Inhibiting SLC26A4 reverses cardiac hypertrophy in H9C2 cells and in rats

Background It has been confirmed that mutations in solute carrier family 26 member 4 (SLC26A4) contribute to pendred syndrome. However, the role of SLC26A4 in cardiac hypertrophy and the signaling pathways remain unclear. Methods Cardiomyocytes were treated by 200 µM phenylephrine (PE) to induce cardiac hypertrophy. Also, the expression of SLC26A4, GSK3, cardiac hypertrophy markers including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was detected through real-time quantitative polymerase chain reaction (RT-qPCR). Flow cytometry assay was used to test the apoptosis of PE-induced cardiomyocytes transfected by small interfere RNA (siRNA)-SLC26A4. Furthermore, we detected the expression of autophagy-related markers including light chain 3 (LC3) and P62. Finally, we established a rat model of abdominal aortic constriction (AAC)-induced cardiac hypertrophy in vivo. Results RT-qPCR results showed that the mRNA expression of SLC26A4 was significantly up-regulated in PE-induced cardiac hypertrophy. After inhibiting SLC26A4, the release of ANP and BNP was significantly decreased and GSK3β was elevated in vivo and in vitro. Furthermore, inhibiting SLC26A4 promoted apoptosis of cardiac hypertrophy cells. In addition, LC3 was down-regulated and P62 was enhanced after transfection of siRNA-SLC26A4. Conclusion Our findings revealed that SLC26A4 increases cardiac hypertrophy, and inhibiting SLC26A4 could decrease the release of ANP/BNP and promote the expression of GSK-3β in vitro and in vivo. Moreover, SLC26A4 silencing inhibits autophagy of cardiomyocytes and induces apoptosis of cardiomyocytes. Therefore, SLC26A4 possesses potential value to be a therapeutic target of cardiac hypertrophy, and our study provides new insights into the mechanisms of cardiac hypertrophy.

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MiR-26a-5p inhibits GSK3β expression and promotes cardiac hypertrophy in vitro

PeerJ ◽

10.7717/peerj.10371 ◽

2020 ◽

Vol 8 ◽

pp. e10371

Author(s):

Liqun Tang ◽

Jianhong Xie ◽

Xiaoqin Yu ◽

Yangyang Zheng

Keyword(s):

Cell Surface ◽

Cardiac Hypertrophy ◽

Surface Area ◽

Myocardial Cell ◽

Immunofluorescence Staining ◽

Cell Surface Area ◽

Direct Target

Background The role of miR-26a-5p expression in cardiac hypertrophy remains unclear. Herein, the effect of miR-26a-5p on cardiac hypertrophy was investigated using phenylephrine (PE)-induced cardiac hypertrophy in vitro and in a rat model of hypertension-induced hypertrophy in vivo. Methods The PE-induced cardiac hypertrophy models in vitro and vivo were established. To investigate the effect of miR-26a-5p activation on autophagy, the protein expression of autophagosome marker (LC3) and p62 was detected by western blot analysis. To explore the effect of miR-26a-5p activation on cardiac hypertrophy, the relative mRNA expression of cardiac hypertrophy related mark GSK3β was detected by qRT-PCR in vitro and vivo. In addition, immunofluorescence staining was used to detect cardiac hypertrophy related mark α-actinin. The cell surface area was measured by immunofluorescence staining. The direct target relationship between miR-26a-5p and GSK3β was confirmed by dual luciferase report. Results MiR-26a-5p was highly expressed in PE-induced cardiac hypertrophy. MiR-26a-5p promoted LC3II and decreased p62 expression in PE-induced cardiac hypertrophy in the presence or absence of lysosomal inhibitor. Furthermore, miR-26a-5p significantly inhibited GSK3β expression in vitro and in vivo. Dual luciferase report results confirmed that miR-26a-5p could directly target GSK3β. GSK3β overexpression significantly reversed the expression of cardiac hypertrophy-related markers including ANP, ACTA1 and MYH7. Immunofluorescence staining results demonstrated that miR-26a-5p promoted cardiac hypertrophy related protein α-actinin expression, and increased cell surface area in vitro and in vivo. Conclusion Our study revealed that miR-26a-5p promotes myocardial cell autophagy activation and cardiac hypertrophy by regulating GSK3β, which needs further research.

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4-Acetyl-Antroquinonol B Suppresses SOD2-Enhanced Cancer Stem Cell-Like Phenotypes and Chemoresistance of Colorectal Cancer Cells by inducing hsa-miR-324 re-Expression

Cancers ◽

10.3390/cancers10080269 ◽

2018 ◽

Vol 10 (8) ◽

pp. 269 ◽

Cited By ~ 10

Author(s):

Oluwaseun Adebayo Bamodu ◽

Ching-Kuo Yang ◽

Wei-Hong Cheng ◽

David T.W. Tzeng ◽

Kuang-Tai Kuo ◽

...

Keyword(s):

Colorectal Cancer ◽

Aberrant Expression ◽

Colon Cells ◽

Colorectal Cancer Cells ◽

Enhanced Expression ◽

Polymerase Chain ◽

Interfering Rna

Background: Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality in both sexes globally. This is not unconnected with the heterogeneity and plasticity of CRC stem cells (CRC-SCs) which stealthily exploit the niche-related and (epi)genetic factors to facilitate metastasis, chemoresistance, tumor recurrence, and disease progression. Despite the accumulating evidence of the role of dysregulated microRNAs in malignancies, the therapeutic efficacy of pharmacological-targeting of CRC-SC-associated microRNAs is relatively under-explored. Experimental approach: In this present study, we employed relatively new bioinformatics approaches, analyses of microarray data, Western blot, real-time polymerase chain reaction (RT-PCR), and functional assays to show that hsa-miR-324-5p expression is significantly suppressed in CRC cells, and inversely correlates with the aberrant expression of SOD2. Results: This converse hsa-miR-324-5p/SOD2 relationship is associated with enhanced oncogenicity, which is effectively inhibited by 4-acetylantroquinonol B (4-AAQB), as evidenced by inhibited cell viability and proliferation, as well as attenuated migration, invasion, and clonogenicity in 4-AAQB-treated DLD1 and HCT116 cells. Interestingly, 4-AAQB did not affect the viability and proliferation of normal colon cells. We also showed that 4-AAQB-induced re-expression of hsa-miR-324-5p, akin to short-interfering RNA, reduced SOD2 expression, correlates with the concurrent down-regulation of SOD2, N-cadherin, vimentin, c-Myc, and BcL-xL2, with concomitant up-regulation of E-cadherin and BAX2 proteins. Enhanced expression of hsa-miR-324-5p in the CRC cells suppressed their tumorigenicity in vitro and in vivo. Additionally, 4-AAQB synergistically potentiates the FOLFOX (folinate (leucovorin), fluorouracil (5FU), and oxaliplatin) anticancer effect by eliciting the re-expression of SOD2-suppressed hsa-miR-324, and inhibiting SOD2-mediated tumorigenicity. Conclusion: Our findings highlight the pre-clinical anti-CSC efficacy of 4-AAQB, with or without FOLFOX in CRC, and suggest a potential novel therapeutic strategy for CRC patients.

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Breast Milk and Saliva Lactoferrin Levels and Postnatal Cytomegalovirus Infection

American Journal of Perinatology ◽

10.1055/s-0040-1701609 ◽

2020 ◽

Author(s):

Kristin E. D. Weimer ◽

Hunter Roark ◽

Kimberley Fisher ◽

C. Michael Cotten ◽

David A. Kaufman ◽

...

Keyword(s):

Breast Milk ◽

Very Low Birth Weight ◽

Quantitative Polymerase Chain Reaction ◽

Neutralization Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Lactating Mothers ◽

Life Threatening ◽

Polymerase Chain

Abstract Objective Very low birth weight preterm infants are at risk for life-threatening infections in the NICU. Breast milk protects against infections but carries the risk of infection by cytomegalovirus (CMV) shed in mother's milk. Lactoferrin is a breast milk and saliva protein with potent neutralizing activity against CMV. Study Design VLBW, maternal breast milk fed infants in the NICU and their lactating mothers were enrolled and followed for 3 months/discharge. Breast milk and infant saliva samples were collected biweekly. Maternal CMV status was determined on breast milk. CMV was measured using quantitative polymerase chain reaction and lactoferrin by enzyme-linked immunosorbent assay. Results In an in vitro neutralization assay, the IC90 of purified human lactoferrin against CMV was 2.08 ng/mL. Bovine lactoferrins were more potent, IC90s > 10-fold higher. Lactoferrin was detected in all breast milk (median: 3.3 × 106 ng/mL) and saliva (median: 84.4 ng/swab) samples. Median CMV load in breast milk was 893 copies/mL. There was no correlation between breast milk lactoferrin concentration and CMV load. Five infants acquired postnatal CMV. There was no difference in saliva or breast milk lactoferrin concentration for mother–infant pairs and postnatal CMV acquisition. Conclusion Lactoferrin neutralizes CMV in vitro, but concentrations in breast milk and saliva are likely too low for effective neutralization in vivo.

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Cyclic nucleotide phosphodiesterase 1C contributes to abdominal aortic aneurysm

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2107898118 ◽

2021 ◽

Vol 118 (31) ◽

pp. e2107898118

Author(s):

Chongyang Zhang ◽

Hongmei Zhao ◽

Yujun Cai ◽

Jian Xiong ◽

Amy Mohan ◽

...

Keyword(s):

Abdominal Aortic Aneurysm ◽

Aortic Aneurysm ◽

Cyclic Nucleotide ◽

Aortic Aneurysms ◽

Cyclic Nucleotide Phosphodiesterase ◽

Causative Role ◽

Abdominal Aortic

Abdominal aortic aneurysm (AAA) is characterized by aorta dilation due to wall degeneration, which mostly occurs in elderly males. Vascular aging is implicated in degenerative vascular pathologies, including AAA. Cyclic nucleotide phosphodiesterases, by hydrolyzing cyclic nucleotides, play critical roles in regulating vascular structure remodeling and function. Cyclic nucleotide phosphodiesterase 1C (PDE1C) expression is induced in dedifferentiated and aging vascular smooth muscle cells (SMCs), while little is known about the role of PDE1C in aneurysm. We observed that PDE1C was not expressed in normal aorta but highly induced in SMC-like cells in human and murine AAA. In mouse AAA models induced by Angiotensin II or periaortic elastase, PDE1C deficiency significantly decreased AAA incidence, aortic dilation, and elastin degradation, which supported a causative role of PDE1C in AAA development in vivo. Pharmacological inhibition of PDE1C also significantly suppressed preestablished AAA. We showed that PDE1C depletion antagonized SMC senescence in vitro and/or in vivo, as assessed by multiple senescence biomarkers, including senescence-associated β-galactosidase activity, γ-H2AX foci number, and p21 protein level. Interestingly, the role of PDE1C in SMC senescence in vitro and in vivo was dependent on Sirtuin 1 (SIRT1). Mechanistic studies further showed that cAMP derived from PDE1C inhibition stimulated SIRT1 activation, likely through a direct interaction between cAMP and SIRT1, which leads to subsequent up-regulation of SIRT1 expression. Our findings provide evidence that PDE1C elevation links SMC senescence to AAA development in both experimental animal models and human AAA, suggesting therapeutical significance of PDE1C as a potential target against aortic aneurysms.

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The circBVES Acts as a Sponge of miR-145-5p to Promote Gastric Cancer Glycolysis and Progression Through Regulating HMGB3 Expression

10.21203/rs.3.rs-76245/v1 ◽

2020 ◽

Author(s):

Lu Jin ◽

Zhiwei He ◽

Changhao Zhu ◽

Guoliang Xiao ◽

Xianjin Yang ◽

...

Keyword(s):

Gastric Cancer ◽

Cancer Cells ◽

Molecular Mechanisms ◽

Quantitative Polymerase Chain Reaction ◽

Gastric Cancer Cells ◽

Repressive Effect ◽

Cancer Tissues

Abstract Background: CircRNA is a new type of non-coding RNA that has attracted much attention for involvement in the development and progression of various human diseases, especially cancer. The most reported role of circRNA in many tumors is ‘MiRNA sponge’. We aimed to investigate the role of circBVES in the proliferation and glycolysis of gastric cancer cells and its molecular mechanisms.Methods: In this study, higher CircBVES expression in gastric cancer tissues was detected by RNA sequencing. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of CircBVES in gastric cancer tissues, and the relationship between the expression of CircBVES and prognosis was further analyzed. Then, the effects of CircBVES on the growth and glycolysis of gastric cancer cells were investigated through in vitro and in vivo functional experiments. The interaction between CircBVES and miR-145-5p was detected by bioinformatics analysis, luciferase activity assay and RNA immunoprecipitation.Results: We found that the expression of CircBVES in gastric cancer tissues was evidently up-regulated, and its level was closely correlated with the prognosis of patients with gastric cancer. Inhibition of CircBVES decreased cell proliferation and glycolysis in vitro. Low expression of CircBVES inhibited tumor growth in vivo. Mechanism analysis showed that CircBVES may serve as a competitive endogenous RNA of miR-145-5p to reduce the expression of miR-145-5p in gastric cancer cells, and relieve the repressive effect of miR-145-5p on target genes HMGB3 and cycle-related proteins CCNE1 and CDK2.Conclusions: Our results suggest that CircAGFG1 may promote the progress of gastric cancer through the CircBVES / miR-145-5p / HMGB3 axis, providing a new target for the treatment of gastric cancer cells.

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Circular RNA hsa_circ_0013958 Functions as an Oncogenic Gene Through Modulating miR-532-3p/WEE1 Axis in Hepatocellular Carcinoma

Frontiers in Oncology ◽

10.3389/fonc.2021.585172 ◽

2021 ◽

Vol 11 ◽

Author(s):

Tao Ma ◽

Yue Ma ◽

Yongjun Du ◽

Zhongheng Wei ◽

Jianchu Wang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Circular Rna ◽

Luciferase Reporter ◽

Western Blot Assay ◽

Polymerase Chain ◽

Oncogenic Gene ◽

Proliferation And Invasion

Backgroundcirc0013958 was identified as a biomarker, which can be used for the diagnosis and screening of lung cancer. However, the role of circ0013958 in hepatocellular carcinoma (HCC) remains unclear.MethodsIn our study, quantitative real-time polymerase chain reaction was performed to determine the levels of circ0013958 in HCC tissues and cell lines. EdU, CCK-8, transwell, flow cytometry and tumorigenesis assays were applied to assess the functions of circ0013958 in HCC in vitro and in vivo. Western blot assay was to detect the expression of WEE1. Luciferase reporter assay, bioinformatics analysis and rescue experiments were used to examine the interaction among circ0013958, miR-532-3p and WEE1.ResultsIt revealed that circ0013958 was significantly up-regulated in HCC, which was positively correlated with poor prognosis of HCC patients. Circ0013958 promoted HCC cell proliferation and invasion, inhibited cell apoptosis in vitro, and promoted tumorigenesis in vivo. Circ0013958 acted as a miR-532-3p sponge to regulate WEE1 expression, thus promoting the progression of HCC.ConclusionsCirc0013958 promotes HCC progression through miR-532-3p/WEE1 axis. Circ0013958 may serve as a potential diagnostic biomarker and therapeutic target of HCC.

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214 USE OF VIRUS ISOLATION AND QUANTITATIVE POLYMERASE CHAIN-REACTION TECHNIQUES TO DETECT BOVINE VIRAL DIARRHEA VIRUS (BVDV) IN SINGLE OR SMALL GROUPS OF PRE-IMPLANTATION BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab214 ◽

2006 ◽

Vol 18 (2) ◽

pp. 214

Author(s):

J. Waldrop ◽

M. Givens ◽

K. Riddell ◽

P. Galik ◽

D. Stringfellow

Keyword(s):

Small Groups ◽

Virus Isolation ◽

Quantitative Polymerase Chain Reaction ◽

Bovine Embryos ◽

Chain Reaction ◽

Diarrhea Virus ◽

Polymerase Chain ◽

Viral Diarrhea Virus

Because of its broad distribution among populations of cattle and its association with materials of animal origin used in embryo production, bovine viral diarrhea virus (BVDV) is a potential problem in applications of embryo technologies. While some isolates of BVDV are known to associate with both in vivo-derived and in vitro-produced bovine embryos, it has yet to be determined if the quantity of virus associated with exposed zona pellucida-intact embryos is sufficient to infect susceptible recipient cows via the intrauterine route. Techniques to detect and quantify BVDV associated with single transferable embryos are important to determine the risk of transmitting BVDV via embryo transfer. The objectives of this study were to define reproducible techniques to detect and quantify BVDV associated with single or small groups of bovine embryos contained in small aliquots of medium using virus isolation (VI) or real time quantitative polymerase chain reaction (Q-PCR) assays. In vivo-derived and in vitro-produced embryos were exposed for 2 h to approximately 106-cell culture infective doses (50% endpoint) per mililiter of a high affinity strain of BVDV, SD-1, and then washed according to IETS guidelines. Embryos were assayed in groups of five or two embryos, or single. There were 5 replicates of the group of five embryos, 4 of the group of two embryos, and 3 of the single embryos for the in vivo-derived embryos undergoing VI; 5, 4, and 2 replicates, respectively, undergoing Q-PCR, and 2, 5, and 2 replicates, respectively, for the in vitro-produced embryo groups undergoing VI and Q-PCR. Those to be assayed by VI were sonicated and the sonicate fluids were layered onto Madin Darby Bovine Kidney (MDBK) cells and passaged to allow for viral replication; an immunoperoxidase monolayer assay was then used for viral detection. A Roche� RNA/DNA extraction kit (Roche Diagnostic Systems, Inc., Somerville, NJ, USA) was used to extract RNA from virally exposed embryos, and extracted samples were assayed in duplicate Q-PCR reactions consisting of 100 �L. The primers used were L1 and U3 which are specific for conserved areas of the 5 prime nontranslated regions of the viral genome of BVDV. The PCR product was detected using hybridization probes s1 and s2 as in Struder et al. 2002 Biologicals 40, 289-296. In vivo-derived groups of five or two embryos, or single embryos, were positive for BVDV 100, 50, and 30% of the time, respectively, when VI was used and 100, 75 and 100%, respectively, when Q-PCR was used. The virus was detected in all of the in vitro-produced embryo groups of five, or two embryos, or single embryos, 100% of the time using VI, and in 100, 80, and 100% respectively, using Q-PCR. The virus isolation technique is highly sensitive but the need to destroy embryos by sonication to identify any embryo-associated virus precludes its use for embryos intended for transfer. Techniques for Q-PCR were sufficiently sensitive to detect and quantify 10 copies of RNA in a sample and to detect BVDV associated with single embryos.

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Effect of Thrombus in AAA on Pressure and Dilation Experienced by the Aneurysm Wall

10.1115/imece2001/bed-23118 ◽

2001 ◽

Author(s):

Mano J. Thubrikar ◽

Francis Robicsek ◽

Michel Labrosse ◽

Vassil Chervenkoff ◽

Brett L. Fowler

Keyword(s):

Maximum Diameter ◽

Pressure Measurements ◽

Indication For Surgery ◽

Intraluminal Thrombus ◽

Aneurysm Wall ◽

Abdominal Aortic ◽

Before And After

Abstract Various factors are considered to play a role in the risk of abdominal aortic aneurysm (AAA) rupture. For instance, a maximum diameter of 7 cm is commonly used as an indication for surgery. There is a need for understanding what makes an aneurysm most likely to rupture. Our focus here is on the role of the intraluminal thrombus and how it affects the pressure and dilation experienced by the aneurysm wall. Since in most of the surgical procedures, the whole aneurysms are almost never removed, the data on the whole aneurysms with thrombus has not been available. The results presented here, therefore, are very important even though they come from a small number of whole aneurysms explored thoroughly. Two types of studies were performed: 1) in vitro and in vivo pressure measurements through the thrombus in three complete AAA, and 2) in vitro dilation measurements during pressurization of two whole aneurysms before and after the thrombus was removed.

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In Vivo Association of Ku with Mammalian Origins of DNA Replication

Molecular Biology of the Cell ◽

10.1091/mbc.12.11.3386 ◽

2001 ◽

Vol 12 (11) ◽

pp. 3386-3401 ◽

Cited By ~ 39

Author(s):

Olivia Novac ◽

Diamanto Matheos ◽

Felipe D. Araujo ◽

Gerald B. Price ◽

Maria Zannis-Hadjopoulos

Keyword(s):

Dna Replication ◽

Quantitative Polymerase Chain Reaction ◽

Polymerase Chain Reaction Analysis ◽

Origin Firing ◽

Origin Binding Protein ◽

M Phase ◽

Polymerase Chain ◽

In Cells

Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association withors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80+/+ and Ku80−/− mouse embryonic fibroblasts. A decrease of ∼70% in in vitro DNA replication was observed when the Ku80−/− extracts were used, compared with the Ku80+/+ extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.

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The JAK2 617V>F mutation triggers erythropoietin hypersensitivity and terminal erythroid amplification in primary cells from patients with polycythemia vera

Blood ◽

10.1182/blood-2006-10-054940 ◽

2007 ◽

Vol 110 (3) ◽

pp. 1013-1021 ◽

Cited By ~ 126

Author(s):

Sabrina Dupont ◽

Aline Massé ◽

Chloé James ◽

Irène Teyssandier ◽

Yann Lécluse ◽

...

Keyword(s):

Polycythemia Vera ◽

Quantitative Polymerase Chain Reaction ◽

Selective Advantage ◽

Erythroid Progenitors ◽

Molecular Events ◽

Polymerase Chain ◽

Late Stages ◽

Early Phases

Abstract The JAK2 617V>F mutation is frequent in polycythemia vera (PV) and essential thrombocythemia (ET). Using quantitative polymerase chain reaction (PCR), we found that high levels of JAK2 617V>F in PV correlate with increased granulocytes and high levels of hemoglobin and endogenous erythroid colony formation. We detected normal progenitors and those that were heterozygous or homozygous for the mutation by genotyping ET and PV clonal immature and committed progenitors. In PV patients, we distinguished homozygous profiles with normal, heterozygous, and homozygous progenitors from heterozygous profiles with only heterozygous and normal progenitors. PV patients with a heterozygous profile had more mutated, committed progenitors than did other PV and ET patients, suggesting a selective amplification of mutated cells in the early phases of hematopoiesis. We demonstrated that mutated erythroid progenitors were more sensitive to erythropoietin than normal progenitors, and that most homozygous erythroid progenitors were erythropoietin independent. Moreover, we observed a greater in vitro erythroid amplification and a selective advantage in vivo for mutated cells in late stages of hematopoiesis. These results suggest that, for PV, erythrocytosis can occur through two mechanisms: terminal erythroid amplification triggered by JAK2 617V>F homozygosity, and a 2-step process including the upstream amplification of heterozygous cells that may involve additional molecular events.

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