Aimin Xue

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.

Poloxamer 188 Attenuates in vitro Traumatic Brain Injury-Induced Mitochondrial and Lysosomal Membrane Permeabilization Damage in Cultured Primary Neurons

Acute membrane damage due to traumatic brain injury (TBI) is a critical precipitating event. However, the subsequent effects of the mechanical trauma, including mitochondrial and lysosomal membrane permeability (MOMP and LMP) remain elusive. The main objective of the current study was to assess the role of a putative membrane-resealing agent poloxamer 188 (P188) in MOMP and LMP in response to a well-defined mechanical insult. Using an in vitro cell shearing device (VCSD), mechanical injury resulted in immediate disruption of membrane integrity in cultured primary neurons, and neurons were treated with P188 or a cathepsin B inhibitor (CBI) after VCSD 10 min. The protective effect of P188 on cultured primary neurons was first detected visually with a light microscope, and measured by MTT assay and LDH assay. The validity of monitoring changes in mitochondrial membrane potential (ΔΨm) was measured by JC-1 staining, and Western blot for cytochrome c and truncated Bid (tBid) in purified mitochondria was also performed. In addition, lysosomal integrity was detected by blotting for cathepsin B and tBid in purified lysosomes. Our results showed post-injury P188 treatment moderated the dissipation of ΔΨm in mitochondria, and inhibited VCSD-induced cytochrome c release from mitochondria as well as cathepsin B from lysosomes. Cathepsin B inhibition (CBI) could also increase cell viability, maintain mitochondrial membrane potential, and repress VCSD-induced release of cytochrome c from mitochondria to cytosol. Both P188 and CBI treatment decreased the cytosolic accumulation of tBid in supernatant of purified lysosomes, and the amount of mitochondrial localized tBid. These data indicate injured neurons have undergone mitochondrial and lysosomal membrane permeability damage, and the mechanism can be exploited with pharmacological interventions. P188s neuroprotection appears to involve a relationship between cathepsin B and tBid-mediated mitochondrial initiation of cell death.

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.

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.

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.

Immunohistochemical analysis of cardiac troponin inhibitor in an experimental model of acute myocardial infarction experimental model and in human tissues

Acute obstruction of coronary arteries leads to acute myocardial infarction (AMI), which causes unexpected death in humans. However, AMI cannot be easily detected in forensic examinations with traditional hematoxylin and eosin (H&E) staining. We analyzed whether cardiac troponin inhibitor (CTnI) could serve as a sensitive and specific early marker for diagnosing AMI in forensic medicine. We established an AMI model in rabbits by ligating the left ventricular branch and observed CTnI expression with immunohistochemistry after different ligation times. We found increased CTnI staining at the 0.5-h time point and depletion of CTnI staining with a 1-h ligation. The areas in which CTnI staining was depleted as seen with immunohistochemical analysis were consistent with the results of H&E staining. Next, human myocardium tissues from 30 persons who died from AMI and were subsequently examined in our forensic center were studied using immunohistochemistry with an antibody to human CTnI. Areas of infarction also showed depletion of CTnI staining. These findings suggested that immunohistochemical detection of CTnI is earlier, more sensitive, and myocardial tissue - specific as compared with H&E staining. CTnI may serve as an ideal marker for diagnosing AMI in forensic investigations.

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.

MicroRNA-19b Downregulates Gap Junction Protein Alpha1 and Synergizes with MicroRNA-1 in Viral Myocarditis

Viral myocarditis (VMC) is a life-threatening disease that leads to heart failure or cardiac arrhythmia. A large number of researches have revealed that mircroRNAs (miRNAs) participate in the pathological processes of VMC. We previously reported that miR-1 repressed the expression of gap junction protein α1 (GJA1) in VMC. In this study, miR-19b was found to be significantly upregulated using the microarray analysis in a mouse model of VMC, and overexpression of miR-19b led to irregular beating pattern in human cardiomyocytes derived from the induced pluripotent stem cells (hiPSCs-CMs). The upregulation of miR-19b was associated with decreased GJA1 in vivo. Furthermore, a miR-19b inhibitor increased, while its mimics suppressed the expression of GJA1 in HL-1 cells. When GJA1 was overexpressed, the miR-19b mimics-mediated irregular beating was reversed in hiPSCs-CMs. In addition, the effect of miR-19b on GJA1 was enhanced by miR-1 in a dose-dependent manner. These data suggest miR-19b contributes to irregular beating through regulation of GJA1 by cooperating with miR-1. Based on the present and our previous studies, it could be indicated that miR-19b and miR-1 might be critically involved in cardiac arrhythmia associated with VMC.

Demographic, clinical and pathological features of sudden deaths due to myocarditis: Results from a state-wide population-based autopsy study

Causes of sudden cardiac deaths have been widely reported with limited data focused specifically on myocarditis. A retrospective review of cases from the Office of the Chief Medical Examiner (OCME), State of Maryland yielded a total of 103 sudden unexpected deaths (SUDs) due to myocarditis (0.17% of all SUDs and 0.70% of autopsied SUDs) from 2005 through 2014. Most deaths occurred in patients <30 years of age with a male:female ratio 1.3:1. Of the 103 cases, 45 (43.7%) patients were witnessed collapsed. Four deaths occurred during exertion, such as exercising at the gym or performing heavy physical work, and 2 deaths were associated with emotional stress. The common cardiac macroscopic findings included ventricular dilatation (39.8%), mild coronary stenosis (17.5%), mottled myocardial appearance (15.5%), and myocardial fibrosis (10.7%). The histological classification of myocarditis was based on the predominant type of inflammatory cell infiltration. In our study group, lymphocytic myocarditis was most common, accounting for 56 cases (54.4%), followed by neutrophilic (32 cases, 31.7%), eosinophilic (13 cases, 12.6%) and giant cell type (2 cases, 1.9%). Microscopic examination revealed myocyte necrosis in 69 cases (67.0%) and interstitial or perivascular fibrosis in 48 cases (46.6%). The percentage of myocyte necrosis was 75.0% (42/58 cases) in lymphocytic, 65.6% (21/31 cases) in neutrophilic, 30.8% (4/13 cases) in eosinophilic, and 100% (2/2 cases) in giant cell myocarditis. Determination of myocarditis as cause of death continues to present a major challenge to forensic pathologists, because histopathologic findings can be subtle and the diagnosis of myocarditis remains difficult.

Axin1 up-regulated 1 accelerates stress-induced cardiomyocytes apoptosis through activating Wnt/β-catenin signaling

ABSTRACT Stress‐induced cardiomyocyte apoptosis contributes to the pathogenesis of a variety of cardiovascular diseases, but how stress induces cardiomyocyte apoptosis remains largely unclear. The present study aims to investigate the effects of Axin1 up‐regulated 1 (Axud1), a novel pro‐apoptotic protein, on the cardiomyocyte survival and the underlying mechanisms. To this end, a rat model under restraint stress (RS) was established and in vitro stress‐induced cardiomyocytes culture was achieved. Our data showed that Axud1 was upregulated in the rat myocardia after exposure to RS. Anti‐apoptotic Bcl‐2 was decreased, whereas pro‐apoptotic Bax and Cleaved caspase‐3 (Cc3) were increased in a time‐dependent manner. The Wnt/&bgr;‐catenin signaling was observed to be interestingly activated in heart undergoing RS. In addition, the treatment of norepinephrine (NE) to in vitro cardiomyocytes increased Axud1 level and induced cell apoptosis. Wnt/&bgr;‐catenin signaling was consistently activated. Knockdown of Axud1 using specific siRNA blunted NE‐induced cardiomyocytes apoptosis and also inactivated the Wnt/&bgr;‐catenin signaling. XAV‐939, an inhibitor of Wnt/&bgr;‐catenin signaling, partially reversed the pro‐apoptotic effect of NE. In conclusion, Axud1 accelerated stress‐induced cardiomyocytes apoptosis through activation of Wnt/&bgr;‐catenin signaling pathway. Our data provided novel evidence that therapeutic strategies against Axud1 or Wnt/&bgr;‐catenin signaling might be promising in relation to RS‐induced myocardial injury. HIGHLIGHTSStress induces apoptosis and upregulates Axud1 expression in both rat myocardia and stressed HL‐1 cells.Suppression of Axud1 expression using special siRNA markedly reduces NE‐induced apoptosis in cardiomyocytes.Axud1 accelerates stress‐induced cardiomyocytes apoptosis by activating Wnt/&bgr;‐catenin signaling pathway.