RING Finger Protein Research Articles

N6-methyladenosine-related single-nucleotide polymorphism analyses identify oncogene RNFT2 in bladder cancer

BackgroundSingle-nucleotide polymorphisms (SNPs) in N6-methyladenosine (m6A) related genetic locus play significant roles in tumorigenesis and development. The expression level of many oncogenes and tumour suppressor genes changed because of m6A-associated SNPs. In addition, the relationship between m6A-SNP and bladder cancer (BCa) has not been well studied.MethodsWe screened m6A-SNPs in BCa by combining m6A-SNPs data and GWAS-SNPs data. Expression quantitative trait loci (eQTL) and differential expression gene (DEGs) analyses were performed. In ring finger protein, transmembrane 2 (RNFT2), rs3088107 (C > G) was found to have significant eQTL signals and make RNFT2 gene differentially-regulated mostly in BCa. We validated the expression level of RNFT2 in 32 pairs of BCa tissues and eight BCa cell lines by quantitative real-time PCR (qRT-PCR). Functional assays were performed to investigate the role of rs3088107 and RNFT2 in BCa in vitro.ResultsWe identified 673 m6A-SNPs, which were associated with BCa. Of these m6A-SNPs, 221 showed eQTL signals, amongst which, rs3088107 in RNFT2 showed significant eQTL signals. Results of bioinformatic analyses showed that 11 genes with m6A-SNPs had a differential expression level in BCa. RNFT2 was predicted to be significantly up-regulated in BCa. The qRT-PCR results validated that RNFT2 was highly expressed in our own BCa tissues and cell lines. High expression of RNFT2 also indicated a worse overall survival. We also revealed that rs3088107 (C > G) could inhibit the expression and m6A modification of RNFT2 by qRT-PCR, western-blot and m6A-RIP assays. Moreover, the results of functional assays indicated that RNFT2 promoted BCa cell proliferation and migration.ConclusionThis research found that m6A-SNPs were associated with oncogene RNFT2 in BCa. Furthermore, m6A-SNPs showed great application potential as a new BCa diagnostic biomarker and prognostic indicator.

Relation between CarS expression and activation of carotenogenesis by stress in Fusarium fujikuroi.

Fusarium fujikuroi, a model organism for secondary metabolism in fungi, produces carotenoids, terpenoid pigments with antioxidant activity. Previous results indicate that carotenoid synthesis in F. fujikuroi is stimulated by light or by different stress conditions and downregulated by a RING finger protein encoded by carS gene. Here, we have analyzed the effects of three stressors, nitrogen scarcity, heat shock, and oxidative stress. We compared them with the effect of light in the wild type, a carS mutant that overproduces carotenoids, and its complemented strain. The assayed stressors increase the synthesis of carotenoids in the three strains, but mRNA levels of structural genes of carotenogenesis, carRA and carB, are only enhanced in the presence of a functional carS gene. In the wild-type strain, the four conditions affect in different manners the mRNA levels of carS: greater in the presence of light, without significant changes in nitrogen starvation, and with patent decreases after heat shock or oxidative stress, suggesting different activation mechanisms. The spores of the carS mutant are more resistant to H2O2 than those of the wild type; however, the mutant shows a greater H2O2 sensitivity at the growth level, which may be due to the participation of CarS in the regulation of genes with catalase domains, formerly described. A possible mechanism of regulation by heat stress has been found in the alternative splicing of the intron of the carS gene, located close to its 3′ end, giving rise to the formation of a shorter protein. This action could explain the inducing effect of the heat shock, but not of the other inducing conditions, which may involve other mechanisms of action on the CarS regulator, either transcriptionally or post-transcriptionally.

Pulmonary arterial angioplasty in peripheral pulmonary arterial stenosis in RNF213 vasculopathy: effective but high-risk treatment with reperfusion injury

Abstract Background p.Arg4810Lys variant of the ring finger protein 213 (RNF213) vasculopathy is a nonsyndromatic vasculopathy involving multisystemic organs including pulmonary arteries. Peripheral pulmonary arterial stenosis (PPAS) with diffuse stenosis and beaded appearance is a unique feature of this disease and no treatment option has been tried before. Methods We performed BPA for PPAS in RNF213 patients (n=7). Severity of pulmonary hypertension was assessed by echocardiography and right heart catheterization at the baseline. Several clinical and hemodynamic parameters were followed up after each BPA sessions. Results All of the patients had dyspnea on exertion. Right ventricular dysfunction was observed in six patients and NT-proBNP was elevated (949.2±1148.1 pg/mL). Baseline echocardiography showed severe pulmonary hypertension (70.1±19.2 mmHg). Pulmonary arterial ballooning was performed to all patients, but stenting (n=6) and cutting balloon (n=1) was necessary due to elastic recoil. Improvement of clinical and hemodynamic parameters was achieved in six patients after 5.3 sessions of BPA in average. Reperfusion edema was seen in four patients, eventually leading to death in one patient. Conclusions Pulmonary arterial angioplasty, when coupled with stenting, is an effective treatment for PPAS with RNF213 vasculopathy. Although reperfusion injury was common and fatal in a few cases, it is the only modality to improve the patient's symptom and hemodynamics till now. Careful selection of the target population should be preceded to perform the intervention. Funding Acknowledgement Type of funding sources: None.

LncRNA KCNQ1OT1 enhances the radioresistance of lung squamous cell carcinoma by targeting the miR-491-5p/TPX2-RNF2 axis.

Lung cancer, especially lung squamous cell carcinoma (LUSC), is one of the most common malignant tumors worldwide. Currently, radiosensitization research is a vital direction for the improvement of LUSC therapy. Long non-coding RNAs (lncRNAs) can be novel biomarkers due to their multiple functions in cancers. However, the function and mechanism of lncRNA KCNQ1OT1 in the radioresistance of LUSC remain to be elucidated. The clonogenic assay was employed to determine the radioresistance of SK-MES-1R and NCI-H226R cells. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot were conducted for the detection of gene expression. Cell proliferation was determined by the methyl thiazolyl tetrazolium (MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine (EdU) staining, and cell apoptosis was assessed by flow cytometry. The relationships between genes were also evaluated by applying the luciferase reporter and radioimmunoprecipitation (RIP) assays. Radioresistant LUSC cells (SK-MES-1R and NCI-H226R) had strong resistance to X-ray irradiation, and lncRNA KCNQ1OT1 was highly expressed in SK-MES-1R and NCI-H226R cells. Moreover, knockdown of lncRNA KCNQ1OT1 prominently suppressed proliferation, attenuated radioresistance, and accelerated the apoptosis of SK-MES-1R and NCI-H226R cells. More importantly, we verified that miR-491-5p was a regulatory target of lncRNA KCNQ1OT1, and Xenopus kinesin-like protein 2 (TPX2) and RING finger protein 2 (RNF2) were the target genes of miR-491-5p. The rescue experiment results also demonstrated that miR-491-5p was involved in the inhibition of cell proliferation and the downregulation of TPX2 and RNF2 expression mediated by lncRNA KCNQ1OT1 knockdown in SK-MES-1R and NCI-H226R cells. LncRNA KCNQ1OT1 was associated with the radioresistance of radioresistant LUSC cells, and the lncRNA KCNQ1OT1/miR-491-5p/TPX2-RNF2 axis might be used as a therapeutic target to enhance the radiosensitivity of radioresistant LUSC cells.

Foot-and-mouth disease virus non-structural protein 2B downregulates the RLR signaling pathway via degradation of RIG-I and MDA5.

Foot-and-mouth disease virus (FMDV) is a single-stranded, positive-sense RNA virus containing at least 13 proteins. Many of these proteins show immune modulation capabilities. As a non-structural protein of the FMDV, 2B is involved in the rearrangement of the host cell membranes and the disruption of the host secretory pathway as a viroporin. Previous studies have also shown that FMDV 2B plays a role in the modulation of host type-I interferon (IFN) responses through the inhibition of expression of RIG-I and MDA5, key cytosolic sensors of the type-I IFN signaling. However, the exact molecular mechanism is poorly understood. Here, we demonstrated that FMDV 2B modulates host IFN signal pathway by the degradation of RIG-I and MDA5. FMDV 2B targeted the RIG-I for ubiquitination and proteasomal degradation by recruiting E3 ubiquitin ligase ring finger protein 125 (RNF125) and also targeted MDA5 for apoptosis-induced caspase-3- and caspase-8-dependent degradation. Ultimately, FMDV 2B significantly inhibited RNA virus-induced IFN-β production. Importantly, we identified that the C-terminal amino acids 126-154 of FMDV 2B are essential for 2B-mediated degradation of the RIG-I and MDA5. Collectively, these results provide a clearer understanding of the specific molecular mechanisms used by FMDV 2B to inhibit the IFN responses and a rational approach to virus attenuation for future vaccine development.

The Systematic Analyses of RING Finger Gene Signature for Predicting the Prognosis of Patients with Hepatocellular Carcinoma.

RING finger (RNF) proteins are frequently dysregulated in human malignancies and are tightly associated with tumorigenesis. However, the expression profiles of RNF genes in hepatocellular carcinoma (HCC) and their relations with prognosis remain undetermined. Here, we aimed at constructing a prognostic model according to RNF genes for forecasting the outcomes of HCC patients using the data from The Cancer Genome Atlas (TCGA) program. We collected HCC datasets to validate the values of our model in predicting prognosis of HCC patients from International Cancer Genome Consortium (ICGC) platform. Then, functional experiments were carried out to explore the roles of the representative RNF in HCC progression. A total of 107 differentially expressed RNFs were obtained between TCGA-HCC tumor and normal tissues. After comprehensive evaluation, a prognostic signature composed of 11 RNFs (RNF220, RNF25, TRIM25, BMI1, RNF216P1, RNF115, RNF2, TRAIP, RNF157, RNF145, and RNF19B) was constructed based on TCGA cohort. Then, the Kaplan-Meier (KM) curves and the receiver operating characteristic curve (ROC) were employed to evaluate predictive power of the prognostic model in testing cohort (TCGA) and validation cohort (ICGC). The KM and ROC curves illustrated the good predictive power in testing and validation cohort. The areas under the ROC curve are 0.77 and 0.76 in these two cohorts, respectively. Among the prognostic signature genes, BMI1 was selected as a representative for functional study. We found that BMI1 protein level was significantly upregulated in HCC tissues. Moreover, the inhibitor of BMI1, PTC-209, displayed an excellent anti-HCC effect in vitro. Enrichment analysis of BMI1 downstream targets showed that BMI1 might be involved in tumor immunotherapy. Together, our overall analyses revealed that the 11-RNFs prognostic signature might provide us latent chances for evaluating HCC prognosis and developing novel HCC therapy.

Identification and molecular analysis of RNF31 Q622H germline polymorphism.

The linear ubiquitin chain assembly complex (LUBAC), which is composed of RING finger protein 31 (RNF31), RANBP2-type and C3HC4-type zinc finger containing 1 and SHANK-associated RH domain interactor subunits, is the only ubiquitin ligase to generate Met1-linked linear ubiquitin chains. Linear ubiquitin chains regulate canonical NF-κB activation and cell death. Single nucleotide polymorphisms in RNF31, such as Q584H and Q622L, are known to cause the activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL) because of enhanced LUBAC-mediated NF-κB activation. The present study identified a novel Q622H polymorphism of RNF31 in two patients with lung cancer, one of whom had concurrent ABC-DLBCL. Immunohistochemical analyses revealed that although the expression of RNF31 was elevated in both patients, only the ABC-DLBCL specimen showed increased NF-κB activation. Cancer panel analysis showed that the Q622H-related ABC-DLBCL did not harbor co-mutations that were previously reported in Q584H-/Q622L-related ABC-DLBCL. Furthermore, in contrast to Q584H and Q622L, Q622H showed no enhancement effects on LUBAC and NF-κB activity in vitro compared with wild-type RNF31. The present study's structural prediction suggested that the electrostatic interaction related to the Q622 residue may not have had an important role in LUBAC formation. In conclusion, the molecular mechanism and mutational background of RNF31 Q622H differed from that of RNF31 Q584H or Q622L. Furthermore, RNF31 Q622H appeared not to induce NF-κB activation in lung cancer.

Ginsenoside Rd ameliorates muscle wasting by suppressing the signal transducer and activator of transcription 3 pathway.

The effects of some drugs, aging, cancers, and other diseases can cause muscle wasting. Currently, there are no effective drugs for treating muscle wasting. In this study, the effects of ginsenoside Rd (GRd) on muscle wasting were studied. Tumour necrosis factor-alpha (TNF-α)/interferon-gamma (IFN-γ)-induced myotube atrophy in mouse C2C12 and human skeletal myoblasts (HSkM) was evaluated based on cell thickness. Atrophy-related signalling, reactive oxygen species (ROS) level, mitochondrial membrane potential, and mitochondrial number were assessed. GRd (10mg/kg body weight) was orally administered to aged mice (23-24months old) and tumour-bearing (Lewis lung carcinoma [LLC1] or CT26) mice for 5weeks and 16days, respectively. Body weight, grip strength, inverted hanging time, and muscle weight were assessed. Histological analysis was also performed to assess the effects of GRd. The evolutionary chemical binding similarity (ECBS) approach, molecular docking, Biacore assay, and signal transducer and activator of transcription (STAT) 3 reporter assay were used to identify targets of GRd. GRd significantly induced hypertrophy in the C2C12 and HSkM myotubes (average diameter 50.8±2.6% and 49.9%±3.7% higher at 100nM, vs. control, P≤0.001). GRd treatment ameliorated aging- and cancer-induced (LLC1 or CT26) muscle atrophy in mice, which was evidenced by significant increases in grip strength, hanging time, muscle mass, and muscle tissue cross-sectional area (1.3-fold to 4.6-fold, vs. vehicle, P≤0.05; P≤0.01; P≤0.001). STAT3 was found to be a possible target of GRd by the ECBS approach and molecular docking assay. Validation of direct interaction between GRd and STAT3 was confirmed through Biacore analysis. GRd also inhibited STAT3 phosphorylation and STAT3 reporter activity, which led to the inhibition of STAT3 nuclear translocation and the suppression of downstream targets of STAT3, such as atrogin-1, muscle-specific RING finger protein (MuRF-1), and myostatin (MSTN) (29.0±11.2% to 84.3±30.5%, vs. vehicle, P≤0.05; P≤0.01; P≤0.001). Additionally, GRd scavenged ROS (91.7±1.4% reduction at 1nM, vs. vehicle, P≤0.001), inhibited TNF-α-induced dysregulation of ROS level, and improved mitochondrial integrity (P≤0.05; P≤0.01; P≤0.001). GRd ameliorates aging- and cancer-induced muscle wasting. Our findings suggest that GRd may be a novel therapeutic agent or adjuvant for reversing muscle wasting.

Extracellular fibrinogen-binding protein released by intracellular Staphylococcus aureus suppresses host immunity by targeting TRAF3

Many pathogens secrete effectors to hijack intracellular signaling regulators in host immune cells to promote pathogenesis. However, the pathogenesis of Staphylococcus aureus secretory effectors within host cells is unclear. Here, we report that Staphylococcus aureus secretes extracellular fibrinogen-binding protein (Efb) into the cytoplasm of macrophages to suppress host immunity. Mechanistically, RING finger protein 114, a host E3 ligase, mediates K27-linked ubiquitination of Efb at lysine 71, which facilitates the recruitment of tumor necrosis factor receptor associated factor (TRAF) 3. The binding of Efb to TRAF3 disrupts the formation of the TRAF3/TRAF2/cIAP1 (cellular-inhibitor-of-apoptosis-1) complex, which mediates K48-ubiquitination of TRAF3 to promote degradation, resulting in suppression of the inflammatory signaling cascade. Additionally, the Efb K71R mutant loses the ability to inhibit inflammation and exhibits decreased pathogenicity. Therefore, our findings identify an unrecognized mechanism of Staphylococcus aureus to suppress host defense, which may be a promising target for developing effective anti-Staphylococcus aureus immunomodulators.

MKRN3 role in regulating pubertal onset: the state of art of functional studies.

Puberty is a critical process characterized by several physical and psychological changes that culminate in the achievement of sexual maturation and fertility. The onset of puberty depends on several incompletely understood mechanisms that certainly involve gonadotropin-releasing hormone (GnRH) and its effects on the pituitary gland. The role of makorin ring finger protein 3 (MKRN3) in the regulation of pubertal timing was revealed when loss-of-function mutations were identified in patients with central precocious puberty (CPP), which to date, represent the most commonly known genetic cause of this condition. The MKRN3 gene showed ubiquitous expression in tissues from a broad spectrum of species, suggesting an important cellular role. Its involvement in the initiation of puberty and endocrine functions has just begun to be studied. This review discusses some of the recent approaches developed to predict MKRN3 functions and its involvement in pubertal development.

Deficiency of a novel lncRNA-HRAT protects against myocardial ischemia reperfusion injury by targeting miR-370-3p/RNF41 pathway.

Accumulating evidence indicates that long non-coding RNAs (lncRNAs) contribute to myocardial ischemia/reperfusion (I/R) injury. However, the underlying mechanisms by which lncRNAs modulate myocardial I/R injury have not been thoroughly examined and require further investigation. A novel lncRNA named lncRNA-hypoxia/reoxygenation (H/R)-associated transcript (lncRNA-HRAT) was identified by RNA sequencing analysis. The expression of lncRNA-HRAT exhibited a significant increase in the I/R mice hearts and cardiomyocytes treated with H/R. LncRNA-HRAT overexpression facilitates H/R-induced cardiomyocyte apoptosis. Furthermore, cardiomyocyte-specific deficiency of lncRNA-HRAT in vivo after I/R decreased creatine kinase (CK) release in the serum, reduced myocardial infarct area, and improved cardiac dysfunction. Molecular mechanistic investigations revealed that lncRNA-HRAT serves as a competing endogenous RNA (ceRNA) of miR-370-3p, thus upregulating the expression of ring finger protein 41 (RNF41), thereby aggravating apoptosis in cardiomyocytes induced by H/R. This study revealed that the lncRNA-HRAT/miR-370-3p/RNF41 pathway regulates cardiomyocyte apoptosis and myocardial injury. These findings suggest that targeted inhibition of lncRNA-HRAT may offer a novel therapeutic method to prevent myocardial I/R injury.

Combining expression of RNF43 and infiltration level of CD163+ tumor associated macrophage predicts prognosis of clear cell renal cell carcinoma.

Searching for reliable indicators for evaluating prognosis diagnosed with clear cell renal cell carcinoma (ccRCC) is crucial for improving clinical therapies. However, current researches have looked mainly at the prognostic value of a single intratumoral indicator, neglecting tumor-infiltrating immune cells (TIICs) in the microenvironment. This study examined whether the integration of Ring finger protein 43 (RNF43) expression and CD163+ tumor-associated macrophage (TAM) infiltration in combination with clinical indexes forecast ccRCC patient outcome with relatively high accuracy. Firstly, the expression of RNF43 and CD163 were detected with immunohistochemistry. Totally, 346 ccRCC patients were random separated evenly into training and validation datasets to make further analyses. We found that RNF43 expression was negatively correlated with infiltration level of CD163+ TAM in ccRCC, which was closely associated with the TNM stage and outcome of these patients. The multiple regression analysis demonstrated that RNF43, CD163, and TNM stage could function as independent risk factors in overall survival (OS) and progression-free survival (PFS) prediction of ccRCC. Furthermore, a better postoperative prognosis index for ccRCC patients was obtained by combining RNF43 and CD163+ TAMs, which assessed with time-dependent C-index analyses and a nomogram. Consequently, combining RNF43 and CD163+ TAMs along with TNM stage acquired robust accuracy in forecasting outcome of patients with ccRCC. In conclusion, combining intratumoral RNF43 expression, CD163+ TAM infiltration, and TNM stage could significantly enhance the veracity in forecasting postoperative outcomes.

QTL analysis of early flowering of female flowers in zucchini (Cucurbita pepo L.)

Early flowering promotes early maturity, production, and the capacity to counteract biotic and abiotic stresses, making it an important agronomic trait in zucchini. The present study demonstrated that the zucchini inbred line ‘19’ consistently flowered early, taking significantly fewer days to bloom the first female flower (DFF) than the inbred line ‘113’. Genetic analysis revealed that DFF, an inheritable quantitative trait, is controlled by multiple genes. Based on the strategy of quantitative trait locus (QTL) sequencing (QTL-seq) combined with linkage analysis, three QTLs for DFF were identified on chromosomes 4, 11, and 20. This study used additional F2 populations grown under different environmental conditions for QTL mapping analysis of DFF with insertion/deletion (InDel) markers to validate these results. Using the composite interval mapping (CIM) method of R/qtl software, we only identified one major locus under all environmental conditions, located in a 117-kb candidate region on chromosome 20. Based on gene annotation, gene sequence alignment, and qRT-PCR analysis, we found that the Cp4.1LG20g08050 gene encoding a RING finger protein may be a candidate gene for the opposite regulation of early flowering in zucchini. In summary, these results lay a foundation for a better understanding of early flowering and improving early flowering-based breeding strategies in zucchini.

RNF126 contributes to stem cell-like properties and metastasis in hepatocellular carcinoma through ubiquitination and degradation of LKB1.

Hepatocellular carcinoma (HCC) is one of the malignant tumors with the worst prognosis, and tumor recurrence and metastasis are the main factors leading to poor prognosis of HCC patients. Accumulating studies show that RNF126, ring finger protein 126, is involved in the pathological process of many tumors. However, the biological function and exact molecular mechanism of RNF126 in HCC remain unclear. In this study, we investigated the role of RNF126 in the pathogenesis of HCC. By analyzing database and verifying with our clinical specimens, it was found that RNF126 was highly expressed in HCC tissues, which is associated with shorter overall survival and higher recurrence rate. Overexpressed RNF126 can significantly promote the proliferation, migration, invasion and angiogenesis of HCC cells, whereas knockdown RNF126 can reverse this effect. Mechanically, RNF126 down-regulates liver kinase B1 (LKB1) expression by ubiquitination of LKB1 to weaken its stability, thereby significantly promoting stem-cell-like activity, migration, and angiogenesis of HCC. Notably, consistent with in vitro results, RNF126 was stably transformed in Hep3B and subcutaneously injected into nude mice. In established mouse xenograft models, tumor growth can be effectively inhibited and the occurrence of lung metastasis is reduced. In HCC, RNF126 may down-regulate LKB1 through ubiquitination, thus becoming a powerful prognostic biomarker and a recognized tumor suppressor. Therefore, our study may provide a promising new therapeutic strategy for targeting RNF126 for HCC patients.

Hypothalamic Overexpression of Makorin Ring Finger Protein 3 Results in Delayed Puberty in Female Mice.

Makorin ring finger protein 3 (MKRN3) is an important neuroendocrine player in the control of pubertal timing and upstream inhibitor of gonadotropin-releasing hormone secretion. In mice, expression of Mkrn3 in the hypothalamic arcuate and anteroventral periventricular nucleus is high early in life and declines before the onset of puberty. Therefore, we aimed to explore if the persistence of hypothalamic Mkrn3 expression peripubertally would result in delayed puberty. Female mice that received neonatal bilateral intracerebroventricular injections of a recombinant adeno-associated virus expressing Mkrn3 had delayed vaginal opening and first estrus compared with animals injected with control virus. Subsequent estrous cycles and fertility were normal. Interestingly, male mice treated similarly did not exhibit delayed puberty onset. Kiss1, Tac2, and Pdyn mRNA levels were increased in the mediobasal hypothalamus in females at postnatal day 28, whereas kisspeptin and neurokinin B protein levels in the arcuate nucleus were decreased, following Mkrn3 overexpression, compared to controls. Cumulatively, these data suggest that Mkrn3 may directly or indirectly target neuropeptides of Kiss1 neurons to degradation pathways. This mouse model suggests that MKRN3 may be a potential contributor to delayed onset of puberty, in addition to its well-established roles in central precocious puberty and the timing of menarche.

Systemic vasculopathy associated with an RNF213 p.Arg4810Lys variant in moyamoya disease: A review

Moyamoya disease (MMD) is a steno-occlusive disease of the distal cerebral arteries. Ring finger protein 213 (RNF213) p.Arg4810Lys is a susceptibility gene of MMD for which related vasculopathies are not well characterized. Heterozygous patients were mostly asymptomatic or exhibited isolated MMD. Homozygous patients showed a very unique pattern of diffuse narrowing of the entire aorta along with stenosis of the splanchnic, renal, coronary, iliofemoral, and/or peripheral pulmonary arteries, regardless of presence or absence of MMD. RNF213 p.Arg4810Lys is associated with high penetrance of systemic vasculopathy in homozygous patients and low penetrance of intracranial stenosis, i.e., MMD, in heterozygous patients, which suggests a gene-dosage effect.

RNF2 mediates pulmonary fibroblasts activation and proliferation by regulating mTOR and p16-CDK4-Rb1 signaling pathway.

Pulmonary fibrosis (PF) is a chronic, progressive interstitial lung disease with unknown etiology, associated with increasing morbidity and pessimistic prognosis. Pulmonary fibroblasts (PFbs) are the key effector cells of PF, in which abnormal activation and proliferation is an important pathogenesis of PF. Ring finger protein 2 (RNF2), is identified as the catalytic subunit of poly-comb repressive complex 1, which is closely related to occurrence and development of lung cancer, but its function in PF has not been revealed. In this paper, we sought to identify the regulatory role of RNF2 in lung fibrogenesis and its underlying mechanisms. The expression of RNF2 in lung fibrosis tissue (human and Bleomycin-induced mouse) and cell model (TGF-β1-induced HFL1 cells) was examined by immunoblotting analysis and immunofluorescence. Western blot, qRT-PCR were performed to evaluate the expression of pro-fibrogenic cytokines (including α-SMA, ECM and MMPs/ TIMPs) induced by TGF-β1 in HFL1 cells. Cell proliferation, cycle progression and apoptosis were examined by fow cytometric. Molecular interactions were tested by Co-IP assays. RNF2 expression was elevated in PF tissues compared to normal adjacent tissues and in PFbs (HFL1) induced by TGF-β1. Furthermore, knockdown of RNF2 could evidently inhibit the abnormal expression of pro-fibrogenic cytokines (including α-SMA, ECM and MMPs/TIMPs) induced by TGF-β1 in HFL1 cells. Functionally, RNF2 silencing could significantly suppress TGF-β1-induced anomalous proliferation, cell cycle progression, apoptosis and autophagy in HFL1 cells. Mechanistically, RNF2 deficiency could effectively inhibit the abnormal activation of mTOR signaling pathway in TGF-β1-induced HFL1 cells, and mTOR pathway had feedback regulation on the expression of RNF2. Further studies RNF2 could regulate the phosphorylation level of RB1 through interacting with p16 to destroy the binding of p16 and CDK4 competitively. Simultaneously, overexpression of RNF2 could show the opposite results. These results indicated that RNF2 is a potent pro-fibrogenic molecule for PFbs activation and proliferation through mTOR and p16-CDK4-Rb signaling pathways, and RNF2 inhibition will be a potential therapeutic avenue for treating PF.

Cattle breed type and anabolic implants impact calpastatin expression and abundance of mRNA associated with protein turnover in the longissimus thoracis of feedlot steers.

Two methods that the beef cattle industry can use to improve efficiency, sustainability, and economic viability are growth promotants and crossbreeding cattle of different breed types. In the United States, over 90% of cattle receive an anabolic implant at some point during production resulting in an overall increase in skeletal muscle growth. Recent research suggests that the two main cattle breed types, Bos indicus and Bos taurus, respond differently to anabolic implants. The objective of this study was to characterize changes that occur in skeletal muscle following implanting in Bos indicus influenced steers or Bos taurus steers. Twenty steers were stratified by initial weight in a 2 × 2 factorial design examining two different breeds: Angus (AN; n = 10) or Santa Gertrudis influenced (SG; n = 10), and two implant strategies: no implant (CON; n = 10) or a combined implant containing 120 mg TBA and 24 mg E2 (IMP; n = 10; Revalor-S, Merck Animal Health). Skeletal muscle biopsies were taken from the longissimus thoracis (LT) 2 and 10 d post-implantation. The mRNA abundance of 24 genes associated with skeletal muscle growth were examined, as well as the protein expression of µ-calpain and calpastatin. Succinate dehydrogenase mRNA abundance was impacted (P = 0.05) by a breed × treatment interaction 2 d post-implanting, with SG-CON having a greater increased abundance than all other steers. A tendency for a breed × treatment interaction was observed for calpain-6 mRNA (P = 0.07), with SG-CON having greater abundance than AN-CON and SG-IMP. Additionally, calpastatin protein expression was altered (P = 0.01) by a breed × treatment interaction, with SG-CON and SG-IMP steers having increased expression (P = 0.01) compared with AN-CON steers. At 2 d post-implanting, a breed × treatment interaction was observed with SG-CON steers having greater (P = 0.05) mRNA abundance of mitogen-activated protein kinase compared with AN-CON steers. Furthermore, breed affected (P = 0.05) calpastatin abundance with AN steers having increased (P = 0.05) abundance 2 d post-implanting compared with SG steers. Meanwhile, implants tended to affect (P = 0.09) muscle RING finger protein-1 mRNA abundance, with CON steers having increased (P = 0.09) abundance compared with that of IMP steers. These findings suggest that cattle breed type and anabolic implants impact calpastatin expression and mRNA abundance associated with protein turnover in the LT of feedlot steers 2 and 10 d post-implantation.

Whole exome sequencing identifies novel inherited genetic variants in tetralogy of Fallot

BackgroundTetralogy of Fallot (TOF) is the most common neonatal cyanotic heart defect, and genetic variation is an important risk factor for the etiology of TOF. Identifying TOF-associated genetic variants is critical to understanding susceptibility and outcome in patients with TOF and may help delineate pathological mechanisms.MethodsWhole exome sequencing (WES) was performed 19 patients with sporadic TOF and 3 healthy controls. The dbSNP, GnomAD, Denovo-db, and ClinVar databases were used to annotate the mutations. PolyPhen, SIFT, MutationTaster, and FATHMM softwares were used for mutation pathogenicity analysis. Sanger sequencing was used to validate candidate variants.ResultsWe identified 21 genetic variants involving 16 genes were found in 12 patients with sporadic TOF. The types of mutations were missense and splicing variants. None of these genes were detected in samples from the 3 healthy controls. These variants include 9 pathogenic variants, 6 suspected pathogenic variants, and 6 variants of unknown significance (VUS). Further analysis showed that the patients with apolipoprotein B (APOB) and ring finger protein 135 (RNF135) variants had more serious clinical symptoms. Sanger sequencing confirmed that the two variants were heterozygous in TOF patients.ConclusionsWe identified several genetic variants associated with TOF and confirmed that RNF135 and ABOB variants were associated with TOF severity. These findings provide new evidence for exploring the genetic mechanism of TOF.

CircRNA RNF10 inhibits tumorigenicity by targeting miR-942-5p/GOLIM4 axis in breast cancer.

We aimed to explore the action of a circRNA produced by ring finger protein 10 (circ_RNF10; hsa_circ_0028899) in the malignant behaviors of breast cancer (BC) and to explore its potential action-of-mechanism. The levels of circ_RNF10, miR-942-5p and Golgi integral membrane protein 4 (GOLIM4) were measured through quantitative real-time polymerase chain reaction, western blot, or immunohistochemistry, and the competing endogenous RNA (ceRNA) relationship among them was verified by dual-luciferase reporter assay. Cell counting kit-8, 5-ethynyl-2'-deoxyuridine, and colony formation assays, transwell assays, and flow cytometry were used to examine cell proliferation, migration and invasion, and apoptosis, respectively. Levels of proliferation and invasion-related markers were determined by western blot. Xenograft assay was performed to assess tumor growth. Circ_RNF10 level was significantly reduced in BC tissues and cells. Elevation of circ_RNF10 blocked BC cell proliferation, migration and invasion while promoted the apoptosis in vitro, companied with decreased PCNA and Twist1 and increased E-cadherin. Furthermore, upregulating circ_RNF10 delayed tumor growth of BC cells in nude mice. Mechanistically, circ_RNF10 acted as a ceRNA for miR-942-5p, and miR-942-5p could target GOLIM4. In addition, miR-942-5p overexpression reversed the influence of circ_RNF10 overexpression on BC progression. Furthermore, GOLIM4 silencing attenuated the inhibitory effect of miR-942-5p knockdown on BC progression. We found that circ_RNF10 suppressed BC malignant behavior by targeting miR-942-5p/GOLIM4 axis.