensis peel extracts as treatments for microbial infections. This study aimed to investigate the influence of Anthemis hyaline, Nigella sativa, and Citrus sinensis extracts on coronavirus replication and apoptosis-related pathways. HeLa-CEACAM1a cells were exposed to mouse hepatitis virus–A59. After viral inoculation, the mRNA levels of 36 genes were quantified using a Fluidigm Dynamic Array nanofluidic chip. IL-8 level and intracellular Ca2+ concentration was measured, and viral titer was assessed by the TCID50/ml assay to detect the extent of infection. Treatment with Nigella sativa extract surged the inflammatory cytokine IL-8 level at both 24 and 48-hour. Changes in gene expression were notable for RHOA, VAV3, ROCK2, CFL1, RASA1, and MPRIP genes following treatment with any of the extracts. The addition of Anthemis hyaline, Nigella sativa, or Citrus sinensis extracts to coronavirus-infected cells reduced viral presence, with Anthemis hyaline extract leading to a virtually undetectable viral load at 6- and 8-hours after infection. While all treatments influenced IL-8 production and viral levels, Anthemis hyaline extract displayed the most pronounced reduction in viral load. Consequently, Anthemis hyaline extract emerges as the most promising agent, harboring potential therapeutic compounds.

Comprehensive Atlas of Alternative Splicing Reveals NSRP1 Promoting Adipogenesis through CCDC18.

Alternative splicing (AS) plays a crucial role in regulating gene expression, function, and diversity. However, limited reports exist on the identification and comparison of AS in Eastern and Western pigs. Here, we analyzed 243 transcriptome data from eight tissues, integrating information on transcription factors (TFs), selection signals, splicing factors (SFs), and quantitative trait loci (QTL) to comprehensively study alternative splicing events (ASEs) in pigs. Five ASE types were identified, with Mutually Exclusive Exon (MXE) and Skipped Exon (SE) ASEs being the most prevalent. A significant portion of genes with ASEs (ASGs) showed conservation across all eight tissues (63.21-76.13% per tissue). Differentially alternative splicing genes (DASGs) and differentially expressed genes (DEGs) exhibited tissue specificity, with blood and adipose tissues having more DASGs. Functional enrichment analysis revealed coDASG_DEGs in adipose were enriched in pathways associated with adipose deposition and immune inflammation, while coDASG_DEGs in blood were enriched in pathways related to immune inflammation and metabolism. Adipose deposition in Eastern pigs might be linked to the down-regulation of immune-inflammation-related pathways and reduced insulin resistance. The TFs, selection signals, and SFs appeared to regulate ASEs. Notably, ARID4A (TF), NSRP1 (SF), ANKRD12, IFT74, KIAA2026, CCDC18, NEXN, PPIG, and ROCK1 genes in adipose tissue showed potential regulatory effects on adipose-deposition traits. NSRP1 could promote adipogenesis by regulating alternative splicing and expression of CCDC18. Conducting an in-depth investigation into AS, this study has successfully identified key marker genes essential for pig genetic breeding and the enhancement of meat quality, which will play important roles in promoting the diversity of pork quality and meeting market demand.

Effects of inhibition of Rho/ROCK pathway on proliferation and migration of airway smooth muscle cells and related mechanisms

To investigate the effects and molecular mechanisms of inhibition of the Ras homolog gene (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) pathway on the proliferation and migration of airway smooth muscle cells involving myocardin (MYOCD). Human airway smooth muscle cells were infected with the adenoviral vector Ad-ZsGreen-shRNA-hROCK1 in vitro. The cells were randomly divided into four groups: ROCK1 gene silencing control (shNC) group, shNC + arachidonic acid (AA, Rho/ROCK pathway activator) group, ROCK1 gene silencing (shROCK1) group, and shROCK1 + AA group (n=3 each). Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression levels of ROCK1 and MYOCD mRNA and protein. ELISA was employed to measure the levels of globular actin and filamentous actin, while immunofluorescent staining and scratch assays were utilized to assess cell proliferation and migration. Compared to the shNC + AA group, the shROCK1 + AA group exhibited decreased levels of ROCK1 and MYOCD mRNA and protein expression, reduced expression levels of globular actin and filamentous actin, and diminished cell proliferation and migration capabilities (P<0.05). Inhibition of the Rho/ROCK pathway suppresses the proliferation and migration of airway smooth muscle cells, which may be associated with the downregulation of MYOCD.

Differentially expressed platelet activation-related genes in dogs with stage B2 myxomatous mitral valve disease

BackgroundPeripheral blood carries a reservoir of mRNAs that regulate cardiac structure and function potential. Although it is well recognized that the typical symptoms of Myxomatous Mitral Valve Disease (MMVD) stage B2 are long-standing hemodynamic disorder and cardiac structure remodeling caused by mitral regurgitation, the transcriptomic alterations in blood from such dogs are not understood.ResultsIn the present study, comparative high-throughput transcriptomic profiling of blood was performed from normal control (NC) and naturally-occurring MMVD stage B2 (MMVD) dogs. Using Weighted Gene Co-expression Network Analyses (WGCNA), Gene Ontology (GO), and Kyoto Encyclopedia of Gene and Genomes (KEGG), we identified that the turquoise module was the most highly correlated with echocardiographic features and found 64 differentially expressed genes (DEGs) that were significantly enriched in platelet activation related pathways. Therefore, from the turquoise module, we selected five DEGs (MDM2, ROCK1, RIPK1, SNAP23, and ARHGAP35) that, according to real-time qPCR, exhibited significant enrichment in platelet activation related pathways for validation. The results showed that the blood transcriptional abundance of MDM2, ROCK1, RIPK1, and SNAP23 differed significantly (P < 0.01) between NC and MMVD dogs. On the other hand, Correlation Analysis revealed that MDM2, ROCK1, RIPK1, and SNAP23 genes negatively regulated the heart structure parameters, and followed the same trend as observed in WGCNA.ConclusionWe screened four platelet activation related genes, MDM2, ROCK1, RIPK1, and SNAP23, which may be considered as the candidate biomarkers for the diagnosis of MMVD stage B2. These findings provided new insights into MMVD pathogenesis.

The combination of phosphoinositol with inositol suppresses colorectal cancer growth and metastasis by regulating CLDN23/ RhoA /ROCK1 pathway

Colorectalcancerisa prevalentmalignanttumor, with liver metastases being the primary cause of death. The combination of inositol hexaphosphate (IP6) and inositol (INS) has been shown to inhibit colorectal cancer metastasis.We identified a correlation between CLDN23 expression and the occurrence of liver metastases in colorectal cancer patients. Following the transfection with the lentiviral vector (LV-CLDN23-RNAi), proteomic analysis of SW620 revealed significant alterations in the occludin, ZO-1, RhoA, and ROCK-1 genes expression by western blotting. We found that both SW620 and SW480 cells showed inhibited proliferation, invasion, and migration ability after the treatment with IP6 and INS. Additionally, this combination upregulated occludin and ZO-1, while downregulated CLDN23, RhoA, ROCK-1, and p-MLC2. In conclusion, the combination of IP6 and INS suppressed the proliferation, invasion, and migration of colorectal cancer cells and regulated CLDN23 expression by enhancing tightjunction proteins, such as occludin and ZO1, via the RhoA /ROCK1 pathway.

Photodynamic therapy with zinc phthalocyanine enhances the anti-cancer effect of tamoxifen in breast cancer cell line: Promising combination treatment against triple-negative breast cancer?

Photodynamic therapy (PDT) is a light-based anti-neoplastic therapeutic approach. Growing evidence indicates that combining conventional anti-cancer therapies with PDT can be a promising approach to treat malignancies. Herein, we aimed to investigate anti-cancer effects of the combination treatment of zinc phthalocyanine (ZnPc)-PDT with tamoxifen (TA) on MDA-MB-231 cells (as a triple-negative breast cancer (TNBC) cell line). For this purpose, we investigated the cytotoxicity of TA and ZnPc-PDT on MDA-MB-231 cells performing the MTT assay. The effect of TA and ZnPc-PDT on the apoptosis of MDA-MB-231 cells was studied using Annexin V/PI and DAPI staining. The wound-healing assay, and colony formation assay were performed to study the effect of TA and ZnPc-PDT on the migration, and clonogenicity of MDA-MB-231 cells, respectively. The qRT-PCR was done to study the gene expression of caspase-8, caspase-9, caspase-3, ZEB1, ROCK1, SNAIL1, CD133, CD44, SOX2, and ABCG2 (ATP-binding cassette sub-family G member 2). Based on our results, monotherapies with TA and ZnPc-PDT can remarkably increase cell cytotoxicity effects, stimulate apoptosis via downregulating Bcl-2 and upregulating caspase-3 and caspase-9, inhibit migration via downregulating SNAIL1 and ZEB1, and suppress clonogenicity via downregulating SOX2 and CD44 in MDA-MB-231 cells. Besides, these monotherapies can downregulate the expression of ABCG2 in MDA-MB-231 cells. Nevertheless, the combination treatment can potentiate the above-mentioned anti-cancer effects compared to monotherapy with TA. Of interest, the combined treatment of TA with ZnPc-PDT can synergically increase cell cytotoxicity effects on MDA-MB-231 cells. In fact, synergistic effects were estimated by calculation of Combination Index (CI); that synergistic outcomes were observed in all groups. Also, this combination treatment can significantly upregulate the caspase-8 gene expression and downregulate ROCK1 and CD133 gene expression in MDA-MB-231 cells. Overall, our results show that ZnPc-PDT can more sensitize the MDA-MB-231 cells to TA treatment. Based on our knowledge and experiment, the synergistic effects of ZnPc-PDT and TA deserve further evaluation in cancer research.

Characterization of lncRNA/circRNA-miRNA-mRNA network to reveal potential functional ceRNAs in the skeletal muscle of chicken.

Skeletal muscle, comprising approximately 40% of body mass, is a highly complex and heterogeneous tissue serving a multitude of functions in the organism. Non-coding RNAs (ncRNAs) are known to participate in skeletal muscle development as critical regulators. However, the regulatory mechanisms of ncRNAs on chicken muscle traits are not well understood. In the present study, we collected the leg muscle from male embryos of Tibetan chicken at embryonic (E) 10 and E18 for RNA sequencing. A total of 6,583 differentially expressed mRNAs (DEMs) including 3,055 down-regulated and 3,528 up-regulated were identified in E18. We identified 695 differentially expressed lncRNAs (DELs) (187 down-regulated and 508 up-regulated) and 1,906 differentially expressed circRNAs (DECs) (1,224 down-regulated and 682 up-regulated) in E18. Among the 130 differentially expressed miRNAs (DEMIs), 59 were up-regulated and 71 were down-regulated in E18. Numerous DEMs and target genes for miRNAs/lncRNAs were significantly enriched in the muscle system process and cell cycle. We constructed a miRNA-gene-pathway network by considering target relationships between genes related to skeletal muscle development and miRNAs. A competing endogenous RNA (ceRNA) network was also constructed by integrating competing relationships between DEMs, DELs, and DECs. Several DELs and DECs were predicted to regulate the ADRA1B, ATP2A2, ATP2B1, CACNA1S, CACNB4, MYLK2, and ROCK2 genes. We discovered the crosstalk between the ncRNAs and their competing mRNAs, which provides insights into ceRNA function and mechanisms in the skeletal muscle development of chicken.

TIP60 acts as a regulator of genes involved in filopodia formation and cell migration during wound healing

Wound healing is a complex phenomenon that requires coordination of numerous molecular and cellular changes to facilitate timely and efficient repair of the damaged tissue. Although many of these molecular pathways have been detailed, others remain to be elucidated. In the present work, we show for the first time, roles for the acetyltransferase TIP60 and nuclear receptor transcription factor PXR in this process, participating in wound healing by altering actin dynamics and cellular motility. We found that in response to wound-injury, TIP60 induces rapid formation of filopodia at the wounded cell front, leading to enhanced cell migration and faster closure of the wound. Further, qPCR analysis revealed heightened expression of Cdc42 and ROCK1 genes, key regulators involved in filopodia formation and actin reorganization, exclusively in TIP60-PXR-expressing cells upon wound-induction. We also performed ChIP assays to confirm the context-specific binding of TIP60 on the ROCK1 promoter and demonstrated that the TIP60 chromodomain is essential for loading of the TIP60–PXR complex onto the chromatin. Results from immunoprecipitation assays revealed that during the wounded condition, TIP60 alters the chromatin microenvironment by specifically acetylating histones H2B and H4, thereby modulating the expression of target genes. Overall, findings of this study show that TIP60 is a novel regulator of the wound healing process by regulating the expression of wound repair-related genes.

Evaluation of the rs35996865 polymorphism of the ROCK1 gene in sepsis.

Sepsis is a complex and serious medical condition resulting from the activation of an innate host response to infections. The etiology of sepsis is complex and can be influenced by genetic susceptibility. The purpose of the present study was to investigate a possible association of Rho-kinase 1 (ROCK1) gene polymorphism with sepsis in a Turkish population. The study group consisted of 100 unrelated patients with sepsis and 100 healthy controls. Genomic DNA was isolated from peripheral leukocytes from EDTA-containing blood using the QIAamp DNA Blood Mini Kit. ROCK1 gene rs35996865 and rs112130712 (Lys1054Arg) polymorphisms were analyzed in genomic DNA using the LightCycler 480 II real-time polymerase chain reaction. There were no significant differences in allele and genotype frequencies for ROCK1 gene rs35996865 polymorphism between the patients with sepsis and control group (p>0.05). Additionally, no association was detected between the rs35996865 polymorphism and mortality in the patient group. No polymorphism was detected with ROCK1 gene rs112130712 (Lys1054Arg) in our study groups. Our data showed that there is no marked association between the rs35996865 polymorphism and sepsis. Therefore, these results suggest that ROCK1 gene rs35996865 polymorphism is not risk factor for the development of sepsis in the Turkish population.

Is mir125-a rs12976445 a significant predictor of the development of breast cancer in women?

Cancer is one of the main causes of mortality worldwide and the cancer deaths figure is estimated to reach 11 million by 2030 (Momenimovahed and Salehiniya, 2019). Among the cancers, breast cancer is the most common malignancy in women worldwide affecting more than 1 million women annually (Stuckey, 2011; Ghoncheh, et al., 2016). Breast cancer incidence varies widely, ranging from 27/100,0002 (Central-East Asia and Africa) to 85-94/100,0002 (Australia, North America and Western Europe) indicating that the environmental and genetic factors play significant role in incidence of breast cancer occurrence worldwide (Sancho-Garnier and Colonna, 2019). Considering the development and pathology of breast cancer, it is a highly heterogeneous cancer in its pathological characteristics, some cases showing slow growth with excellent prognosis, while others being aggressive tumors (Tao, et al., 2015). Despite many studies carried out to investigate the etiology of breast cancer, much of the etiology of this disease is unknown, however, many gene polymorphisms of breast cancer have been described as possible neoplasm etiologic factors (Bugano et al., 2008).MicroRNAs (miRNAs) have been reported to have a pivotal role in gene regulation and the control of cancer-related mechanisms such as apoptosis and metastasis. miRNAs are short non-coding RNAs that are involved in post-transcriptional regulation of gene expression. miRNAs are transcribed by RNA polymerase II. The primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce pre-miRNA, which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA. The mature miRNA is incorporated into a RNA-induced silencing complex, which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in translational inhibition or destabilization of the target mRNA. Studies have shown that the miR-125 family acts as a tumor suppressor in breast cancer and inhibits ERBB2, ERBB3 and other target genes at post-transcriptional level. The miR-125 family has three homologues, hsa-miR-125a, hsa-miR-125b-1 and hsa-miR-125b-2 located at (19q.13.41), (11q.23) and (21q.21), respectively. Research shows ERBB2, HUR, ROCK-1, KLF13 and ARID3 genes are controlled by miR-125a. However, increased expression level of the genes is related to decreased expression of miR-125a, in breast cancer. Single nucleotide polymorphisms (SNP) of the miRNA encoding genes are believed to be involved in cancer development (Bahreini et al., 2020). Single nucleotide polymorphisms such as rs143525273, rs12975333, rs10404453 and rs12976445 are effective in miR-125a expression. Due to the increasing incidence of breast cancer and important role of miR-125a rs12976445 in the disease many studies have cited this single nucleotide polymorphism (Tang et al., 2015; Tang et al., 2019; Duna et al., 2007; Inoue et al., 2014; Deng et al., 2017; Bahreini et al., 2021).In this study, blood samples were prepared from two groups: patients with breast cancern (approved by an oncologist) (n=51) and control healthy women (n=50) after obtaining written informed consent form. PCR-RFLP technique was used to determine miR-125a genotype. The data were analyzed using Chi-Square.Our findings indicated that there was significant difference regarding rs12976445 genotype distribution between the control group and patients with breast cancer indicating that rs12976445 might be a predictor of the development of breast cancer in women.

Vascular smooth muscle ROCK1 contributes to hypoxia-induced pulmonary hypertension development in mice

Rho kinase (ROCK) is implicated in the development of pulmonary arterial hypertension (PAH) in which abnormal pulmonary vascular smooth muscle (VSM) contractility and remodeling lead to right heart failure. Pharmacologic ROCK inhibitors block experimental pulmonary hypertension (PH) development in rodents but can have off-target effects and do not distinguish between the two ROCK forms, ROCK1 and ROCK2, encoded by separate genes. An earlier study using gene knock out (KO) in mice indicated that VSM ROCK2 is required for experimental PH development, but the role of ROCK1 is not well understood. Here we investigated the in vivo role of ROCK1 in PH development by generating a VSM-targeted homozygous ROCK1 gene KO mouse strain. Adult control mice exposed to Sugen5416 (Su)/hypoxia treatment to induce PH had significantly increased right ventricular systolic pressures (RVSP) and RV hypertrophy versus normoxic controls. In contrast, Su/hypoxia-exposed VSM ROCK1 KO mice did not exhibit significant RVSP elevation, and RV hypertrophy was blunted. Su/hypoxia-induced pulmonary small vessel muscularization was similarly elevated in both control and VSM ROCK1 KO animals. siRNA-mediated ROCK1 knock-down (KD) in human PAH pulmonary arterial SM cells (PASMC) did not affect cell growth. However, ROCK1 KD led to reduced AKT and MYPT1 signaling in serotonin-treated PAH PASMC. The findings suggest that like VSM ROCK2, VSM ROCK1 actively contributes to PH development, but in distinction acts via nonproliferative pathways to promote hypoxemia, and thus may be a distinct therapeutic target in PH.

ROCK2 Polymorphism and Expression Contribute to Increased Susceptibility and Poor Prognosis in Hepatocellular Carcinoma.

AimTo analyze the association between ROCK2 gene polymorphism/expression and susceptibility/prognosis of hepatocellular carcinoma (HCC).MethodsPeripheral venous blood from 204 hepatocellular carcinoma patients (HCC group), 242 chronic hepatitis B patients (CHB group), 215 liver cirrhosis patients (LC group) and 201 healthy volunteers (normal group) were collected. ROCK2 gene (including the rs9808232 locus) was amplified by PCR and the products were sequenced. Tumor tissues from patients with HCC and liver tissues from CHB, LC and normal groups were collected, and mRNA and protein expression of ROCK2 gene in liver tissues were tested by RT-qPCR and immunofluorescence, respectively.ResultsCompared with the normal group, the mRNA and protein expression levels of ROCK2 in the HCC group increased significantly (P < 0.05), but there are no obvious increases in the CHB and LC groups (P > 0.05). ROCK2 gene rs9808232 polymorphism was associated with an increased susceptibility of HCC, and genotypes AC and CC may be risk factors for HCC (P < 0.001). There was a significant association between the ROCK2 gene rs9808232 polymorphism and the route of metastasis in HCC patients (P < 0.05). According to the Kaplan–Meier analysis, the overall survival of patients with AC and CC genotypes of ROCK2 rs9808232 was much shorter than that of patients with AA genotype (median overall survival, 25.5 months vs 16.0 months; Log rank P = 0.006). The overall survival time of patients with high ROCK2 expression is much shorter than that of patients with low ROCK2 expression (median overall survival time, 15.0 months vs 20.5 months; Log rank P = 0.008). Compared with genotype AA, the ROCK2 protein levels of genotypes AC and CC were distinctly increased in the HCC group, especially genotype CC (P< 0.05).ConclusionROCK2 gene rs9808232 polymorphism may contribute to its expression in liver tissue, which may increase the susceptibility and poor prognosis of HCC.

The Effect of miR-4800 Restoration on Proliferation and Migration of Human Breast Cancer Cells In Vitro.

Purpose: MicroRNAs (miRNAs) can contribute to cancer initiation, development, and progression. In this study, the effect of miRNA-4800 restoration on the growth and migration inhibition of human breast cancer (BC) cells was investigated. Methods: For this purpose, transfection of miR-4800 was performed into MDA-MB-231 BC cells using jetPEI. Subsequently, the expression levels of miR-4800 and CXCR4, ROCK1, CD44, and vimentin genes were measured using quantitative real-time polymerase chain reaction (q-RT-PCR) and specific primers. Also, the proliferation inhibition and apoptosis induction of cancer cells were evaluated by MTT and flow cytometry (Annexin V-PI method) techniques, respectively. Additionally, cancer cell migration after miR-4800 transfection was assessed by wound-healing (scratch) assay. Results: The restoration of miR-4800 in MDA-MB-231 cells resulted in the decreased expression level of CXCR4 (P ˂ 0.01), ROCK1 (P ˂ 0.0001), CD44 (P ˂ 0.0001), and vimentin (P ˂ 0.0001) genes. Also, MTT results showed restoration of miR-4800 could significantly reduce cell viability rate (P ˂ 0.0001) compared with the control group. Cell migration remarkably inhibited (P ˂ 0.001) upon miR-4800 transfection in treated BC cells. Flow cytometry data demonstrated that miR-4800 replacement considerably induced apoptosis in cancer cells (P ˂ 0.001) compared with control cells. Conclusion: Taken together, it seems that miR-4800 can act as a tumor suppressor miRNA in BC and play an essential role in modulating apoptosis, migration, and metastasis in BC. Therefore, it may be suggested as a potential therapeutic target in treating BC by performing additional tests in the future.

RhoA, ROCK-1, and ROCK-2 Gene Expression and Polymorphisms in Cholesteatoma Patients.

Background: The aim of this study was to evaluate the gene polymorphism and expressions of Rho-A, ROCK-1, and ROCK-2 in cholesteatoma.Methods: In this study, 120 healthy control group patients and 120 cholesteatoma patients were enrolled. Venous blood was taken from all of the cholesteatoma and control group patients. The genotyping of ROCK-1(G/T)rs35996865, ROCK-2(A/C)rs10178332, and Rho-A(A/T)rs2177268 polymorphisms was performed using predesigned TaqMan SNP Genotyping Assays. Assays-on-Demand SNP genotyping kit was used for the real-time polymerase chain reaction. The expression levels of Rho-A(Hs00357608_m1), ROCK-1(Hs01127699_m1), and ROCK-2(Hs00178154_m1) genes were determined.Results: The expression of Rho-A, ROCK-1, and ROCK-2 was lower in cholesteatoma patients than in the control group. There was no difference in Rho-AAT/TT and ROCK-1GT/TT variation in cholesteatoma patients compared to the control. However, ROCK-2 AC/CC variance was lower in cholesteatoma patients.Conclusion: The expression of Rho-A, ROCK-1, and ROCK-2 genes may be decreased in cholesteatoma. Furthermore, since ROCK-2 AC/CC genotype is also lower in cholesteatoma, having C allele seems to decrease the risk of developing this disease.

Risk and Protective Factors of Lifetime Cocaine-Associated Chest Pain.

Introduction: Cocaine users often present with repetitive events of cocaine-associated chest pain (CACP), clinically resembling acute coronary syndromes. The aim of the study is to describe the specific risk factors for CACP.Method: Cocaine users (n = 316) were recruited for a multicenter cross-sectional study. Lifetime CACP history, sociodemographic factors, and lifetime use of cocaine and other substances were assessed. Thirty single nucleotide polymorphisms (SNPs) of NOS3, ROCK2, EDN1, GUCY1A3, and ALDH2 genes, suggested by the literature on coronary spasms, were selected. The associations with CACP history were tested using the chi-square test, Student's t-test and logistic regression.Results: Among the 316 subjects [78.5% men, mean age 37.5 years, (standard-deviation ±8.7)], 190 (60.1%) were daily cocaine users and 103 (32.6%) reported a lifetime CACP history. Among those with a lifetime CACP history, the median was 10 events per individual. In multivariate analysis, lifetime CACP history was associated with daily cocaine use [odds-ratio (OR) 3.24; 95% confidence intervals (1.29–9.33)], rapid route of cocaine use [OR 2.33 (1.20–4.64) vs. intranasal use], and lifetime amphetamine use [daily amphetamine use: OR 2.80 (1.25–6.32) and non-daily amphetamine use: OR 2.14 (1.15–4.04) vs. never used]. Patients with lifetime opioid maintenance treatment (OMT) reported significantly less lifetime CACP history [OR 0.35 (0.16–0.76)]. None of the selected SNPs was associated with CACP history after multiple testing corrections.Conclusions: Clinical variables describing the intensity of stimulant use were positively associated with lifetime CACP history, while OMT was negatively associated with it. Specific harm reduction strategies can target these risk factors.