Lincai Ye

Long noncoding RNA HOTAIR, a hypoxia-inducible factor-1α activated driver of malignancy, enhances hypoxic cancer cell proliferation, migration, and invasion in non-small cell lung cancer

Despite the fact that great advances have been made in the management of non-small cell lung cancer (NSCLC), the prognosis of advanced NSCLC remains very poor. HOX transcript antisense intergenic RNA (HOTAIR) has been identified as an oncogenic long noncoding RNA (lncRNA) that is involved in the progression of a variety of carcinomas and acts as a negative prognostic biomarker. Yet, little is known about the effect of HOTAIR in the hypoxic microenvironment of NSCLC. The expression and promoter activity of HOTAIR were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and luciferase reporter assay. The function of the hypoxia-inducible factor-1α (HIF-1α) binding site to hypoxia-responsive elements (HREs) in the HOTAIR promoter region was tested by luciferase reporter assay with nucleotide substitutions. The binding of HIF-1α to the HOTAIR promoter in vivo was confirmed by chromatin immunoprecipitation assay (CHIP) and electrophoretic mobility shift assay (EMSA). The effect of HIF-1α suppression by small interference RNA or YC-1 on HOTAIR expression was also determined. In the present study, we demonstrated that HOTAIR was upregulated by hypoxia in NSCLC cells. HOTAIR is a direct target of HIF-1α through interaction with putative HREs in the upstream region of HOTAIR in NSCLC cells. Furthermore, HIF-1α knockdown or inhibition could prevent HOTAIR upregulation under hypoxic conditions. Under hypoxic conditions, HOTAIR enhanced cancer cell proliferation, migration, and invasion. These data suggested that suppression of HOTAIR upon hypoxia of NSCLC could be a novel therapeutic strategy.

Label-free quantitative proteomic analysis of right ventricular remodeling in infant Tetralogy of Fallot patients.

Tetralogy of Fallot (TOF) results in chronic progressive right ventricular (RV) pressure overload and shunt hypoxemia. We investigated the global changes in the proteome of RV among infant patients with and without TOF to gain an insight into early RV remodeling. One hundred and thirty-six differentially expressed proteins were identified using label-free LC-ESI-MS/MS analysis. Western blot results revealed that the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) increased significantly in TOF patients; and levels of lysocardiolipin acyltransferase 1 (LCLAT1), lumican (LUM), and versican (VCAN) decreased significantly. QRT-PCR analysis showed that levels of PFKFB2 mRNA were markedly increased, but those of LCLAT1 and LUM were significantly decreased. VCAN mRNA showed no significant change in response to pathophysiology of TOF. The results of immunohistochemical staining were similar to those of Western blot analysis. Results of the proteomic analysis indicated that the level of glycolysis-related proteins had increased and levels of lipid-metabolism-related proteins had decreased. ECM proteins were found to be more down-regulated in TOF in the present study than in previous reports. Taken together, our findings may provide clues to both the metabolic inflexibility and ECM remodeling during the early RV remodeling, which occur in response to chronic hypoxia and long-term pressure overload in TOF patients.

Cardiomyocytes in Young Infants With Congenital Heart Disease: a Three-Month Window of Proliferation

Perinatal reduction in cardiomyocyte cell cycle activity is well established in animal models and humans. However, cardiomyocyte cell cycle activity in infants with congenital heart disease (CHD) is unknown, and may provide important information to improve treatment. Human right atrial specimens were obtained from infants during routine surgery to repair ventricular septal defects. The specimens were divided into three groups: group A (age 1–3 months); group B (age, 4–6 months); and group C (age 7–12 months). A dramatic fall in the number of Ki67 -positive CHD cardiac myocytes occurred after three months. When cultured in vitro, young CHD myocytes (≤3 months) showed more abundant Ki67-positive cardiomyocytes and greater incorporation of EdU, indicating enhanced proliferation. YAP1 and NICD—important transcript factors in cardiomyocyte development and proliferation—decreased with age and β-catenin increased with age. Compared with those of older infants, cardiomyocytes of young CHD infants (≤3 months) have a higher proliferating capacity in vivo and in vitro. From the perspective of cardiac muscle regeneration, CHD treatment at a younger age (≤3 months) may be more optimal.

Decreased Yes-Associated Protein-1 (YAP1) Expression in Pediatric Hearts with Ventricular Septal Defects

Background Ventricular septal defects (VSDs) are the most common and simplest type of congenital heart diseases (CHDs). Animal studies have suggested that the downregulation of Yes-associated protein 1 (YAP1) during embryonic development causes VSD-associated CHDs. However, how YAP1 contributes to isolated VSD (iVSD) is unclear. Methods and Results Twenty right atrial specimens were obtained from iVSD patients during routine congenital cardiac surgery and we assessed YAP1 expression in these specimens. For controls, six right atrial specimens were obtained from normal hearts of children without heart disease, three of whom died from cerebral palsy, and three who underwent heart transplants. YAP1 mRNA and protein levels and nuclear localization were significantly reduced in iVSD specimens compared to normal heart specimens. Concomitantly, mRNA levels of YAP1 downstream targets CTGF and AXL were also significantly decreased in iVSD specimens. Although Ki67-positive cardiomyocytes in iVSD specimens were comparable to normal heart specimens, Ki67-positive non-cardiomyocytes were significantly decreased. Conclusions YAP1 expression was markedly decreased in hearts of iVSD children. Given the important role of YAP1 during heart development, downregulation of YAP1 expression may contribute to iVSD and affect the proliferation of non-cardiomyocytes.

A neonatal rat model of increased right ventricular afterload by pulmonary artery banding

Objective: To construct a neonatal rat model of increased right ventricular (RV) afterload for studying the pathophysiological remodeling of the right ventricle in patients with congenital heart disease with increased RV afterload. Methods: Surgery was performed within 6 hours after birth. Horizontal thoracotomy was performed by dissecting the intercostal muscles and splitting the sternum. The PA was then banded with 11‐0 nylon thread. At postnatal day 7 (P7), constriction of PA was confirmed by echocardiography. The RV systolic and diastolic pressures were measured by cardiac catheterization. The RV end‐systolic volume, end‐diastolic volume, end‐diastolic diameter, and free wall thickness were assessed by magnetic resonance imaging. The histological changes in sham‐operated and PA‐banding (PAB) hearts were evaluated by hematoxylin and eosin staining. Results: Increased RV afterload was established by constriction of the PA in neonatal rats within 6 hours after birth. The survival rate was 75% at P7. Relative to the sham group, the peak pressure gradient across the PA constriction and RV systolic and diastolic pressures, end‐systolic volume, end‐diastolic volume, end‐diastolic diameter, and free wall thickness were significantly increased in the PAB group at P7 (P < .01). Consistently, histological examination showed that the RV free wall was significantly hypertrophic in the PAB group. Conclusions: We successfully established a neonatal RV afterload increase model through PAB within 6 hours after birth, which can be used to study the pathophysiological changes in congenital heart diseases with increased RV afterload.

A gain-of-function ACTC1 3′UTR mutation that introduces a miR-139-5p target site may be associated with a dominant familial atrial septal defect

The ostium secundum atrial septal defect (ASDII) is the most common type of congenital heart disease and is characterized by a left to right shunting of oxygenated blood caused by incomplete closure of the septum secundum. We identified a familial form of isolated ASDII that affects four individuals in a family of five and shows autosomal dominant inheritance. By whole genome sequencing, we discovered a new mutation (c.*1784T > C) in the 3′-untranslated region (3′UTR) of ACTC1, which encodes the predominant actin in the embryonic heart. Further analysis demonstrated that the c.*1784T > C mutation results in a new target site for miRNA-139-5p, a microRNA that is involved in cell migration, invasion, and proliferation. Functional analysis demonstrated that the c.*1784T > C mutation specifically downregulates gene expression in a luciferase assay. Additionally, miR-139-5p mimic causes further decrease, whereas miR-139-5p inhibitor can dramatically rescue the decline in gene expression caused by this mutation. These findings suggest that the familial ASDII may be a result of an ACTC1 3′UTR gain-of-function mutation caused by the introduction of a new miR-139-5p target site. Our results provide the first evidence of a pathogenic mutation in the ACTC1 3′UTR that may be associated with familial isolated ASDII.

The Role of Na+/Ca2+ Exchanger 1 in Maintaining Ductus Arteriosus Patency

Patency of the ductus arteriosus (DA) is crucial for both fetal circulation and patients with DA-dependent congenital heart diseases (CHD). The Na+/Ca2+ exchanger 1 (NCX1) protein has been shown to play a key role in the regulation of vascular tone and is elevated in DA-dependent CHD. This current study was conducted to investigate the mechanisms underpinning the role of NCX1 in DA patency. Our data showed NCX1 expression was up-regulated in the DA of fetal mice. Up-regulation of NCX1 expression resulted in a concomitant decrease in cytosolic Ca2+ levels in human DA smooth muscle cells (DASMCs) and an inhibition of the proliferation and migration capacities of human DASMCs. Furthermore, treatment of DASMCs with KB-R7943, which can reduce Ca2+ influx, resulted in the inhibition of both cell proliferation and migration. These findings indicate that NCX1 may play a role in maintaining patent DA not only by preventing DA functional closure through reducing cytosolic Ca2+ level in DASMC but also by delaying the anatomical closure process. The latter delay is facilitated by the down-regulation of human DASMC proliferation and migration. It is also likely that a reduction in cytosolic Ca2+ levels inhibits the proliferation and migration capacities of human DASMCs in vitro.

Age-Dependent Oxidative DNA Damage Does Not Correlate with Reduced Proliferation of Cardiomyocytes in Humans.

Background Postnatal human cardiomyocyte proliferation declines rapidly with age, which has been suggested to be correlated with increases in oxidative DNA damage in mice and plays an important role in regulating cardiomyocyte proliferation. However, the relationship between oxidative DNA damage and age in humans is unclear. Methods Sixty right ventricular outflow myocardial tissue specimens were obtained from ventricular septal defect infant patients during routine congenital cardiac surgery. These specimens were divided into three groups based on age: group A (age 0–6 months), group B (age, 7–12 months), and group C (>12 months). Each tissue specimen was subjected to DNA extraction, RNA extraction, and immunofluorescence. Results Immunofluorescence and qRT-PCR analysis revealed that DNA damage markers—mitochondrial DNA copy number, oxoguanine 8, and phosphorylated ataxia telangiectasia mutated—were highest in Group B. However immunofluorescence and qRT-PCR demonstrated that two cell proliferation markers, Ki67 and cyclin D2, were decreased with age. In addition, wheat germ agglutinin-staining indicated that the average size of cardiomyocytes increased with age. Conclusions Oxidative DNA damage of cardiomyocytes was not correlated positively with age in human beings. Oxidative DNA damage is unable to fully explain the reduced proliferation of human cardiomyocytes.

GSK-3β Inhibitor CHIR-99021 Promotes Proliferation Through Upregulating β-Catenin in Neonatal Atrial Human Cardiomyocytes.

Background: The renewal capacity of neonate human cardiomyocytes provides an opportunity to manipulate endogenous cardiogenic mechanisms for supplementing the loss of cardiomyocytes caused by myocardial infarction or other cardiac diseases. GSK-3&bgr; inhibitors have been recently shown to promote cardiomyocyte proliferation in rats and mice, thus may be ideal candidates for inducing human cardiomyocyte proliferation. Methods: Human cardiomyocytes were isolated from right atrial specimens obtained during routine surgery for ventricle septal defect and cultured with either GSK-3&bgr; inhibitor (CHIR-99021) or &bgr;-catenin inhibitor (IWR-1). Immunocytochemistry was performed to visualize 5-ethynyl-2′-deoxyuridine (EdU)–positive or Ki67-positive cardiomyocytes, indicative of proliferative cardiomyocytes. Results: GSK-3&bgr; inhibitor significantly increased &bgr;-catenin accumulation in cell nucleus, whereas &bgr;-catenin inhibitor significantly reduced &bgr;-catenin accumulation in cell plasma. In parallel, GSK-3&bgr; inhibitor increased EdU-positive and Ki67-positive cardiomyocytes, whereas &bgr;-catenin inhibitor decreased EdU-positive and Ki67-positive cardiomyocytes. Conclusions: These results indicate that GSK-3&bgr; inhibitor can promote human atrial cardiomyocyte proliferation. Although it remains to be determined whether the observations in atrial myocytes could be directly applicable to ventricular myocytes, the current findings imply that Wnt/&bgr;-catenin pathway may be a valuable pathway for manipulating endogenous human heart regeneration.

Proliferation of cardiomyocytes in young infants, future implication in human heart regeneration

Background- Human heart is a limited-mitotic organ that responds to injury with limited cell renewal. In young infants, study of the regenerative capacity of cardiomyocytes may provide an important approach for heart regeneration. Methods and results- Human right atrial specimens were obtained during routine surgery for ventricle septal defect(VSD) and were divided into two groups: Young infant group (age, 1-3 months) and Old infant group (age, 4-6 months). Results showed that Ki67 is expressed in proliferating cardiac myocytes, and that the number of Ki67-positive cells in young infant group is significantly higher than old group. The Notch pathway was found to regulate cardiomyocyte proliferation and apoptosis during development. Current data showed that NICD expression is significantly higher in young age group and NICD was mainly expressed in cardiomyocyte nuclei. Conclusions- Proliferating cardiac myocytes are more abundant in the young infant period (<3 months) and the Notch pathway is conserved in humans. Further understanding of their proliferative ability at different ages may provide novel therapeutic targets that can be used to enhance cardiovascular regenerative capacity.