Mingchang Zhang

Effects of RNA interference-mediated gene silencing of JMJD2A on human breast cancer cell line MDA-MB-231 in vitro

Previous data demonstrate that JMJD2A is a cancer-associated gene and may be involved in human breast cancer by demethylation of H3K9me3. The aim of this study was to investigate depressive effects on JMJD2A by transfection with JMJD2A-sepcific siRNA in human breast cancer cell line MDA-MB-231 and effects on cell proliferation, invasion and migration. JMJD2A-specific siRNA was chemically synthesised and transfected into human breast cancer cell line MDA-MB-231. Expression levels of JMJD2A were detected by quantitative real-time PCR and Western blot analysis. Cells proliferation was evaluated by using flow cytometric anlysis and MTT assay. The abilities of invasion and migration were evaluated by cell migration and invasion assay with Boyden chambers. The results showed that the transfection was successful and expression levels of JMJD2A mRNA and protein in siRNA group were both down-regulated. By MTT assay, the mean actual absorbance in siRNA group was significantly lower than that in blank control group (P < 0.05) and negative control group (P < 0.05). In addition, the percentage of cells in G0/G1 phase in siRNA group was significantly more than that in blank control group (P < 0.05) and negative control group (P < 0.05). Furthermore, by cell invasion and migration assay, the decreased number of migrated cells in siRNA group was observed (P < 0.05). These data imply that silencing JMJD2A gene could result in cell cycle change and proliferation inhibition, and lead to suppress tumor cell invasion and migration. It provides a new perspective in understanding the pleiotropic functions of JMJD2A and its contribution to human breast cancer.

JMJD2A contributes to breast cancer progression through transcriptional repression of the tumor suppressor ARHI

Breast cancer is a worldwide health problem and the leading cause of cancer death among females. We previously identified Jumonji domain containing 2A (JMJD2A) as a critical mediator of breast cancer proliferation, migration and invasion. We now report that JMJD2A could promote breast cancer progression through transcriptional repression of the tumor suppressor aplasia Ras homolog member I (ARHI). Immunohistochemistry was performed to examine protein expressions in 155 cases of breast cancer and 30 non-neoplastic tissues. Spearman correlation analysis was used to analyze the correlation between JMJD2A expression and clinical parameters as well as several tumor regulators in 155 cases of breast cancer. Gene and protein expressions were monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Results from knockdown of JMJD2A, overexpression of JMJD2A, Co-immunoprecipitation (Co-IP) assay, dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) elucidated molecular mechanisms of JMJD2A action in breast cancer progression. Furthermore, the effects of ARHI overexpression on JMJD2A-mediated tumor progression were investigated in vitro and in vivo. For in vitro experiments, cell proliferation, wound-healing, migration and invasion were monitored by cell counting, scratch and Boyden Chamber assays. For in vivo experiments, control cells and cells stably expressing JMJD2A alone or together with ARHI were inoculated into mammary fat pads of mice. Tumor volume, tumor weight and metastatic nodules were measured by caliper, electronic balance and nodule counting, respectively. JMJD2A was highly expressed in human breast cancers and positively correlated with tumor progression. Knockdown of JMJD2A increased ARHI expression whereas overexpression of JMJD2A decreased ARHI expression at both protein and mRNA levels. Furthermore, E2Fs and histone deacetylases were involved in the transcriptional repression of ARHI expression by JMJD2A. And the aggressive behavior of JMJD2A in breast cancers could be reversed by re-expression of ARHI in vitro and in vivo. We demonstrated a cancer-promoting effect of JMJD2A and defined a novel molecular pathway contributing to JMJD2A-mediated breast cancer progression.IntroductionBreast cancer is a worldwide health problem and the leading cause of cancer death among females. We previously identified Jumonji domain containing 2A (JMJD2A) as a critical mediator of breast cancer proliferation, migration and invasion. We now report that JMJD2A could promote breast cancer progression through transcriptional repression of the tumor suppressor aplasia Ras homolog member I (ARHI).MethodsImmunohistochemistry was performed to examine protein expressions in 155 cases of breast cancer and 30 non-neoplastic tissues. Spearman correlation analysis was used to analyze the correlation between JMJD2A expression and clinical parameters as well as several tumor regulators in 155 cases of breast cancer. Gene and protein expressions were monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Results from knockdown of JMJD2A, overexpression of JMJD2A, Co-immunoprecipitation (Co-IP) assay, dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) elucidated molecular mechanisms of JMJD2A action in breast cancer progression. Furthermore, the effects of ARHI overexpression on JMJD2A-mediated tumor progression were investigated in vitro and in vivo. For in vitro experiments, cell proliferation, wound-healing, migration and invasion were monitored by cell counting, scratch and Boyden Chamber assays. For in vivo experiments, control cells and cells stably expressing JMJD2A alone or together with ARHI were inoculated into mammary fat pads of mice. Tumor volume, tumor weight and metastatic nodules were measured by caliper, electronic balance and nodule counting, respectively.ResultsJMJD2A was highly expressed in human breast cancers and positively correlated with tumor progression. Knockdown of JMJD2A increased ARHI expression whereas overexpression of JMJD2A decreased ARHI expression at both protein and mRNA levels. Furthermore, E2Fs and histone deacetylases were involved in the transcriptional repression of ARHI expression by JMJD2A. And the aggressive behavior of JMJD2A in breast cancers could be reversed by re-expression of ARHI in vitro and in vivo.ConclusionWe demonstrated a cancer-promoting effect of JMJD2A and defined a novel molecular pathway contributing to JMJD2A-mediated breast cancer progression.

Distribution of biventricular disease in arrhythmogenic cardiomyopathy: an autopsy study

Arrhythmogenic cardiomyopathy is a rare cardiomyopathy characterized by fibrofatty replacement primarily of the right ventricular myocardium. It is a major cause of sudden death in the young and in athletes. There are few autopsy studies of the ventricular distribution of the disease. Fifty cases of sudden cardiac death with fibrofatty replacement in either ventricle from a single medical examiners office were studied. Distribution of disease as determined grossly and microscopically was correlated with activity at time of death, race, and presence of inflammation. Extent of disease was right ventricular in 6 cases (12%; age, 25 ± 5 years), biventricular in 25 (50%; age, 36 ± 3 years), and left ventricular in 19 (38%; age, 37 ± 3 years) (P = .13). Inflammation was present in 44% of biventricular arrhythmogenic cardiomyopathy versus 74% of left ventricular arrhythmogenic cardiomyopathy and 83% of right ventricular arrhythmogenic cardiomyopathy (P = .06). Arrhythmogenic cardiomyopathy, when presenting with sudden death, is usually biventricular. There is a trend that univentricular involvement occurs at an earlier age and that right ventricular involvement shows more inflammation, suggesting different stages of disease.

Effects of siRNA-mediated knockdown of jumonji domain containing 2A on proliferation, migration and invasion of the human breast cancer cell line MCF-7

Jumonji domain containing 2A (JMJD2A) is a potential cancer-associated gene that may be involved in human breast cancer. The present study aimed to investigate suppressive effects on the MCF-7 human breast cancer cell line by transfection with JMJD2A-specific siRNA. Quantitative real-time PCR and western blot analysis were used to detect the expression levels of JMJD2A. Flow cytometric (FCM) analysis and WST-8 assay were used to evaluate cell proliferation. Boyden chambers were used in cell migration and invasion assays to evaluate the cell exercise capacity. Expression levels of JMJD2A mRNA and protein in the siRNA group were both downregulated successfully by transfection. FCM results showed that the percentage of cells in the G0/G1 phase in the siRNA group was significantly greater than that in the blank (P<0.05) and negative control groups (P<0.05). Additionally, the mean absorbance in the siRNA group was significantly lower (P<0.05), as observed by WST-8 assay. Moreover, a decreased number of migrated cells in the siRNA group was observed (P<0.05) using a cell migration and invasion assay. These data indicated that knockdown of JMJD2A may cause inhibition of proliferation, migration and invasion of MCF-7 cells. This study provides a new perspective in understanding the molecular mechanisms underlying the progression of breast cancer and offers a potential therapeutic target for breast cancer.

Allograft pathology in patients transplanted for idiopathic dilated cardiomyopathy.

There are few morphologic studies on idiopathic dilated cardiomyopathy (CM) treated with transplant. We prospectively correlated gross, histologic, and clinical findings pertaining to hearts explanted in a 5-year period from patients with a clinical diagnosis of nonischemic CM and also correlated left ventricular diameter with preoperative echocardiographic reports. Of 64 patients with a clinical diagnosis of dilated cardiomyopathy (DC), 42 were men (age, 51±13 y) and 22 were women (age, 42±18 y). The pathologic diagnosis was idiopathic (dilated) cardiomyopathy (DC) in 55 patients (86%) and features of specific CM in 9 patients (14%). Specific diagnoses were fibrofatty change consistent with arrhythmogenic right ventricular cardiomyopathy (n=6), amyloidosis (n=2), and sarcoidosis (n=1), none of which were suspected clinically. The 55 hearts with idiopathic DC had a mean heart weight of 508 (range, 220 to 980) g. Pathologic subsets of the DC group included 4 hearts without enlargement, cavity dilatation, or significant histologic findings (minimal DC); 3 hearts with histologic evidence of healed myocarditis; and 5 hearts with mildly noncompacted left ventricle with hypertrabeculation. Five patients had prior mitral or tricuspid valve replacement/repairs to manage heart failure. There were 7 postpartum DC cases, 1 with a histologic pattern of healed myocarditis and 1 alcoholism-associated DC. Familial DC comprised 16% (9 of 55) of patients. In patients without prior assist device placement, pathologic left ventricular cavity diameter correlated with echocardiographic end-diastolic volume (r 2, 0.8, P<0.0001). Morphologically, DC is a heterogeneous group. Areas of fibrofatty change and features of noncompaction are not uncommon. Left ventricular measurement at explant correlates well with echocardiographic findings, with a relatively consistent underestimation of the diameter.

Deregulation of RGS17 Expression Promotes Breast Cancer Progression.

Objective: A high level of RGS17 expression is observed in diverse human cancers and correlates with tumor progression. Herein, we aim to investigate its expression and function in breast cancer. Methods: The expression of RGS17 was detected by immunohistochemical analysis and western blot analysis. The level of miR-32 expression was investigated by qRT-PCR. Western blot analysis was used to determine the relationship between RGS17 and miR-32. A series of loss or gain of function assays was performed to measure the effects of RGS17 or miR-32 on tumor migration, invasion, and proliferation. Results: Compared to that in normal breast specimen, the expression of RGS17 had a significantly higher expression level in breast cancer tissues and cell lines. Although the potential relationship of RGS17 expression with clinicopathological features was not observed, there was a significant correlation of RGS17 expression with p63 expression. In cells, inhibition of RGS17 expression impaired cell migration, invasion, and proliferation. Further, RGS17 was identified as a direct and functional target of miR-32. Overexpression of miR-32 in cells could decrease the expression of RGS17 and inhibit cell migration, invasion, and proliferation. In contrast, ectopic expression of RGS17 could attenuate phenotypes caused by miR-32 overexpression. Conclusion: The expression of RGS17 was upregulated in breast cancer, which could enhance cell migration, invasion, and proliferation. Moreover, the RGS17 was identified as a target of miR-32. Our results suggest that RGS17 might play an important role in breast cancer progression and could be a potential target for human breast cancer treatment.

In-stent restenosis is associated with neointimal angiogenesis and macrophage infiltrates.

Restenosis after stenting occurs secondary to the neointima formation. Neovessels have been found in the neointima within stents. However, there are few studies correlating neointimal angiogenesis and in-stent restenosis in humans. We analyzed 65 post-mortem stented arteries from 33 patients with duration >3 months. Cause of death was determined incidental to the coronary findings in every case. Stented segments were embedded in paraffin and stained immunohistochemically for CD68 (macrophages), and endothelial marker PECAM-1 (CD31). Computerized morphometry was performed to quantitate neovessel density for CD31, macrophage infiltrates, as well as plaque and neointimal area. In-stent restenosis was defined as luminal narrowing ≥ 75% cross-section of the stented area. Underlying plaque morphology was classified as fibrous or atheromatous. Neovessels were present in the neointima of 57 stented segments (88%). Mean neovessel density was greater in restenotic vs. non-restenotic neointimas (p = 0.009) and macrophage density was also greater (p = 0.006). Neointimal area correlated positively with density of neointimal vessels (p = 0.002), as well as neointimal macrophage density (p = 0.006), but not type of stent, underlying plaque type, or underlying plaque macrophage score. We conclude that in-stent restenosis is associated with neointimal angiogenesis which is accompanied by macrophage inflammation. The relevance of these findings to treatment and prevention of in-stent restenosis needs to be further explored.

Quantitative Immunohistochemistry of Desmosomal Proteins (Plakoglobin, Desmoplakin and Plakophilin), Connexin-43, and N-cadherin in Arrhythmogenic Cardiomyopathy: An Autopsy Study

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder related to mutations in desmosomal proteins. The current study tests the hypothesis that immunohistochemical staining for desmosomal proteins is of diagnostic utility by studying autopsy-confirmed cases of ARVC. Methods and Results: We studied 23 hearts from patients dying suddenly with ARVC. Control subject tissues were 21 hearts from people dying from non-cardiac causes (n=15), dilated cardiomyopathy (n=3) and coronary artery disease (n=3). Areas free of fibrofatty change or scarring were assessed on 50 sections from ARVC (24 left ventricle, 26 right ventricle) and 28 sections from controls. Immunohistochemical stains against plakoglobin, plakophilin, desmoplakin, connexin-43, and N-cadherin were applied and area expression analyzed by computerized morphometry. Desmin was stained as a control for fixation and similarly analyzed. The mean area of desmin expression was similar in controls and ARVC (86% vs. 85%, p=0.6). Plakoglobin expression was 4.9% ± 0.3% in controls, vs. 4.6% ± 0.3% in ARVC (p=0.3). Plakophilin staining was 4.8% ± 0.3% in controls vs. 4.4% ± 03% in ARVC (p=0.3). Desmoplakin staining was 3.4% in controls vs. 3.2 ± 0.2% in ARVC (p=0.6). There were no significant differences when staining was compared between right and left ventricles (all p > 0.1). For non-desmosomal proteins, the mean area of connexin-43 staining showed no significant difference by presence of disease. Conclusions: The small and insignificant decrease in junction protein expression in ARVC suggests that immunohistochemistry is not a useful tool for the diagnosis.

Caspase-8 deficiency presenting as late-onset multi-organ lymphocytic infiltration with granulomas in two adult siblings

Caspase-8 deficiency (CED) was originally described in 2002 in two pediatric patients presenting with clinical manifestations resembling autoimmune lymphoproliferative syndrome (ALPS) accompanied by infections, and T, B and NK cell defects. Since then, no new CED patients were published. Here we report two adult siblings (Pt1 and Pt2) presenting in their late thirties with pulmonary hypertension leading to lung transplant (Pt1), and a complex neurological disease leading to multiple cranial nerves palsies (Pt2) as their main manifestations. A thorough clinical and immunological evaluation was performed at the Primary Immunodeficiency Clinic at NIH, followed by whole exome sequencing. The patients had multiorgan lymphocytic infiltration and granulomas, as well as clinical signs of immune deficiency/ immune dysregulation. Both siblings carried homozygous mutations in CASP8, c.1096C > T, p.248R > W. This was the same mutation described on the previously published CED patients, to whom these new patients were likely distantly related. We report two new CED patients presenting during adulthood with life-threatening end-organ lymphocyte infiltrates affecting the lungs, liver, spleen, bone marrow and central nervous system. This phenotype broadens the clinical spectrum of manifestations associated with this disease and warrants the search of CASP8 mutations in other cohorts of patients.

Mutations of desmoglein-2 in sudden death from arrhythmogenic right ventricular cardiomyopathy and sudden unexplained death

Desmoglein-2 (DSG2), a member of the desmosomal cadherin superfamily, has been linked to arrhythmogenic right ventricular cardiomyopathy (ARVC)which may cause life-threatening ventricular arrhythmias and sudden death. Fatal arrhythmias resulting in sudden death also occur in the absence of morphologic cardiac abnormalities at autopsy. We sequenced all 15 exons of DSG2 in DNA extracted from post-mortem heart tissues of 25 patients dying with ARVC and 25 from sudden unexplained death (SUD). The primers were designed using the Primer Express 3.0 software. Direct sequencing for both sense and antisense strands was performed with a BigDye Terminator DNA sequencing kit on a 3130 xl Genetic Analyzer. Mutation damage prediction was made using Mutation Taster, Polyphen and SIFT software. 2 DSG2 mutations (p. S1026Q fsX12, p. G678R)in two ARVC samples and 2 DSG2 mutations(p. E 896K, p. A858 V) in two SUD samples were identified, all the mutations were novel. We concluded that DSG2 mutations may not specific for ARVC and may be related to the fatal arrhythmic events even in patients with a morphological normal heart.