BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts.

Cardiac dysfunction is a hallmark of aging in humans and mice. Here we report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the aging phenotype. Our data indicate that age-associated overexpression of BIN1 occurs alongside dysregulated endosomal recycling and disrupted trafficking of cardiac CaV1.2 and type 2 ryanodine receptors. These deficiencies affect channel function at rest and their upregulation during acute stress. In vivo echocardiography reveals reduced systolic function in old mice. BIN1 knockdown using an adeno-associated virus serotype 9 packaged shRNA-mBIN1 restores the nanoscale distribution and clustering plasticity of ryanodine receptors and recovers Ca2+ transient amplitudes and cardiac systolic function toward youthful levels. Enhanced systolic function correlates with increased phosphorylation of the myofilament protein cardiac myosin binding protein-C. These results reveal BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium.

[Microtubule-associated tumor suppressor 1 inhibits hemin-induced apoptosis of vascular endothelial cells via hemeoxygenase 1].

This study aimed to investigate the effects of microtubule associated tumor suppressor 1 (MTUS1) on hemeoxygenase 1 (HMOX1) expression and hemin-induced apoptosis of vascular endothelial cells and its regulatory mechanism. RNA sequencing, RT-qPCR and Western blot were used to assess altered genes of hemin binding proteins, the expression of cAMP response element-binding protein (CREB) and nuclear respiratory factor 2 (NRF2), hemin-induced HMOX1 expression in MTUS1 knockdown human umbilical vein endothelial cells (HUVEC), and the effect of overexpression of CREB and NRF2 on HMOX1 expression in MTUS1 knockdown 293T cells. The effect of MTUS1 or HMOX1 knockdown on hemin-induced apoptosis in HUVEC, and the overexpression of NRF2 on hemin-induced apoptosis in MTUS1 knockdown 293T cells were assayed with CCK8 and Western blot. The results showed that MTUS1 was knocked down significantly in HUVEC by siRNA (P < 0.01), accompanied by decreased HMOX1 expression (P < 0.01). The increased HMOX1 expression induced by hemin was also inhibited by MTUS1 knockdown (P < 0.01). And the apoptosis of HUVEC induced by hemin was amplified by MTUS1 or HMOX1 knockdown (P < 0.01). Moreover the expression of CREB and NRF2 were both inhibited by MTUS1 knockdown in HUVEC (P < 0.01). The decreased HMOX1 regulated by MTUS1 knockdown could be rescued partly by overexpression of NRF2 (P < 0.01), however, not by overexpression of CREB. And the MTUS1 knockdown mediated decreased 293T cells viability induced by hemin could be partly rescued by NRF2 overexpression (P < 0.01). These results suggest that MTUS1 can inhibit hemin-induced apoptosis of HUVEC, and the mechanism maybe related to MTUS1/NRF2/HMOX1 pathway.

Discovery and technical validation of high-performance methylated DNA markers for the detection of cervical lesions at risk of malignant progression in low- and middle-income countries.

BACKGROUND: Cervical cancer remains a leading cause of death, particularly in developing countries. WHO screening guidelines recommend human papilloma virus (HPV) detection as a means to identify women at risk of developing cervical cancer. While HPV testing identifies those at risk, it does not specifically distinguish individuals with neoplasia. We investigated whether a quantitative molecular test that measures methylated DNA markers could identify high-risk lesions in the cervix with accuracy. RESULTS: Marker discovery was performed in TCGA-CESC Infinium Methylation 450 K Array database and verified in three other public datasets. The panel was technically validated using Quantitative Multiplex-Methylation-Specific PCR in tissue sections (N = 252) and cervical smears (N = 244) from the USA, South Africa, and Vietnam. The gene panel consisted of FMN2, EDNRB, ZNF671, TBXT, and MOS. Cervical tissue samples from all three countries showed highly significant differential methylation in squamous cell carcinoma (SCC) with a sensitivity of 100% [95% CI 74.12-100.00], and specificity of 91% [95% CI 62.26-99.53] to 96% [95% CI 79.01-99.78], and receiver operating characteristic area under the curve (ROC AUC) = 1.000 [95% CI 1.00-1.00] compared to benign cervical tissue, and cervical intraepithelial neoplasia 2/3 with sensitivity of 55% [95% CI 37.77-70.84] to 89% [95% CI 67.20-98.03], specificity of 93% [95% CI 84.07-97.38] to 96% [95% CI 79.01-99.78], and a ROC AUC ranging from 0.793 [95% CI 0.68-0.89] to 0.99 [95% CI 0.97-1.00] compared to CIN1. In cervical smears, the marker panel detected SCC with a sensitivity of 87% [95% CI 77.45-92.69], specificity 95% [95% CI 88.64-98.18], and ROC AUC = 0.925 [95% CI 0.878-0.974] compared to normal, and high-grade squamous intraepithelial lesion (HSIL) at a sensitivity of 70% (95% CI 58.11-80.44), specificity of 94% (95% CI 88.30-97.40), and ROC AUC = 0.884 (95% CI 0.822-0.945) compared to low-grade intraepithelial lesion (LSIL)/normal in an analysis of pooled data from the three countries. Similar to HPV-positive, HPV-negative cervical carcinomas were frequently hypermethylated for these markers. CONCLUSIONS: This 5-marker panel detected SCC and HSIL in cervical smears with a high level of sensitivity and specificity. Molecular tests with the ability to rapidly detect high-risk HSIL will lead to timely treatment for those in need and prevent unnecessary procedures in women with low-risk lesions throughout the world. Validation of these markers in prospectively collected cervical smear cells followed by the development of a hypermethylated marker-based cervical cancer detection test is warranted.

Identification and validation of a costimulatory molecule-related signature to predict the prognosis for uveal melanoma patients.

Uveal melanoma (UVM) is the most common primary tumor in adult human eyes. Costimulatory molecules (CMs) are important in maintaining T cell biological functions and regulating immune responses. To investigate the role of CMs in UVM and exploit prognostic signature by bioinformatics analysis. This study aimed to identify and validate a CMs associated signature and investigate its role in the progression and prognosis of UVM. The expression profile data of training cohort and validation cohort were downloaded from The Cancer Genome Atlas (TCGA) dataset and the Gene Expression Omnibus (GEO) dataset. 60 CM genes were identified, and 34 genes were associated with prognosis by univariate Cox regression. A prognostic signature was established with six CM genes. Further, high- and low-risk groups were divided by the median, and Kaplan-Meier (K-M) curves indicated that high-risk patients presented a poorer prognosis. We analyzed the correlation of gender, age, stage, and risk score on prognosis by univariate and multivariate regression analysis. We found that risk score was the only risk factor for prognosis. Through the integration of the tumor immune microenvironment (TIME), it was found that the high-risk group presented more immune cell infiltration and expression of immune checkpoints and obtained higher immune scores. Enrichment analysis of the biological functions of the two groups revealed that the differential parts were mainly related to cell-cell adhesion, regulation of T-cell activation, and cytokine-cytokine receptor interaction. No differences in tumor mutation burden (TMB) were found between the two groups. GNA11 and BAP1 have higher mutation frequencies in high-risk patients. Finally, based on the Genomics of Drug Sensitivity in Cancer 2 (GDSC2) dataset, drug sensitivity analysis found that high-risk patients may be potential beneficiaries of the treatment of crizotinib or temozolomide. Taken together, our CM-related prognostic signature is a reliable biomarker that may provide ideas for future treatments for the disease.

USP19 regulates DNA methylation damage repair and confers temozolomide resistance through MGMT stabilization.

OBJECTIVE: To elucidate the relationship between USP19 and O(6)-methylguanine-DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance. METHODS: Screening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management. RESULTS: USP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients. CONCLUSION: The regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients' temozolomide chemotherapy response.

MELK aggravates lung adenocarcinoma by regulating EZH2 ubiquitination and H3K27me3 histone methylation of LATS2.

We tried to elucidate the possible roles of maternal embryonic leucine pull chain kinase (MELK) in lung adenocarcinoma (LUAD) growth and metastasis. Differentially expressed genes in LUAD samples were analysed by the GEPIA database. Clinical tissue samples and cells were collected for MELK, EZH2 and LATS2 expression determination. Co-IP assay was used to verify the interaction between EZH2 and MELK; CHX tracking assay and ubiquitination assay detected the degradation of MELK on EZH2 ubiquitination. ChIP assay detected the enrichment of EZH2 and H3K27me3 on the LATS2 promoter region. LUAD cells were selected for in vitro validation, and the tumorigenic ability of LUAD cells was also observed in a transplantation tumour model of LUAD nude mice. MELK and EZH2 were highly expressed in LUAD samples, while LATS2 was lowly expressed. MELK interacted with EZH2 to inhibit its ubiquitination degradation; EZH2 elevated H3K27me3 modification in the LATS2 promoter to lower LATS2 expression. Silencing MELK or EZH2 or overexpressing LATS2 restrained LUAD cell proliferation and invasion, and facilitated their apoptosis. Silencing MELK or EZH2 or overexpressing LATS2 suppressed tumour formation in nude mice. This study demonstrated that MELK aggravated LUAD by upregulating EZH2 and downregulating LATS2.

A LATS2 and ALKBH5 positive feedback loop supports their oncogenic roles.

N(6)-methyladenosine (m6A) critically regulates RNA dynamics in various biological processes. The m6A demethylase ALKBH5 promotes tumorigenesis of glioblastoma, while the intricate web that orchestrates its regulation remains enigmatic. Here, we discover that cell density affects ALKBH5 subcellular localization and m6A dynamics. Mechanistically, ALKBH5 is phosphorylated by the large tumor suppressor kinase 2 (LATS2), preventing its nuclear export and enhancing protein stability. Furthermore, phosphorylated ALKBH5 reciprocally erases m6A from LATS2 mRNA, thereby stabilizing this transcript. Unexpectedly, LATS2 depletion suppresses glioblastoma stem cell self-renewal independent of yes-associated protein activation. Additionally, deficiency in either LATS2 or ALKBH5 phosphorylation impedes tumor progression in mouse xenograft models. Moreover, high levels of LATS2 expression and ALKBH5 phosphorylation are associated with tumor malignancy in patients with gliomas. Collectively, our study unveils an oncogenic positive feedback loop between LATS2 and ALKBH5, revealing a non-canonical branch of the Hippo pathway for RNA processing and suggesting potential anti-cancer interventions.

ZNF554 Inhibits Endometrial Cancer Progression via Regulating RBM5 and Inactivating WNT/ß-Catenin Signaling Pathway.

OBJECTIVE: Uterine corpus endometrial carcinoma (UCEC), a kind of gynecologic malignancy, poses a significant risk to women's health. The precise mechanism underlying the development of UCEC remains elusive. Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein superfamily, was reported to be dysregulated in various illnesses, including malignant tumors. This study aimed to examine the involvement of ZNF554 in the development of UCEC. METHODS: The expression of ZNF554 in UCEC tissues and cell lines were examined by qRT-PCR and Western blot assay. Cells with stably overexpressed or knocked-down ZNF554 were established through lentivirus infection. CCK-8, wound healing, and Transwell invasion assays were employed to assess cell proliferation, migration, and invasion. Propidium iodide (PI) staining combined with fluorescence-activated cell sorting (FACS) flow cytometer was utilized to detect cell cycle distribution. qRT-PCR and Western blotting were conducted to examine relative mRNA and protein levels. Chromatin immunoprecipitation assay and luciferase reporter assay were used to explore the regulatory role of ZNF554 in RNA binding motif 5 (RBM5). RESULTS: The expression of ZNF554 was found to be reduced in both UCEC samples and cell lines. Decreased expression of ZNF554 was associated with higher tumor stage, decreased overall survival, and reduced disease-free survival in UCEC. ZNF554 overexpression suppressed cell proliferation, migration, and invasion, while also inducing cell cycle arrest. In contrast, a decrease in ZNF554 expression resulted in the opposite effect. Mechanistically, ZNF554 transcriptionally regulated RBM5, leading to the deactivation of the Wingless (WNT)/ß-catenin signaling pathway. Moreover, the findings from rescue studies demonstrated that the inhibition of RBM5 negated the impact of ZNF554 overexpression on ß-catenin and p-glycogen synthase kinase-3ß (p-GSK-3ß). Similarly, the deliberate activation of RBM5 reduced the increase in ß-catenin and p-GSK-3ß caused by the suppression of ZNF554. In vitro experiments showed that ZNF554 overexpression-induced decreases in cell proliferation and migration were counteracted by RBM5 knockdown. Additionally, when RBM5 was overexpressed, it hindered the improvements in cell proliferation and migration caused by reducing the ZNF554 levels. CONCLUSION: ZNF554 functions as a tumor suppressor in UCEC. Furthermore, ZNF554 regulates UCEC progression through the RBM5/WNT/ß-catenin signaling pathway. ZNF554 shows a promise as both a prognostic biomarker and a therapeutic target for UCEC.

STUB1 is acetylated by KAT5 and alleviates myocardial ischemia-reperfusion injury through LATS2-YAP-ß-catenin axis.

Myocardial ischemia-reperfusion injury (MIRI) is involved in the pathogenesis of multiple cardiovascular diseases. This study elucidated the biological function of lysine acetyltransferase 5 (KAT5) in cardiomyocyte pyroptosis during MIRI. Oxygen-glucose deprivation/reoxygenation and left anterior descending coronary artery ligation were used to establish MIRI models. Here we show, KAT5 and STIP1 homology and U-box-containing protein 1 (STUB1) were downregulated, while large tumor suppressor kinase 2 (LATS2) was upregulated in MIRI models. KAT5/STUB1 overexpression or LATS2 silencing repressed cardiomyocyte pyroptosis. Mechanistically, KAT5 promoted STUB1 transcription via acetylation modulation, and subsequently caused ubiquitination and degradation of LATS2, which activated YAP/ß-catenin pathway. Notably, the inhibitory effect of STUB1 overexpression on cardiomyocyte pyroptosis was abolished by LATS2 overexpression or KAT5 depletion. Our findings suggest that KAT5 overexpression inhibits NLRP3-mediated cardiomyocyte pyroptosis to relieve MIRI through modulation of STUB1/LATS2/YAP/ß-catenin axis, providing a potential therapeutic target for MIRI.

Tumor suppressor role of the complement inhibitor CSMD1 and its role in TNF-induced neuroinflammation in gliomas.

BACKGROUND: The complement inhibitor CSMD1 acts as a tumor suppressor in various types of solid cancers. Despite its high level of expression in the brain, its function in gliomas, malignant brain tumors originating from glial cells, has not been investigated. METHODS: Three cohorts of glioma patients comprising 1500 patients were analyzed in our study along with their clinical data. H4, U-118 and U-87 cell lines were used to investigate the tumor suppressor function of CSMD1 in gliomas. PDGFB-induced brain tumor model was utilized for the validation of in vitro data. RESULTS: The downregulation of CSMD1 expression correlated with reduced overall and disease-free survival, elevated tumor grade, wild-type IDH genotype, and intact 1p/19q status. Moreover, enhanced activity was noted in the neuroinflammation pathway. Importantly, ectopic expression of CSMD1 in glioma cell lines led to decreased aggressiveness in vitro. Mechanically, CSMD1 obstructed the TNF-induced NF-kB and STAT3 signaling pathways, effectively suppressing the secretion of IL-6 and IL-8. There was also reduced survival in PDGFB-induced brain tumors in mice when Csmd1 was downregulated. CONCLUSIONS: Our study has identified CSMD1 as a tumor suppressor in gliomas and elucidated its role in TNF-induced neuroinflammation, contributing to a deeper understanding of glioma pathogenesis.

ENO1 promotes trophoblast invasion regulated by E2F8 in recurrent miscarriage.

Recurrent miscarriage (RM) is related to the dysfunction of extravillous trophoblast cells (EVTs), but the comprehensive mechanisms remain largely unexplored. We analyzed single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing and microarray datasets obtained from Gene Expression Omnibus (GEO) database to explore the hub genes in the mechanisms of RM. We identified 1724 differentially expressed genes (DEGs) in EVTs from the RM, and they were all expressed along the trajectory of EVTs. These DEGs were associated with hypoxia and glucose metabolism. Single-cell Regulatory Network Inference and Clustering (SCENIC) analysis revealed that E2F transcription factor (E2F) 8 (E2F8) was a key transcription factor for these DEGs. And the expression of ENO1 can be positively regulated by E2F8 via RNA sequencing analysis. Subsequently, we performed immunofluorescence assay (IF), plasmid transfection, western blotting, chromatin immunoprecipitation (ChIP), real-time quantitative polymerase chain reaction (qRT-PCR), and transwell assays for validation experiments. We found that the expression of alpha-Enolase 1 (ENO1) was lower in the placentas of RM. Importantly, E2F8 can transcriptionally regulate the expression of ENO1 to promote the invasion of trophoblast cells by inhibiting secreted frizzled-related protein 1/4 (SFRP1/4) to activate Wnt signaling pathway. Our results suggest that ENO1 can promote trophoblast invasion via an E2F8-dependent manner, highlighting a potential novel target for the physiological mechanisms of RM.

MGMT ProFWise: Unlocking a New Application for Combined Feature Selection and the Rank-Based Weighting Method to Link MGMT Methylation Status to Serum Protein Expression in Patients with Glioblastoma.

Glioblastoma (GBM) is a fatal brain tumor with limited treatment options. O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status is the central molecular biomarker linked to both the response to temozolomide, the standard chemotherapy drug employed for GBM, and to patient survival. However, MGMT status is captured on tumor tissue which, given the difficulty in acquisition, limits the use of this molecular feature for treatment monitoring. MGMT protein expression levels may offer additional insights into the mechanistic understanding of MGMT but, currently, they correlate poorly to promoter methylation. The difficulty of acquiring tumor tissue for MGMT testing drives the need for non-invasive methods to predict MGMT status. Feature selection aims to identify the most informative features to build accurate and interpretable prediction models. This study explores the new application of a combined feature selection (i.e., LASSO and mRMR) and the rank-based weighting method (i.e., MGMT ProFWise) to non-invasively link MGMT promoter methylation status and serum protein expression in patients with GBM. Our method provides promising results, reducing dimensionality (by more than 95%) when employed on two large-scale proteomic datasets (7k SomaScan® panel and CPTAC) for all our analyses. The computational results indicate that the proposed approach provides 14 shared serum biomarkers that may be helpful for diagnostic, prognostic, and/or predictive operations for GBM-related processes, given further validation.

Specific CpG sites methylation is associated with hematotoxicity in low-dose benzene-exposed workers.

Benzene is a broadly used industrial chemicals which causes various hematologic abnormalities in human. Altered DNA methylation has been proposed as epigenetic biomarkers in health risk evaluation of benzene exposure, yet the role of methylation at specific CpG sites in predicting hematological effects remains unclear. In this study, we recruited 120 low-level benzene-exposed and 101 control male workers from a petrochemical factory in Maoming City, Guangdong Province, China. Urinary S-phenylmercapturic acid (SPMA) in benzene-exposed workers was 3.40-fold higher than that in control workers (P < 0.001). Benzene-induced hematotoxicity was characterized by reduced white blood cells counts and nuclear division index (NDI), along with an increased DNA damage and urinary 8-hydroxy-2'-deoxyguanosine (all P < 0.05). Methylation levels of TRIM36, MGMT and RASSF1a genes in peripheral blood lymphocytes (PBLCs) were quantified by pyrosequencing. CpG site 6 of TRIM36, CpG site 2, 4, 6 of RASSF1a and CpG site 1, 3 of MGMT methylation were recognized as hot CpG sites due to a strong correlation with both internal exposure and hematological effects. Notably, integrating hot CpG sites methylation of multiple genes reveal a higher efficiency in prediction of integrative damage compared to individual genes at hot CpG sites. The negative dose-response relationship between the combined methylation of hot CpG sites in three genes and integrative damage enabled the classification of benzene-exposed individuals into high-risk or low-risk groups using the median cut-off value of the integrative index. Subsequently, a prediction model for integrative damage in benzene-exposed populations was built based on the methylation status of the identified hot CpG sites in the three genes. Taken together, these findings provide a novel insight into application prospect of specific CpG site methylation as epi-biomarkers for health risk assessment of environmental pollutants.

The impact of CREBRF rs373863828 Pacific-variant on infant body composition.

In Maori and Pacific adults, the CREBRF rs373863828 minor (A) allele is associated with increased body mass index (BMI) but reduced incidence of type-2 and gestational diabetes mellitus. In this prospective cohort study of Maori and Pacific infants, nested within a nutritional intervention trial for pregnant women with obesity and without pregestational diabetes, we investigated whether the rs373863828 A allele is associated with differences in growth and body composition from birth to 12-18 months' corrected age. Infants with and without the variant allele were compared using generalised linear models adjusted for potential confounding by gestation length, sex, ethnicity and parity, and in a secondary analysis, additionally adjusted for gestational diabetes. Carriage of the rs373863828 A allele was not associated with altered growth and body composition from birth to 6 months. At 12-18 months, infants with the rs373863828 A allele had lower whole-body fat mass [FM 1.4 (0.7) vs. 1.7 (0.7) kg, aMD -0.4, 95% CI -0.7, 0.0, P = 0.05; FM index 2.2 (1.1) vs. 2.6 (1.0) kg/m2 aMD -0.6, 95% CI -1.2,0.0, P = 0.04]. However, this association was not significant after adjustment for gestational diabetes, suggesting that it may be mediated, at least in part, by the beneficial effect of CREBRF rs373863828 A allele on maternal glycemic status.

CNS tumors with PLAGL1-fusion: beyond ZFTA and YAP1 in the genetic spectrum of supratentorial ependymomas.

A novel methylation class, "neuroepithelial tumor, with PLAGL1 fusion" (NET-PLAGL1), has recently been described, based on epigenetic features, as a supratentorial pediatric brain tumor with recurrent histopathological features suggesting an ependymal differentiation. Because of the recent identification of this neoplastic entity, few histopathological, radiological and clinical data are available. Herein, we present a detailed series of nine cases of PLAGL1-fused supratentorial tumors, reclassified from a series of supratentorial ependymomas, non-ZFTA/non-YAP1 fusion-positive and subependymomas of the young. This study included extensive clinical, radiological, histopathological, ultrastructural, immunohistochemical, genetic and epigenetic (DNA methylation profiling) data for characterization. An important aim of this work was to evaluate the sensitivity and specificity of a novel fluorescent in situ hybridization (FISH) targeting the PLAGL1 gene. Using histopathology, immunohistochemistry and electron microscopy, we confirmed the ependymal differentiation of this new neoplastic entity. Indeed, the cases histopathologically presented as "mixed subependymomas-ependymomas" with well-circumscribed tumors exhibiting a diffuse immunoreactivity for GFAP, without expression of Olig2 or SOX10. Ultrastructurally, they also harbored features reminiscent of ependymal differentiation, such as cilia. Different gene partners were fused with PLAGL1: FOXO1, EWSR1 and for the first time MAML2. The PLAGL1 FISH presented a 100% sensitivity and specificity according to RNA sequencing and DNA methylation profiling results. This cohort of supratentorial PLAGL1-fused tumors highlights: 1/ the ependymal cell origin of this new neoplastic entity; 2/ benefit of looking for a PLAGL1 fusion in supratentorial cases of non-ZFTA/non-YAP1 ependymomas; and 3/ the usefulness of PLAGL1 FISH.

The possible correlation between miR-762, Hippo signaling pathway, TWIST1, and SMAD3 in lung cancer and chronic inflammatory diseases.

MicroRNAs are small RNA molecules that have a significant role in translational repression and gene silencing through binding to downstream target mRNAs. MiR-762 can stimulate the proliferation and metastasis of various types of cancer. Hippo pathway is one of the pathways that regulate tissue development and carcinogenesis. Dysregulation of this pathway plays a vital role in the progression of cancer. This study aimed to evaluate the possible correlation between miR-762, the Hippo signaling pathway, TWIST1, and SMAD3 in patients with lung cancer, as well as patients with chronic inflammatory diseases. The relative expression of miR-762, MST1, LATS2, YAP, TWIST1, and SMAD3 was determined in 50 lung cancer patients, 30 patients with chronic inflammatory diseases, and 20 healthy volunteers by real-time PCR. The levels of YAP protein and neuron-specific enolase were estimated by ELISA and electrochemiluminescence immunoassay, respectively. Compared to the control group, miR-762, YAP, TWIST1, and SMAD3 expression were significantly upregulated in lung cancer patients and chronic inflammatory patients, except SMAD3 was significantly downregulated in chronic inflammatory patients. MST1, LATS2, and YAP protein were significantly downregulated in all patients. MiR-762 has a significant negative correlation with MST1, LATS2, and YAP protein in lung cancer patients and with MST1 and LATS2 in chronic inflammatory patients. MiR-762 may be involved in the induction of malignant behaviors in lung cancer through suppression of the Hippo pathway. MiR-762, MST1, LATS2, YAP mRNA and protein, TWIST1, and SMAD3 may be effective diagnostic biomarkers in both lung cancer patients and chronic inflammatory patients. High YAP, TWIST1, SMA3 expression, and NSE level are associated with a favorable prognosis for lung cancer.

Immunohistochemical and molecular evaluation of TUSC2 expression in breast cancer.

OBJECTIVE: Tumor suppressor candidate 2 has shown to be deleted in lung, colon, and bladder cancer types. In the present study, we aimed to investigate the expression of TUSC2 in breast cancer. MATERIALS AND METHODS: A total of thirty patients with breast cancer were included in the study. Normal and tumor tissue samples from fresh mastectomy materials were stored at -80 C until the number of cases was completed for gene expression analysis. Histopathological examination was carried out with routine hematoxylin & eosin method. TUSC2 staining was performed for immunohistochemical analysis. RESULTS: The tumors of thirteen patients were Luminal A, fourteen patients were Luminal B, one patient was cerbB2(+), and tumors of two patients were triple-negative. Ki67 proliferation index was less than 14% in fifteen cases and tumor size was less than 2 cm in seven cases. Lymphovascular invasion and lymph node metastasis were present in thirteen cases. Statistically, TUSC2 expression significantly decreased or was lost in breast tumor tissues compared to normal tissues (p < 0.0001). TUSC2 expression decreased as the Ki67 proliferation index increased (p = 0.0003), and TUSC2 expression decreased as tumor size increased (p = 0.0483). The loss or decrease in the TUSC2 expression was significant as the tumor grade increased (p = 0.3740). Gene expression analysis correlated with immunohistochemistry results. CONCLUSION: The results of the present study demonstrated a decrease or loss of TUSC2 expression in breast cancer tissue compared to normal tissue. A correlation was found between TUSC2 expression and Ki67 proliferation index and tumor size.

A systematic review of high impact CpG sites and regions for MGMT methylation in glioblastoma [A systematic review of MGMT methylation in GBM].

BACKGROUND: MGMT (O 6 -methylguanine-DNA methyltransferase) promoter methylation is a commonly assessed prognostic marker in glioblastoma (GBM). Epigenetic silencing of the MGMT gene by promoter methylation is associated with greater overall and progression free survival with alkylating agent regimens. To date, there is marked heterogeneity in how MGMT promoter methylation is tested and which CpG sites are interrogated. METHODS: To further elucidate which MGMT promoter CpG sites are of greatest interest, we performed comprehensive searches in PubMed, Web of Science, and Embase and reviewed 2,925 article abstracts. We followed the GRADE scoring system to assess risk of bias and the quality of the studies we included. RESULTS: We included articles on adult glioblastoma that examined significant sites or regions within MGMT promoter for the outcomes: overall survival, progression free survival, and/or MGMT expression. We excluded systemic reviews and articles on lower grade glioma. fifteen articles met inclusion criteria with variable overlap in laboratory and statistical methods employed, as well as CpG sites interrogated. Pyrosequencing or BeadChip arrays were the most popular methods utilized, and CpG sites between CpG's 70-90 were most frequently investigated. Overall, there was moderate concordance between the CpG sites that the studies reported to be highly predictive of prognosis. Combinations or means of sites between CpG's 73-89 were associated with improved OS and PFS. Six studies identified CpG sites associated with prognosis that were closer to the transcription start site: CpG's 8, 19, 22, 25, 27, 32,38, and CpG sites 21-37, as well as low methylation level of the enhancer regions. CONCLUSION: The following systematic review details a comprehensive investigation of the current literature and highlights several potential key CpG sites that demonstrate significant association with OS, PFS, and MGMT expression. However, the relationship between extent of MGMT promoter methylation and survival may be non-linear and could be influenced by potential CpG hotspots, the extent of methylation at each CpG site, and MGMT enhancer methylation status. There were several limitations within the studies such as smaller sample sizes, variance between methylation testing methods, and differences in the various statistical methods to test for association to outcome. Further studies of high impact CpG sites in MGMT methylation is warranted.

CPLX2 is a novel tumor suppressor and improves the prognosis in glioma.

BACKGROUND: Glioma is a type of malignant cancer that affect the central nervous system. New predictive biomarkers have been investigated in recent years, but the clinical prognosis for glioma remains poor. The function of CPLX2 in glioma and the probable molecular mechanism of tumor suppression were the focus of this investigation. METHODS: The glioma transcriptome profile was downloaded from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases for analysis of CPLX2 expression in glioma. RT-qPCR was performed to detect the expression of CPLX2 in 68 glioma subjects who have been followed up. Kaplan-Meier survival analyses were conducted to assess the effect of CPLX2 on the prognosis of glioma patients. The knockdown and overexpressed cell lines of CPLX2 were constructed to investigate the impact of CPLX2 on glioma. The cell growth, colony formation, and tumor formation in xenograft were performed. RESULTS: The expression of CPLX2 was downregulated in glioma and was negatively correlated with the grade of glioma. The higher expression of CPLX2 predicted a longer survival, as indicated by the analysis of Kaplan-Meier survival curves. Overexpressed CPLX2 impaired tumorigenesis in glioma progression both in vivo and in vitro. Knocking down CPLX2 promoted the proliferation of glioma cells. The analysis of GSEA and co-expression analysis revealed that CPLX2 may affect the malignancy of glioma by regulating the hypoxia and inflammation pathways. CONCLUSIONS: Our data indicated that CPLX2 functions as a tumor suppressor and could be used as a potential prognostic marker in glioma.

Netrin-1 Role in Nociceptive Neuron Sprouting through Activation of DCC Signaling in a Rat Model of Bone Cancer Pain.

BACKGROUND: Bone cancer pain (BCP) is a common primary or metastatic bone cancer complication. Netrin-1 plays an essential role in neurite elongation and pain sensitization. This study aimed to determine the role of netrin-1 from the metastatic bone microenvironment in BCP development and identify the associated signaling pathway for the strategy of BCP management. METHODS: The rat BCP model was established by intratibial implantation of Walker 256 cells. Von Frey filaments measured the mechanical pain threshold. Movement-induced pain was assessed using limb use scores. Expressions of associated molecules in the affected tibias or dorsal root ganglia (DRG) were measured by immunofluorescence, immunohistochemistry, real-time quantitative polymerase chain reaction, or western blotting. Transduction of deleted in colorectal cancer (DCC) signaling was inhibited by intrathecal injection of DCC-siRNA. RESULTS: In BCP rats, the presence of calcitonin gene-related peptide (CGRP)-positive nerve fibers increased in the metastatic bone lesions. The metastatic site showed enrichment of well-differentiated osteoclasts and expressions of netrin-1 and its attractive receptor DCC. Upregulation of DCC and increased phosphorylation levels of focal adhesion kinase (FAK) and Rac family small GTPase 1/Cell division cycle 42 (Rac1/Cdc42) were found in the DRG. Intrathecal administration of DCC-siRNA led to a significant reduction in FAK and Rac1/Cdc42 phosphorylation levels in the DRG, decreased nociceptive nerve innervation, and improved pain behaviors. CONCLUSIONS: Netrin-1 may contribute to the activation of the BCP by inducing nociceptive nerve innervation and improving pain behaviors.