LncRNA HOTAIR regulates anoikis-resistance capacity and spheroid formation of ovarian cancer cells by recruiting EZH2 and influencing H3K27 methylation.

This study aims to investigate the role of the long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) in the regulation of anoikis resistance of ovarian cancer cells, a prerequisite for metastasis and chemoresistance in ovarian cancer cells. Ovarian cancer SKOV3 cells were cultured in an ultra-low attachment system to establish an anoikis model. The relationship between cellular anoikis capability and HOTAIR expression level was studied by flow cytometry and RT-PCR. The ability of spheroid formation, migration, and invasion of the suspended cells was assessed following the knockdown of HOTAIR expression. The expression of EZH2, H3K27me3, representative targets of EZH2, and anoikis-related biomarkers was also detected. An increase in the duration of suspension culture time rendered the SKOV3 cells anoikis-resistant with a significantly lower apoptotic rate compared to the adherent cells. HOTAIR expression in the suspension cells increased significantly, while that in the adherent cells did not. Following small interfering RNA (siRNA)-mediated knockdown of HOTAIR expression, the abilities of anoikis resistance, migration, and invasion decreased in the suspension cells. Knockdown of HOTAIR levels also reduced the spheroid forming ability of the tumor cells in continuous suspension cultures. Moreover, EZH2 expression correlated with HOTAIR expression, thus regulating the expression of miR-193a and DOK2 via introducing H3K27me3. Western blot analysis of anoikis-related markers showed that N-cadherin, ZEB1, and TWIST1 were downregulated following inhibition of HOTAIR, while E-cadherin and ErbB3 were upregulated. In conclusion, HOTAIR enhances the anoikis resistance and spheroid forming ability of ovarian cancer cells by recruiting EZH2 and influencing H3K27 methylation that may contribute to migration, invasion, and chemoresistance of ovarian cancer cells.

MiR-210-3p accelerates tumor-relevant cell functions of endometrial carcinoma by repressing RUNX1T1.

BACKGROUND: Biological mechanism of miR-210-3p in endometrial carcinoma (EC) remains unclear. Here, our purpose is to study effects of miR-210-3p on malignant progression of EC. METHODS: Bioinformatics analysis showed miRNA and mRNA are abnormally expressed in EC tissues. Quantitative real-time fluorescence polymerase chain reaction (qRT-PCR) was utilized to compare miR-210-3p mRNA level in EC cells and tissues. qRT-PCR and western blot were used to measure RUNX1T1 and NCAM1 at mRNA and protein levels, and western blot for p-AKT and AKT proteins related to PI3K/AKT signaling pathway. Furthermore, EC cell behaviors were assayed via Cell Counting Kit-8, cell colony formation assay, wound healing, transwell and flow cytometry experiments. Interaction between RUNX1T1 and miR-210-3p was verified through dual-luciferase assay. Immunohistochemistry was used to analyze RUNX1T1 expression in clinical samples RESULTS: MiR-210-3p was considerably upregulated and RUNX1T1 was significantly under-expressed in EC. Overexpression of miR-210-3p stimulated cell proliferation, migration, invasion, and restrained cell apoptosis in EC. Dual-luciferase assay proved that RUNX1T1 was a target gene of miR-210-3p. The level of RUNX1T1 in EC was downregulated after overexpressing miR-210-3p. Rescue assay showed that overexpression of RUNX1T1 had an inhibitory impact on tumor-relevant cell behaviors, whereas overexpression of miR-210-3p rescued such inhibition. Overexpression of RUNX1T1 reduced p-AKT expression, which was restored with concomitantly overexpressed miR-210-3p. CONCLUSION: In general, miR-210-3p behaves as an oncogene in EC by down-regulating the expression of RUNX1T1. This study elucidates a new functional mechanism in EC, and indicates miR-210-3p an underlying target.

Crosstalks between mTORC1 and mTORC2 variagate cytokine signaling to control NK maturation and effector function.

The metabolic checkpoint kinase mechanistic/mammalian target of rapamycin (mTOR) regulates natural killer (NK) cell development and function, but the exact underlying mechanisms remain unclear. Here, we show, via conditional deletion of Raptor (mTORC1) or Rictor (mTORC2), that mTORC1 and mTORC2 promote NK cell maturation in a cooperative and non-redundant manner, mainly by controlling the expression of Tbx21 and Eomes. Intriguingly, mTORC1 and mTORC2 regulate cytolytic function in an opposing way, exhibiting promoting and inhibitory effects on the anti-tumor ability and metabolism, respectively. mTORC1 sustains mTORC2 activity by maintaining CD122-mediated IL-15 signaling, whereas mTORC2 represses mTORC1-modulated NK cell effector functions by restraining STAT5-mediated SLC7A5 expression. These positive and negative crosstalks between mTORC1 and mTORC2 signaling thus variegate the magnitudes and kinetics of NK cell activation, and help define a paradigm for the modulation of NK maturation and effector functions.

Profiling analysis of long non-coding RNAs in early postnatal mouse hearts.

Mammalian cardiomyocytes undergo a critical hyperplastic-to-hypertrophic growth transition at early postnatal age, which is important in establishing normal physiological function of postnatal hearts. In the current study, we intended to explore the role of long non-coding (lnc) RNAs in this transitional stage. We analyzed lncRNA expression profiles in mouse hearts at postnatal day (P) 1, P7 and P28 via microarray. We identified 1,146 differentially expressed lncRNAs with more than 2.0-fold change when compared the expression profiles of P1 to P7, P1 to P28, and P7 to P28. The neighboring genes of these differentially expressed lncRNAs were mainly involved in DNA replication-associated biological processes. We were particularly interested in one novel cardiac-enriched lncRNA, ENSMUST00000117266, whose expression was dramatically down-regulated from P1 to P28 and was also sensitive to hypoxia, paraquat, and myocardial infarction. Knockdown ENSMUST00000117266 led to a significant increase of neonatal mouse cardiomyocytes in G0/G1 phase and reduction in G2/M phase, suggesting that ENSMUST00000117266 is involved in regulating cardiomyocyte proliferative activity and is likely associated with hyperplastic-to-hypertrophic growth transition. In conclusion, our data have identified a large group of lncRNAs presented in the early postnatal mouse heart. Some of these lncRNAs may have important functions in cardiac hyperplastic-to-hypertrophic growth transition.

Downregulation of ATG5-dependent macroautophagy by chaperone-mediated autophagy promotes breast cancer cell metastasis.

Recent data have shown that the expression of lysosome-associated membrane protein type 2 A (LAMP2A), the key protein in the chaperone-mediated autophagy (CMA) pathway, is elevated in breast tumor tissues. However, the exact effects and mechanisms of CMA during breast cancer metastasis remain largely unknown. In this study, we found that the LAMP2A protein level was significantly elevated in human breast cancer tissues, particularly in metastatic carcinoma. The increased LAMP2A level was also positively correlated with the histologic grade of ductal breast cancer. High LAMP2A levels also predicted shorter overall survival of breast cancer patients. Downregulation of CMA activity by LAMP2A knockdown significantly inhibited the growth and metastasis of both MDA-MB-231 and MDA-MB-468 breast cancer cells in vivo and in vitro, while upregulation of CMA activity by LAMP2A overexpression had the opposite effect. Mechanistically, we found that elevated CMA activity mediated increased growth and metastasis of human breast cancer cells by downregulating the activity of autophagy-related gene 5 (ATG5)-dependent macroautophagy. Collectively, these results indicate that the anti-macroautophagic property is a key feature of CMA-mediated tumorigenesis and metastasis and may, in some contexts, serve as an attractive target for breast cancer therapies.

Maternal inflammation activated ROS-p38 MAPK predisposes offspring to heart damages caused by isoproterenol via augmenting ROS generation.

Maternal inflammation contributes to the increased incidence of adult cardiovascular disease. The current study investigated the susceptibility of cardiac damage responding to isoproterenol (ISO) in adult offspring that underwent maternal inflammation (modeled by pregnant Sprague-Dawley rats with lipopolysaccharides (LPS) challenge). We found that 2 weeks of ISO treatment in adult offspring of LPS-treated mothers led to augmented heart damage, characterized by left-ventricular systolic dysfunction, cardiac hypertrophy and myocardial fibrosis. Mechanistically, prenatal exposure to LPS led to up-regulated expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, antioxidant enzymes, and p38 MAPK activity in left ventricular of adult offspring at resting state. ISO treatment exaggerated ROS generation, p38 MAPK activation but down-regulated reactive oxygen species (ROS) elimination capacity in the left ventricular of offspring from LPS-treated mothers, while antioxidant N-acetyl-L-cysteine (NAC) reversed these changes together with improved cardiac functions. The p38 inhibitor SB202190 alleviated the heart damage only via inhibiting the expression of NADPH oxidases. Collectively, our data demonstrated that prenatal inflammation programs pre-existed ROS activation in the heart tissue, which switches on the early process of oxidative damages on heart rapidly through a ROS-p38 MAPK-NADPH oxidase-ROS positive feedback loop in response to a myocardial hypertrophic challenge in adulthood.

Sustained elevation of NF-κB activity sensitizes offspring of maternal inflammation to hypertension via impairing PGC-1α recovery.

Growing evidence has demonstrated that maternal detrimental factors, including inflammation, contribute to the development of hypertension in the offspring. The current study found that offspring subjected to prenatal exposure of inflammation by lipopolysaccharide (LPS) challenge during the second semester showed significantly increased systolic blood pressure. In addition, these offspring also displayed augmented vascular damage and reactive oxygen species (ROS) levels in thoracic aortas when challenged with deoxycorticosterone acetate and high-salt diet (DOCA-salt). Interestingly, the antioxidant N-acetyl-L-cysteine markedly reversed these changes. Mechanistically, prenatal LPS exposure led to pre-existing elevated peroxisome proliferators-activated receptor-γ co-activator (PGC)-1α, a critical master of ROS metabolism, which up-regulated the ROS defense capacity and maintained the balance of ROS generation and elimination under resting state. However, continued elevation of NF-κB activity significantly suppressed the rapid recovery of PGC-1α expression response to DOCA-salt challenge in offspring that underwent prenatal inflammatory stimulation. This was further confirmed by using a NF-κB inhibitor (N-p-Tosyl-L-phenylalanine chloromethyl ketone) that restored PGC-1α recovery and prevented blood pressure elevation induced by DOCA-salt. Our results suggest that maternal inflammation programmed proneness to NF-κB over-activation which impaired PGC-1α-mediated anti-oxidant capacity resulting in the increased sensitivity of offspring to hypertensive damage.