Scramble Group Research Articles

MicroRNA-29b attenuates fibrosis in a rat model of Peyronie's disease.

Peyronie's disease is characterized by the formation of fibrotic plaques in the penile tunica albuginea. Effective treatments are limited, warranting the investigation of new promising therapies, such as the application of microRNAs that regulate fibrosis-related genes. We aimed to investigate the therapeutic potential of mimicking microRNA-29b in a fibrin-induced rat model of Peyronie's disease. The study was designed in two phases. To establish an optimal Peyronie's disease model, rats received either human fibrin and thrombin or saline solutions into the tunica albuginea on days 0 and 5. The animal model validation was done through expression and histopathological analyses, the latest by an experienced uropathologist. After validation, we performed microRNA-29b treatment on days 14, 21, and 28 of the study. This phase had control (normal saline) and scramble (microRNA scramble) groups. The mid-penile shaft was removed on day 30 for histological examination and molecular analyses in both study stages. The control group displayed typical tunica albuginea histologic architecture in the animal model validation. In Peyronie's disease group, the Hematoxylin and eosin and Masson Trichrome staining methods demonstrated an interstitial inflammatory process with concomitant dense fibrotic plaques as well as disarrangement of collagen fibers. Additionally, we found out that reduced microRNA-29b (p=0.05) was associated with significantly increased COL1A1 and transforming growth factor β1 genes and proteins (p>0.05) in the Peyronie's disease group. After treatment with mimic microRNA-29b stimulation, the Hematoxylin & eosin and Masson Trichrome staining revealed a discrete and less dense fibrotic plaque. This result was associated with significantly decreasing expression of COL1A1, COL3A1, and transforming growth factor β1 genes and proteins (p<0.05). The fibrin-induced animal model showed significant histopathological and molecular changes compared to the Control group, suggesting that our model was appropriate. Previous findings have shown that increased expression of microRNA-29b was associated with decreased pathological fibrosis. In the present study, treatment with microRNA-29b decreased the gene and protein expression of collagens and transforming growth factor β1. This study reveals the therapeutic potential for Peyronie's disease involving molecular targets. MicroRNA-29b application on the rat's tunica albuginea attenuated fibrosis, arising as a novel potential strategy for Peyronie's disease management.

Enhancing RECK Expression Through miR-21 Inhibition: A Promising Strategy for Bladder Carcinoma Control.

Bladder carcinoma (BC) is the tenth most frequent malignancy worldwide, with high morbidity and mortality rates. Despite recent treatment advances, high-grade BC and muscle-invasive BC present with significant progression and recurrence rates, urging the need for alternative treatments. The microRNA-21 (miR-21) has superexpression in many malignancies and is associated with cellular invasion and progression. One of its mechanisms of action is the regulation of RECK, a tumor suppressor gene responsible for inhibiting metalloproteinases, including MMP9. In a high-grade urothelial cancer cell line, we aimed to assess if miR-21 downregulation would promote RECK expression and decrease MMP9 expression. We also evaluated cellular migration and proliferation potential by inhibition of this pathway. In a T24 cell line, we inhibited miR-21 expression by transfection of a specific microRNA inhibitor (anti-miR-21). There were also control and scramble groups, the last with a negative microRNA transfected. After the procedure, we performed a genetic expression analysis of miR-21, RECK, and MMP9 through qPCR. Migration, proliferation, and protein expression were evaluated via wound healing assay, colony formation assay, flow cytometry, and immunofluorescence.After anti-miR-21 transfection, miR-21 expression decreased with RECK upregulation and MMP9 downregulation. The immunofluorescence assay showed a significant increase in RECK protein expression (p < 0.0001) and a decrease in MMP9 protein expression (p = 0.0101). The anti-miR-21 transfection significantly reduced cellular migration in the wound healing assay (p < 0.0001). Furthermore, in the colony formation assay, the anti-miR-21 group demonstrated reduced cellular proliferation (p = 0.0008), also revealed in the cell cycle analysis by flow cytometry (p = 0.0038). Our results corroborate the hypothesis that miR-21 is associated with BC cellular migration and proliferation, revealing its potential as a new effective treatment for this pathology.

Abstract 14651: Noncoding RNA Drug, TY1, Attenuates Cardiac Fibrosis in Two Mouse Models of Systemic Sclerosis

Introduction: Systemic sclerosis (SSc) is a connective tissue disease characterized by inflammation and fibrosis of the skin and vital organs. The disease has no specific treatment options and a high mortality (~30% at 3 years). We have previously demonstrated anti-fibrotic and anti-inflammatory properties of EV-YF1, a small Y RNA abundant in extracellular vesicles secreted by cardiosphere-derived cells (CDCs). Here we describe the therapeutic bioactivity of a shorter, engineered derivative, TY1. Methods: 5-7-week-old female C57BL/6J wild type mice (n=100 total) were injected with phosphate-buffered saline (PBS; WT) or 100 μL bleomycin (1 mg/ml) subcutaneously every other day for 3 weeks. Animals then received either vehicle (formulation only, in PBS), TY1, or scrambled TY1 (as an RNA control) via oral gavage. Endpoints include exercise capacity, lung weight, systolic and diastolic function by echocardiography, and histologic quantification of fibrosis in heart and skin. To confirm the antifibrotic findings in an inflammation-independent model, 4-6-week-old Tight-skin (tsk-1) mice (n=18 total) received oral TY1, scramble or vehicle for 4 weeks. Results: Diastolic dysfunction was evident in bleomycin mice, but was normalized by TY1 ingestion (E/E’ ratios in WT: 18.91, vehicle: 32.29, scramble: 36.19, TY1: 20.65 p &lt;0.0001). Likewise, lung congestion and exercise intolerance were ameliorated by TY1 ingestion (p&lt;0.0001). In both models (bleomycin-induced and tsk-1), skin and cardiac fibrosis were greater in vehicle or scramble groups than in WT, but were normalized by TY1 after 4 weeks (p &lt;0.001). Conclusions: These results demonstrate the therapeutic bioactivity of a bioinspired new chemical entity in two complementary mouse models of SSc. Thus, TY1 merits investigation as a potential novel therapeutic for SSc.

Pre-Leukemic Transformation of the Bone Marrow Microenvironment Induced By AML1-ETO Fusion Protein

Objective: The bone marrow (BM) microenvironment, especially stromal cells (SCs) and cytokines, plays a critical role in supporting normal hematopoiesis. However, in cases of hematological malignancies such as acute myeloid leukemia (AML), leukemia cells can reshape the BM microenvironment to promote their survival. Despite this, there is still limited understanding of the changes in AML1-ETO fusion protein induced pre-leukemia microenvironment niche. In this study, the alterations of the BM microenvironment in the Aml1 Eto/+; Mx1-Cre (AE KI) mouse model are thoroughly explored and analyzed. This investigation will encompass the study of SCs, cytokines, their sources, and their potential impacts on the occurrence and progression of AML induced by AML1-ETO. Methods: After 4 weeks of PIPC induction, proportion changes of endothelial cells (ECs), osteoblasts, mesenchymal stem cells (MSCs) in the BM of Aml1 Eto/+; Mx1-Cre mice (experimental group) and Aml1 Eto/+; w/o Mx1-Cre mice (control group) were analyzed by flow cytometry. Bulk RNA sequencing (RNAseq) and single cell RNA sequencing (scRNAseq) techniques were applied for analysis of abnormal hematopoietic Lin -Sca-1 +c-Kit + (LSK) stem cells. Cytokine levels were detected using ELISA and Luminex bead assay. Additionally, CRISPR-Cas9 technology was used to knock out the target genes and their functions were validated. Results: Following 4 weeks of PIPC induction, the experimental group exhibited a considerable reduction in the proportion and absolute number of ECs, osteoblasts and MSCs, with ECs being the most significantly reduced. The Luminex assay revealed a substantial increase in cytokine release in the BM of the experimental group, including IL4, IL10, TNFa, Cxcl10, Cxcl11, Ccl1, Ccl2, Ccl4, Ccl7, etc. Bulk RNAseq studies of LSK cells showed that the inflammatory pathway in the experimental group was significantly upregulated. To further explore the release sources of cytokines in the BM and heterogeneity in LSK cell cytokine secretion, scRNAseq analysis of LSK cells was conducted in the AE KI group, which were classified into 9 subgroups. Subgroups 1 and 5 exhibited a significant upregulation of Ccl2, Ccl4, Cxcl2, and IL15. Subgroups 4 and 8 showed upregulation of Ccl2 and IL4. Subgroups 0 and 2 exhibited upregulation of Ccl4, while subgroups 6, 7, and 9 showed upregulation of Cxcl2. Notably, subgroups 1 and 5 also displayed increased expression of Ccr5, which is the receptor for Ccl2. To further validate the origins of cytokines, LSK cells were sorted from experimental group and the control group for in vitro culture, respectively. Subsequently, the cell culture supernatants were collected for Luminex assay, revealing a notable upregulation of Ccl2 and Ccl7 in the experimental group. Furthermore, when comparing m-cherry + LSK cells (AML1-ETO positive) with m-cherry - LSK cells (AML1-ETO negative) from the same mouse, a significant increase in the Ccl2 levels in the m-cherry + LSK cell culture supernatant was observed. Flow cytometry analysis of LSK cells further revealed high expression of Ccl2 and its receptor Ccr5 in Ccl2 + LSK cells. In order to gain a deeper understanding of the functions of Ccl2 and Ccl7, CRISPR-Cas9 technology was used to knockout Ccl2 and Ccl7 in c-kit + cells, which were then transplanted into mice for further investigation, respectively. After 3 months, partial restoration of ECs was observed compared to that of the scramble control group. Conclusion: Our findings suggested that significant changes occurred in the BM microenvironment in AML1-ETO-induced pre-leukemia stage. These changes were characterized by a decrease in SCs and an increase in pro-inflammatory cytokine levels. Moreover, our study identified the heterogeneity in cytokine transcriptional profiles among AE KI LSK cells. The secretion of Ccl2 and Ccl7 by AE KI LSK cells with a blockage in hematopoietic differentiation seemed to be involved in the reduction of stromal cell population. These results provided valuable insights for further exploration of the intricate interactions between abnormal hematopoietic stem cells and the BM microenvironment in AML1-ETO-induced pre-leukemic and leukemic states. Understanding these interactions is crucial for advancing our knowledge of the underlying mechanisms involved in the development and progression of AML.

High expression of VARS promotes the growth of multiple myeloma cells by causing imbalance in valine metabolism.

Multiple myeloma (MM) is a plasma cell malignancy occurring in middle and old age. MM is still an incurable disease due to its frequent recurrence and drug resistance. However, its pathogenesis is still unclear. Abnormal amino acid metabolism is one of the important characteristics of MM, and the important metabolic pathway of amino acids participates in protein synthesis as basic raw materials. Aminoacyl transfer ribonucleic acid synthetase (ARS) gene is a key regulatory gene in protein synthesis. This study aims to explore the molecular mechanism for ARS, a key factor of amino acid metabolism, in regulating amino acid metabolism in MM and affecting MM growth. The corresponding gene number was combined with the gene expression profile GSE5900 dataset and GSE2658 dataset in Gene Expression Omnibus (GEO) database to standardize the gene expression data of ARS. GSEA_4.2.0 software was used to analyze the difference of gene enrichment between healthy donors (HD) and MM patients in GEO database. GraphPad Prism 7 was used to draw heat maps and perform data analysis. Kaplan-Meier and Cox regression model were used to analyze the expression of ARS gene and the prognosis of MM patients, respectively. Bone marrow samples from 7 newly diagnosed MM patients were collected, CD138+ and CD138- cells were obtained by using CD138 antibody magnetic beads, and the expression of ARS in MM clinical samples was analyzed by real-time RT-PCR. Human B lymphocyte GM12878 cells and human MM cell lines ARP1, NCI-H929, OCI-MY5, U266, RPMI 8266, OPM-2, JJN-3, KMS11, MM1.s cells were selected as the study objects. The expression of ARS in MM cell lines was analyzed by real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) lentiviruses were used to construct gene knock-out plasmids (VARS-sh group). No-load plasmids (scramble group) and gene knock-out plasmids (VARS-sh group) were transfected into HEK 293T cells with for virus packaging, respectively. Stable expression cell lines were established by infecting ARP1 and OCI-MY5 cells, and the effects of knockout valyl-tRNA synthetase (VARS) gene on proliferation and apoptosis of MM cells were detected by cell counting and flow cytometry, respectively. GEO data were divided into a high expression group and a low expression group according to the expression of VARS. Bioinformatics analysis was performed to explore the downstream pathways affected by VARS. Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) and high performance liquid chromatography (HPLC) were used to detect the valine content in CD138+ cells and ARP1, OCI-MY5 cells and supernatant of knockdown VARS gene in bone marrow samples from patients, respectively. Gene enrichment analysis showed that tRNA processing related genes were significantly enriched in MM compared with HD (P<0.0001). Further screening of tRNA processing-pathway related subsets revealed that cytoplasmic aminoacyl tRNA synthetase family genes were significantly enriched in MM (P<0.0001). The results of gene expression heat map showed that the ARS family genes except alanyl-tRNA synthetase (AARS), arginyl-tRNA synthetase (RARS), seryl-tRNA synthetase (SARS) in GEO data were highly expressed in MM (all P<0.01). With the development of monoclonal gammopathy of undetermined significance (MGUS) to MM, the gene expression level was increased gradually. Kaplan-Meier univariate analysis of survival results showed that there were significant differences in the prognosis of MM patients in methionyl-tRNA synthetase (MARS), asparaginyl-tRNA synthetase (NARS) and VARS between the high expression group and the low expression group (all P<0.05). Cox regression model multivariate analysis showed that the high expression of VARS was associated with abnormal overall survival time of MM (HR=1.83, 95% CI 1.10 to 3.06, P=0.021). The high expression of NARS (HR=0.90, 95% CI 0.34 to 2.38) and MARS (HR=1.59, 95% CI 0.73 to 3.50) had no effect on the overall survival time of MM patients (both P>0.05). Real-time RT-PCR and Western blotting showed that VARS, MARS and NARS were highly expressed in CD138+ MM cells and MM cell lines of clinical patients (all P<0.05). Cell counting and flow cytometry results showed that the proliferation of MM cells by knockout VARS was significantly inhibited (P<0.01), the proportion of apoptosis was significantly increased (P<0.05). Bioinformatics analysis showed that in addition to several pathways including the cell cycle regulated by VARS, the valine, leucine and isoleucine catabolic pathways were upregulated. Non-targeted metabolomics data showed reduced valine content in CD138+ tumor cells in MM patients compared to HD (P<0.05). HPLC results showed that compared with the scramble group, the intracellular and medium supernatant content of ARP1 cells and the medium supernatant of OCI-MY5 in the VARS-shRNA group was increased (all P<0.05). MM patients with abnormal high expression of VARS have a poor prognosis. VARS promotes the malignant growth of MM cells by affecting the regulation of valine metabolism.

P-802 TFAP2C is required for proper segregation of developmental markers regulating trophectoderm commitment during mouse preimplantation development

Abstract Study question What is the effect of CRISPR/Cas9-mediated knock-out of trophectoderm markers TFAP2A/C on polarization and lineage commitment in mouse? Summary answer CRISPR/Cas9-mediated KO revealed discrepancies in TFAP2A/C function, wherein TFAP2C is dispensable for blastocyst formation but controls the segregation of transcription factors in the mouse embryo. What is known already TFAP2C is an important transcription factor that is expressed solely in trophectoderm (TE) in mouse, and both TE and epiblast in human. Recently, genetic ablation of both TFAP2C and TEAD4 (player of the HIPPO-pathway) completely abolished polarization in mouse, which indicates that TFAP2C might already act before the first lineage segregation takes place. In addition, TFAP2C RNA depletion experiments in mouse embryos revealed a compensatory upregulation of TFAP2A, which indicates functional redundancy. Nonetheless, the question remains how exactly TFAP2C regulates polarization on a molecular level in mouse and whether there is also a functional implication of its isoform TFAP2A. Study design, size, duration Guide RNAs were designed targeting exon 5 of Tfap2c and exon 2 of the Tfap2a gene. CRISPR/Cas9 ribonucleoprotein complexes were delivered into mouse zygotes via electroporation. Additionally, appropriate non-targeted and scramble (inactive crRNA) control groups were included. Morphological analysis, immunofluorescence and next-generation sequencing (NGS) were applied to check for gene editing efficiency and the impact of KO on embryonic development and lineage segregation. Participants/materials, setting, methods Targeted mouse embryos and controls were cultured for a maximum of 4.5 days in vitro. They were stained for different developmental markers, including CDX2 (TE), SOX2 (early ICM), NANOG (epibast, EPI) and SOX17 (hypoblast, PrE) at different stages of development, such as polarization (E2.5), first (E3.5) and second (E4.5) lineage segregation. Immunostaining was used to determine cell number, TE/ICM fraction, marker localization and fluorescence intensity. Embryos were subjected to genetic analysis to determine on-target efficiency. Main results and the role of chance CRISPR/Cas9-mediated electroporation of mouse zygotes generated efficiently complete knock-out embryos. Of the 39 mouse zygotes targeted for TFAP2C, 38 (97%) of them were edited. From the 38 embryos edited for TFAP2C, 24 (62%) embryos displayed 100% frameshift mutations, and were considered KO. TFAP2C KO mouse embryos cultured until E4.5 were still able to form blastocysts (13/15, 87%), but were associated with inferior blastocyst quality compared to the control groups. Furthermore, TFAP2c-null blastocysts could hatch but herniated at multiple places, in contrast to wild-type blastocysts, which typically herniate at one place. In full frameshift TFAP2C KO mouse embryos, we observed delayed CDX2 expression at E2.5 (n = 7). At E3.5 (n = 5), however, CDX2 expression could still be pertained in KO embryos, whereas nuclear localization of SOX2 was disturbed. At E4.5 (n = 6), some primitive endoderm cells were observed in the presumed ICM (SOX17-positive), whereas no NANOG-positive (EPI) cells could be detected. However, some cells of presumed TE of E4.5 TFAP2C-KO embryos displayed erroneous SOX17 or NANOG expression, indicating that they adopted another cell fate irrespective of cell localization. Secondly, we generated TFAP2A KO mouse embryos (3/5, 60%) which exhibited complete morula arrest (100%; n = 3) at E4.5. Limitations, reasons for caution CRISPR/Cas9 is limited by the occurrence of mosaicism (more than one genotype present in an embryo) and potential off-target editing, which we will assess at in silico predicted off-target sites via NGS in mouse embryonic stem cells. The observations of the study will be consolidated by increasing the sample size. Wider implications of the findings Gene editing studies enable us to unravel the molecular interactions that are required for human preimplantation development. Obtaining novel insights into the molecular networks of the TFAP2-transcription factor family could improve our fundamental understanding on the spatial segregation of key transcription factors, which is crucial for successful implantation. Trial registration number Not applicable

Abstract 1283: MUC5AC promotes brain metastases and colonization in non small cell lung cancer

Abstract Background: Non-small cell lung adenocarcinoma (NSCLC), constitutes 80-85% of the lung cancers. The 5-year survival of patients with metastatic lung cancer is only 5%. Additionally, the prognosis worsens in NSCLC patients (40-50%) that have developed brain metastasis. Mucin 5AC (MUC5AC) is overexpressed in the primary NSCLC and brain metastatic tissues; however, the mechanism in the MUC5AC-mediated brain metastasis is unclear. Methods: Immunohistochemistry was performed on lung cancer samples. Expression studies were conducted in the brain-tropic cell lines (A549-BrM, PC9-BrM, and HCC4006-BrM cells) and their respective parental lines. CRISPR Cas9 or shRNA was used to either knockout/knockdown MUC5AC in the cell lines. Mass-spectrometry analysis and protein arrays were conducted in the brain trophic cells. In vivo mouse models involving intracardiac injection of cancer cells were performed, and the effect of MUC5AC brain metastasis and survival was analyzed. Results: MUC5AC was significantly increased in NSCLC brain metastatic tissues. Similarly, MUC5AC expression was elevated in NSCLC brain tropic (BrM) cell lines compared to parental lines. Treatment of BrM or parental cells with the conditioned media of normal human astrocytes resulted in increased MUC5AC levels. We observed astrocyte-derived CCL2-induced the MUC5AC expression in both parental and BrM cells. Mass-spectrometry based proteomic analysis indicated decreased cell adhesion molecules such as ALCAM, ITGB4, ITGB5, CDH17, and DSG2 in MUC5AC knockdown tropic cells. Further, Mass-spectrometry based interactome studies revealed MUC5AC interaction with Annexin A2 enabling the cancer cell adhesion. Intracardiac injection of MUC5AC knockdown A549-BrM (A549-BrM-shMUC5AC) cells showed a significant reduction in brain metastasis and increased survival of the mice compared to scramble control group (P&amp;lt;0.001). Conclusion: Our study identified the role of MUC5AC in metastasis and colonization in the brain environment. Mechanistically, MUC5AC interacts with the Annexin A2 during metastasis, while astrocyte derived CCL2 further induced the MUC5AC expression facilitating the colonization of the NSCLC cells in the brain. Hence, targeting MUC5AC and its interactions partner Annexin A2 may prevent NSCLC brain metastasis. Citation Format: Sanjib Chaudhary, Shailendra Kumar Maurya, Jawed Siddiqui, Mohd W. Nasser, Surinder K. Batra, Imayavaramban Lakshmanan, Apar Kishor Ganti. MUC5AC promotes brain metastases and colonization in non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1283.

Deficiency of the RNA-binding protein ELAVL1/HuR leads to the failure of endogenous and exogenous neuroprotection of retinal ganglion cells.

ELAVL1/HuR is a keystone regulator of gene expression at the posttranscriptional level, including stress response and homeostasis maintenance. The aim of this study was to evaluate the impact of hur silencing on the age-related degeneration of retinal ganglion cells (RGC), which potentially describes the efficiency of endogenous neuroprotection mechanisms, as well as to assess the exogenous neuroprotection capacity of hur-silenced RGC in the rat glaucoma model. The study consisted of in vitro and in vivo approaches. In vitro, we used rat B-35 cells to investigate, whether AAV-shRNA-HuR delivery affects survival and oxidative stress markers under temperature and excitotoxic insults. In vivo approach consisted of two different settings. In first one, 35 eight-week-old rats received intravitreal injection of AAV-shRNA-HuR or AAV-shRNA scramble control. Animals underwent electroretinography tests and were sacrificed 2, 4 or 6 months after injection. Retinas and optic nerves were collected and processed for immunostainings, electron microscopy and stereology. For the second approach, animals received similar gene constructs. To induce chronic glaucoma, 8 weeks after AAV injection, unilateral episcleral vein cauterization was performed. Animals from each group received intravitreal injection of metallothionein II. Animals underwent electroretinography tests and were sacrificed 8 weeks later. Retinas and optic nerves were collected and processed for immunostainings, electron microscopy and stereology. Silencing of hur induced apoptosis and increased oxidative stress markers in B-35 cells. Additionally, shRNA treatment impaired the cellular stress response to temperature and excitotoxic insults. In vivo, RGC count was decreased by 39% in shRNA-HuR group 6 months after injection, when compared to shRNA scramble control group. In neuroprotection study, the average loss of RGCs was 35% in animals with glaucoma treated with metallothionein and shRNA-HuR and 11.4% in animals with glaucoma treated with metallothionein and the scramble control shRNA. An alteration in HuR cellular content resulted in diminished photopic negative responses in the electroretinogram. Based on our findings, we conclude that HuR is essential for the survival and efficient neuroprotection of RGC and that the induced alteration in HuR content accelerates both the age-related and glaucoma-induced decline in RGC number and function, further confirming HuR's key role in maintaining cell homeostasis and its possible involvement in the pathogenesis of glaucoma.

Protective effect of pentraxin 3 on pathological retinal angiogenesis in an in vitro model of diabetic retinopathy

BackgroundDiabetic retinopathy (DR) is the most common retinal microvascular disease caused by diabetes. Previous studies indicated that Pentraxin 3 (PTX3), an acute phase reactant, was closely related to the development of DR. But the exact effect of PTX3 in diabetic retinopathy needs more investigations. MethodsReal-time fluorescence quantitative polymerase chain reaction (qRT-PCR) analysis and western blot (WB) were used to detect the expression of PTX3 in vitro. The Ki67 immunofluorescent staining, scratch-wound migration assay, and tube formation experiments were performed to detect the effect of PTX3 knockdown and overexpression on the fibroblast growth factor (FGF)-induced proliferation, migration and tube-forming ability of human retinal microvascular endothelial cells (HRMECs). The phosphorylation levels of extracellular regulated protein kinases (ERK) and fibroblast growth factor receptor (FGFR) in HRMECs were detected by WB. ResultsIn vitro, the mRNA and protein expressions of PTX3 in the high-concentration glucose condition group were upregulated compared with the normal group (p < 0.05). The proliferation, migration and tube-forming abilities of HRMECs exposed to high-concentration glucose were enhanced (p < 0.01, p < 0.01, p < 0.05 respectively), and the phosphorylation of FGFR and ERK1/2 were increased (p < 0.01, p < 0.05 respectively) compared with the normal condition group. Compared with the high glucose condition group, the proliferation, migration and tube-forming abilities of HRMECs in the high glucose + PTX3 siRNA condition group were further strengthened (p < 0.001, p < 0.0001, p < 0.05 respectively), and the phosphorylation of FGFR and ERK1/2 were increased (p < 0.001, p < 0.01 respectively). Compared with the high glucose condition group, the proliferation, migration and tube-forming abilities of HRMECs in the high glucose + PTX3 overexpression condition group were compromised (p < 0.001, p < 0.05, p < 0.01 respectively), and the phosphorylation of FGFR and ERK1/2 were inhibited (p < 0.001, p < 0.0001 respectively). Neither the scramble siRNA condition group nor the blank plasmid condition group showed significant difference on the proliferation, migration and tube-forming abilities of HRMECs compared with the high glucose condition group (p > 0.05). ConclusionsThe upregulated expression of PTX3 may play a protective role on pathological angiogenesis in DR. PTX3 may serve as a new target for the treatment of DR.

Astaxanthin Regulates PPAR-&amp;lt;i&amp;gt;γ&amp;lt;/i&amp;gt;/NF-&amp;lt;i&amp;gt;κ&amp;lt;/i&amp;gt;B Pathway to Mitigate Nerve Injury after Cerebral Ischemia/Reperfusion in Rats

Purpose: This research evaluates the efficacy of astaxanthin (AX) on cerebral ischemia/reperfusion (I/R) injury in rats and elucidates the potential mechanism of its neuronal protective effect. Methods: Rats were subjected to a middle cerebral artery occlusion/reperfusion (MCAO/R) model. Fifty grown male Sprague-Dawley (SD) rats were divided into 5 groups, including sham operation group (Sham), MCAO/R group, MCAO/R+AX group, MCAO/R+ AX+ Scramble group and MCAO/R+AX+ si-PPAR-γ group. The neurological score and cerebral infarction volume were evaluated after surgery. Rat microglia (RM) were stimulated by lipopolysaccharide (LPS) to form an inflammatory environment. LPS-induced RM cells were incubated with different concentrations of AX (1, 5 or 10 μg/mL), then cell viability, the expression of microglial activation markers, including cytokines (IL-1β, IL-6 and TNF-α), cluster of differentiation 68 (CD68), inducible nitric oxide synthase (iNOS) and CD206 and the expression of PPAR-γ and phosphorylated P65 (p-P65) proteins were determined. Cells were treated with pcDNA-PPAR-γ, as well as treatment with si-PPAR-γ or PPAR-γ antagonist GW9662 before AX treatment, and then cell activation mediators were tested. Results: AX inhibits LPS-induced RM cells activation and enhanced the expression level of PPAR-γ protein in way of dose-dependent, and pcDNA-PPAR-γ treatment had the same effect as AX. While si-PPAR-γ transfection or PPAR-γ suppressant GW9662 treatment reversed the effect of AX, and cut down the level of PPAR-γ protein and augmented the level of p-P65 protein. In addition, AX treatment alleviated the infarct volume, and sensorimotor and cognitive functions of MCAO/R model rats. Conclusion: AX alleviates LPS-induced microglial injury and has a protective effect on rat cerebral I/R injury by regulating the PPAR-γ/NF-κB pathway.

Gene Expression Patterns and Functional Characterization of CD36 in Normal Hematopoietic Cells

Metabolic reprogramming is a hallmark of cancer development and progression. Lipid metabolism is among the most deregulated metabolic pathways in cancer contributing to cell survival and invasion. The fatty acid translocase CD36 has attracted attention in cancer research in recent years due to its role in fatty acid transport as a scavenger receptor. We have recently reported that upregulated APOC2 cooperates with CD36 contributing to leukemia growth by promoting the LYN-ERK signaling mediated metabolic activities of acute myeloid leukemia (AML) cells. Consistently, the knockdown of CD36 or treatment with anti-CD36 antibody reduced the leukemia progression and promoted the overall survival of AML xenograft mice. CD36 is also present on various types of normal cells, including monocytes, macrophages, endothelial cells, adipocytes, and platelets and contribute to their normal functions. Thus, whether targeting CD36 in normal hematopoietic cells poses any toxicity warrants investigation to bring the therapeutic strategy of targeting CD36 in AML to maturity. Here, we leveraged public data to assess the expression patterns of CD36 in normal hematopoietic cells. We found that CD36 expression is several folds higher in dendritic cells, monocytes, and NK cells compared with B cells, eosinophils, neutrophils, and T cells (ANOVA P < 0.001). To investigate the role of Cd36 in normal hematopoietic cells, we carried out gain- and loss-of-function approaches in murine normal hematopoietic primary cells. We engineered a tet-on inducible shRNA lentiviral plasmid targeting two different sequences of the mouse Cd36. In both healthy mouse primary spleen and bone marrow cells, the two pairs of shRNAs reduced the Cd36 mRNA levels compared with scramble control group (~70-90% reduction in mRNA expression). We also engineered two different lentivirus ectopic expression plasmid to overexpress murine Cd36 (PLVX-Cd36 virus and PCDH-Cd36). Lentiviral transduction of both spleen and bone marrow mouse cells confirmed by microscopy visualization of GFP-expressing cells showed several folds increase in Cd36 expression for both plasmids measured by qPCR and western blot. We assessed the effect of Cd36 knockdown on the expansion of spleen and bone marrow hematopoietic cells over five days of cell culture. Relative to the 1 st day of cell count, there was no significant decrease in the mean bone marrow cell and spleen count between the two pair of shRNA compared with scramble control group at day 3 and day 5 post doxycycline treatment (P > 0.05). Cells in the different groups showed about 2 folds expansion on day 5 relative to day 1. Our preliminary data suggest that Cd36 knockdown has limited impact on the survival of normal hematopoietic cells. In vivo experiments assessing the effect of Cd36 gain and loss of function on the engraftment of normal hematopoietic cells in mice are ongoing to further establish the functional impact of Cd36 manipulation on normal hematopoietic cells. Altogether, our studies will demonstrate whether CD36 is dispensable for the survival of normal hematopoietic cells and thus may potentially present a viable therapeutic approach in AML. DisclosuresYaghmour: Jazz: Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Astellas: Speakers Bureau; Alexion: Speakers Bureau; BMS: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Agios: Consultancy, Speakers Bureau.

Effects of indoleamine 2, 3-dioxygenase (IDO) silencing on immunomodulatory function and cancer-promoting characteristic of adipose-derived mesenchymal stem cells (ASCs).

Indoleamine 2, 3-dioxygenase (IDO) catabolizes tryptophan, mediates immunomodulatory functions, and is released by stromal cells such as mesenchymal stem cells. The aims of this study were to investigate the effects of IDO silencing on immunosuppressive function of adipose-derived mesenchymal stem cells (ASCs), T cells phenotype, and the proliferation/migration of tumor cells. ASCs isolated from adipose tissues of healthy women were transfected with IDO-siRNA. Galectin-3, transforming growth factor-β1, hepatocyte growth factor, and interleukin-10 as immunomodulators were measured in ASCs using qRT-PCR. T cells phenotype, interferon-γ, and interleukin-17 expression were evaluated in peripheral blood lymphocytes (PBLs) cocultured with IDO silenced-ASCs by flow cytometry and qRT-PCR, respectively. Scratch assay was applied to assess the proliferation/migration of MDA-MB-231 cell line. Galectin-3 was upregulated (p ˂ 0.05) while hepatocyte growth factor was downregulated (p ˂ 0.05) in IDO-silenced ASCs compared to control groups. Regulatory T cells were inhibited in PBLs cocultured with IDO-silenced ASCs; also T helper2 was decreased in PBLs cocultured with IDO-silenced ASCs relative to the scramble group. IDO-silenced ASCs caused interferon-γ overexpression but interleukin-17 downregulation in PBLs. The proliferation/migration of MDA-MB-231 was suppressed after exposing to condition media of IDO-silenced ASCs compared with condition media of untransfected (p < 0.01) and scramble-transfected ASCs (p < 0.05). The results exhibited the weakened capacity of IDO-silenced ASCs for suppressing the immune cells and promoting the tumor cells' proliferation/migration. IDO suppression may be utilized as a strategy for cancer treatment. Simultaneous blocking of immunomodulators along with IDO inhibitors may show more effects on boosting the efficiency of immune-based cancer therapies.

Inhibition of miR-134-5p protects against kainic acid-induced excitotoxicity through Sirt3-mediated preservation of mitochondrial function

Epilepsy is a neurological disorder which is characterized by brain hyper-excitability and manifests as seizure. Due to its complicated pathogenesis, treatment for epilepsy still remains a huge challenge for neurology in the whole world. MciroRNA-134 (miR-134) is one kind of miRNAs which was firstly found abundant in synapses. In this study, we tried to unveil the role of inhibiting MciroRNA-134-5p (miR-134-5p) in excitotoxicity induced by kainic acid (KA) in the hippocampal neurons (HT22) cells. The results showed that treatment of KA increased the expression of miR-134-5p significantly and caused marked neuron excitotoxicity, evidenced by risen cell death rate, higher LDH release and aggravated cell viability. After suppressing miR-134-5p expression via transfecting HT22 cells with miR-134-5p antisense (Anti-134), cell viability was promoted obviously, along with decreased LDH release and cell death rate. In addition, KA-induced lipid peroxidation, cytochrome c release and mitochondrial ROS generation were also attenuated by Anti-134. The level of Sirtuin 3 (Sirt3) and its downstream antioxidant enzymes, such as mitochondrial superoxide dismutase 2 (SOD2), isocitrate dehydrogenase 2 (IDH2) and glutathione peroxidase (GSH-Px), were significantly higher in Anti-134 group compared with the control and scramble group. After inhibiting Sirt3 expression with SiRNA targeting Sirt3 (Si-Sirt3) and 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP), the positive role of Anti-134 was apparently reversed. In conclusion, this research highly suggests that inhibition of miR-134-5p could protect neurons from KA-induced excitotoxicity through Sirt3-mediated preservation of mitochondrial function.

DJ-1 alleviates oxidative stress injury by activating the Nrf2 pathway in rats with cerebral ischemia-reperfusion injury

To investigate the antioxidant effect of DJ-1 (Park7) in rats with cerebral ischemia/reperfusion (IR) injury and its potential mechanism. A total of 108 SD rats were randomly divided into sham-operated group, middle cerebral artery occlusion (MCAO) group, Scramble group, DJ-1 siRNA group, negative control (NC) group and DJ-1 overexpression group. Except for those in the sham group, all the rats were subjected to MCAO to establish models of cerebral IR injury. In DJ-1 siRNA and DJ-1 overexpression group, a DJ-1 siRNA and an adeno-associated virus vector carrying DJ-1 gene was injected into the lateral ventricle of the rats, respectively. In each group, neurological scores and brain water content were determined after the operation, and pathological changes of the brain tissue and neuronal injury in the cortical infarction area were assessed using HE and Nissl staining. Oxidative stress in the brain tissues was analyzed by detecting superoxide dismutase (SOD) and malondialdehyde (MDA). The expression levels of DJ-1, Nrf2, Ho-1 and NQO1 in the brain tissue were detected with Western blotting, and the expression and nucleation of Nrf2 was determined by immunofluorescence staining. Compared with those in MCAO group, the neurological scores (P < 0.001) and brain water content (P < 0.001) were significantly increased in DJ-1 siRNA group. Intracerebral injection of DJ-1 siRNA following MCAO obviously aggravated neuron injury in cerebral ischemia region, further reduced SOD activity and increased MDA content (P < 0.001), and significantly lowered the expression levels of Nrf2 and its downstream proteins HO-1 and NQO1 (P < 0.001). Intracerebral injection of the adenoviral vector for DJ-1 (P=0.003) overexpression significantly upregulated the levels of Nrf2 (P=0.006) and its downstream proteins HO-1 (P=0.004) and NQO1 (P=0.014). As an important neuroprotective factor, DJ-1 alleviates oxidative stress induced by cerebral IR injury in rats by activating the Nrf2 pathway.

Inhibition of miR-155-5p in non-small cell lung cancer, a potential target for induction of autophagy

ObjectivesThe function of autophagy appears to be diverse in cancer. Autophagy also has a complex relationship with apoptosis under different cell conditions. Considering the high mortality and morbidity of Non-small Cell Lung Cancer (NSCLC) around the world, and the potential role of miR-155-5p in cancer progression, we aimed to investigate the autophagy and apoptosis of NSCLC cell line after inhibition of miR-155-5p. Methods and resultsThe antimiR-155-5p and Scramble oligonucleotides were transfected into the A549 human adenocarcinoma lung cancer. After 24 h, the expression level of SIRT1, SIRT2, TP53INP1, and mTOR were evaluated for both groups performing qRT-PCR. After 48 h, the apoptosis assay was also performed using Annexin V-FITC/PI-based flow cytometry. The transfection of antimiR-155-5p decreased the miR-155-5p expression significantly, which increased the expression levels of SIRT1and TP53INP1 as miR-155-5p target genes and expression of SIRT2 as an indirect target. The high expression of SIRT1 inhibited the mTOR pathway leading to enhanced induction of autophagy. The excess autophagy increased the apoptosis rate compared to the scramble group. ConclusionThe exact control of autophagy is an important issue for normal and cancer cells. Here, inhibition of miR-155-5p induced autophagy in a substantial way leading to autophagic cell death. Therefore, targeting miR-155-mediated autophagy might be a potential therapeutic method for NSCLC.

Effect of SRY-Related High Mobility Group Box 9 on Extracellular Signal-Regulated Kinase/p38 Signaling Pathway in Glioma

As a common malignant tumor in neurosurgery, glioma is characterized as high incidence rate, easy to invade, metastasize and recurrent. It is difficult to treat and has a poor prognosis. The gliomas pathogenesis is complex and has not been fully resolved. Therefore, finding effective molecular targets for glioma is beneficial to improve therapeutic effect. The SRY-related high mobility group box 9 (SOX9) gene involves in mammalian development and is significantly increased in glioma. However, SOX9’s role in gliomas is unclear. The glioma cell line U87 was assigned into control group, scramble group that was transfected with siRNA negative control, and SOX9 siRNA group that was transfected with SOX9 siRNA followed by analysis of SOX9 mRNA and protein level by qPCR and Western blot, cell proliferation by MTT assay, cell apoptosis by Caspase 3 activity assay, cell invasion by Transwell assay, and MMP-9 level by ELISA. SOX9 siRNA transfection significantly downregulated SOX9 mRNA and protein expressions, inhibited U87 cell proliferation, enhanced Caspase 3 activity, suppressed cell invasion of U87, decreased the secretion of MMP-9 in the supernatant, and reduced ERK1/2 and P38 phosphorylation levels (P &lt; 0.05). SOX9 can regulate the progression of glioma by regulating ERK/P38 signaling pathway, promoting cell apoptosis, inhibiting cell proliferation, and restraining cell invasion.

NUSAP1 Enhances Drug Resistance By Binding to RAD51 through DNA Damage Repair Pathway in Chronic Lymphocytic Leukemia

Introduction: Enhanced DNA damage repair effect is an important mechanism for drug-resistance in chronic lymphocytic leukemia (CLL). Moreover, the ability of cancer cells to repair under radiation or chemotherapy drug induced DNA damage also serves as one of the mechanisms for therapy resistance. It is reported that nucleolar and spindle associated protein 1 (NUSAP1), a microtubule binding protein, has been involved in DNA damage repair process and plays important roles in the development, progression, and metastasis in several types of cancer. However, its role and mechanism in the development of CLL are still unclear. Methods: Expression levels of NUSAP1 mRNA and protein in CLL cell lines and patient specimens were detected by qRT-PCR and Western blot, and Kaplan-Meier survival curve and overall survival were analyzed by log-rank test. Peripheral blood samples from de novo CLL patients and healthy volunteers were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University (SPHASU). Microarray datasets GSE22762 were obtained from Gene Expression Omnibus. With altering NUSAP1 expression by lentivirus-transfected cells in vitro, the effects of NUSAP1 on cell proliferation, apoptosis and cycle were detected by CCK8, Annexin V-PE /7AAD staining and PI/RNase staining respectively. Bioinformatics analysis, luciferase reporter analysis, immunoprecipitation and were applied to discern and examine the relationship between NUSAP1 and its potential targets. Results: According to clinical specimens and bioinformatics analysis, the expression level of NUSAP1 gene in samples of CLL patients was significantly increased than that of healthy donors (P&amp;lt;0.05) (Figure A). Besides, the results indicated that the OS of patients with highly expressed NUSAP1 was significantly worse than in patients with low expression with the statistical analysis database GSE22762. mRNA and protein expression levels of NUSAP1 were significantly higher in CLL cell lines than in PBMCs from healthy donors (Figure C). Our findings indicated that NUSAP1 knockdown notably inhibited cell proliferation when compared with the Scramble group (Figure D). Moreover, the amounts of DNA fragmentation of the apoptotic cells were remarkably increased by NUSAP1 shRNA in MEC-1 and EHEB cells when compared with the Scramble group (Figure E). In addition, after knocking down NUSAP1, MEC-1 and EHEB cells were blocked in G0/G1 phase (Figure F). Moreover, addition to fludarabine or ibrutinib with shNUSAP1 group showed enhanced cytotoxicity in CLL cells (Figure G). The differential genes were analyzed via RNA-seq between Scramble and ShNUSAP1 group. Intriguingly, annotations of gene ontology (GO) analysis indicated that NUSAP1 was closely related to biological processes including cell cycle and response to drug. Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that NUSAP1 were enriched in pathways in cancer, DNA replication and cell cycle. Gene set enrichment analysis (GSEA) implicated that NUSAP1 was functionally enriched in DNA replication, cell cycle and proteasome (Figure H). Immunofluorescence showed that NUSAP1 was mainly distributed in the cell nucleus, and the expression level of RAD51 was positively correlated with the change of NUSAP1 expression (Figure I). Surppression of NUSAP1 inhibited the action of proteins in DNA damage repair pathway (Figure J). Through COIP, NUSAP1 was identified to bind with RAD51 and play an important role in DNA damage repair pathway (Figure K). Hence, NUSAP1 participates in the DNA damage repair process and enhances the drug resistance in CLL. Conclusions: This study first demonstrated that the high expression of NUSAP1 in CLL patients is associated with poor prognosis through database analysis and experiments in vitro. Interference of NUSAP1 expression led to a slower CLL cell proliferation and a higher apoptosis rate, meanwhile induced the G1 phase arrest. Collectively, our findings demonstrated that NUSAP1 contributes to DNA damage repairing by binding to RAD51 and enhances drug resistance in CLL. Therefore, NUSAP1 is expected to be a potential target for the treatment of CLL with drug-resistance. Figure 1 Disclosures No relevant conflicts of interest to declare.

Retraction of: miR-106a-5p Suppresses the Proliferation, Migration, and Invasion of Osteosarcoma Cells by Targeting HMGA2, by Qian-Yun He, et al. DNA Cell Biol, 35(9) 506-520; DOI: 10.1089/dna.2015.3121.

DNA and Cell BiologyVol. 39, No. 10 RetractionOpen AccessRetraction of: miR-106a-5p Suppresses the Proliferation, Migration, and Invasion of Osteosarcoma Cells by Targeting HMGA2, by Qian-Yun He, et al. DNA Cell Biol, 35(9) 506–520; DOI: 10.1089/dna.2015.3121This article retracts the following:Retracted: miR-106a-5p Suppresses the Proliferation, Migration, and Invasion of Osteosarcoma Cells by Targeting HMGA2Published Online:2 Oct 2020https://doi.org/10.1089/dna.2015.3121.retractAboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail DNA and Cell Biology officially retracts the paper entitled, “miR-106a-5p Suppresses the Proliferation, Migration, and Invasion of Osteosarcoma Cells by Targeting HMGA2,” by Qian-Yun He, Guang-Chao Wang, Hao Zhang, Da-Ke Tong, Chen Ding, Kang Liu, Fang Ji, Xiongbai Zhu, and Shengwu Yang. DNA Cell Biol, 35(9) 506–520; DOI: 10.1089/dna.2015.3121, due to suspicion of scientific misconduct.The corresponding author, Dr. Fang Ji, contacted the journal via email, and requested a correction be made to Figure 8A, indicating, “When I recently collated the results of the project, I found that in the immunofluorescence experiment, as I used several folders to store pictures but the naming was not standardized, the pictures of other groups in Figure 8A scramble group, hmega2 siRNA group, mir-106a-5p + hmega2 group were misused. [sic]”The email also contained a new figure, however, upon examination by the Editor, it was clear that there were differences between the published figure and the replacement figure. There was clear duplication of lanes in the Western blot figure. The author was then asked to provide further explanation, and Dr. Ji's response was, “Figure 8 A scramble group, hmega2 siRNA group and mir-106a-5p + hmega2 group used the wrong pictures of other groups, and provided the three groups of pictures again [sic].” This response did not address the confusion and several other requests for clarification were made, but went unanswered. The lack of response for clarification concerned the Editor who then re-reviewed the request again, which resulted in the discovery that additional problematic images were raised on the PubPeer platform1 and believes that the authors are now eager to correct them as there appears to be clear scientific misconduct with respect to digital manipulation of figures. Considering the available evidence and lack of further details from the authors, the Editor officially retracts the article from the journal.The Editor and Publisher of DNA and Cell Biology are committed to preserving the scientific literature and the community it serves.Reference1. https://pubpeer.com/publications/35F4FADC69A2182180559AC68655CE Last accessed September 3, 2020. Google ScholarFiguresReferencesRelatedDetailsRelated articlesRetracted: miR-106a-5p Suppresses the Proliferation, Migration, and Invasion of Osteosarcoma Cells by Targeting HMGA201 Sep 2016DNA and Cell Biology Volume 39Issue 10Oct 2020 InformationCopyright 2020, Mary Ann Liebert, Inc., publishersTo cite this article:DNA and Cell Biology.Oct 2020.1912-1912.http://doi.org/10.1089/dna.2015.3121.retractPublished in Volume: 39 Issue 10: October 2, 2020Online Ahead of Print:September 21, 2020PDF download

Effect of Long Non-Coding RNA Plasmacytoma Variant Translocation 1 (PVT1) on the Regulation of SRY (Sex Determining Region Y)-Box 2 (SOX2) in Gastric Cancer

The pathogenesis of gastric cancer (GC) is unclear, which has brought major problems in the diagnosis and treatment. LncRNAs is involved in the regulation of tumorigenesis and development. LncRNA PVT1 is abnormally expressed in various tumors, such as liver cancer and ovarian cancer. However, its role and mechanism in GC have not been clarified. The gastric cancer tissue and adjacent tissues were collected for measuring LncRNA PVT1 level by Real-time PCR. The gastric cancer cell line SGC-7901 was cultured in vitro and divided into control group, scramble group, and PVT1 siRNA group. The tetrazolium salt (MTT) colorimetric assay was to analyze cell proliferation. Caspase 3 activity was tested to assess cell apoptosis. Transwell chamber assay was adopted to evaluated cell invasion. Western Blot was for measuring the epithelial-mesenchymal transition (EMT) molecular proteins and SOX2. Significantly upregulated LncRNA PVT1 was found in gastric cancer tissue (P &lt; 0 05) which was associated tumor differentiation and stage and lymph node metastasis (P &lt; 0 05). LncRNA PVT1 siRNA transfection in SGC-7901 significantly downregulated LncRNA PVT1 expression, inhibited tumor cell proliferation, enhanced Caspase 3 activity, restrained gastric cancer cell invasion, elevated E-cadherin level, reduced Vimentin expression, and declined SOX2 expression (P &lt; 0 05). LncRNA PVT1 is upregulated in gastric cancer and related to the clinical pathology. Downregulation of LncRNA PVT1 inhibits cell proliferation, invasion, and EMT by regulating SOX2, thereby delaying the progression of gastric cancer.

Effect of diabetic induced thioredoxin interacting protein (TXNIP) on islet cell senescence

Objective: To investigate whether the increased expression of thioredoxin interacting protein (TXNIP) in diabetes affects the senescence of islet β cells. Methods: Six normal mice (db/m) and six diabetic mice (db/db) were randomly selected. Fasting blood glucose was measured by blood sugar meter, the expression levels of TXNIP protein, p16, p21 and Rb in pancreatic tissues were detected by Western blot, senescence-associated beta-galactosidase activity in pancreatic tissue was determined by immunochemical staining. INS-1 islet beta cells were randomly divided into 7 groups (n=6), and transfected with lentiviruses (30 μl) for 4 to 6 hours, then was screened with puromycin (PM, 3 μg/m) for 7 days to construct normal group, scramble ShRNA group (interference with airborne poison group), TXNIP-ShRNA-1 group (TXNIP silence group-1), TXNIP-ShRNA-2 group (TXNIP silence group 2), TXNIP-ShRNA-3 group (TXNIP silence group 3), Ad-GFP group (overexpression of the air virus group), Ad-TXNIP-GFP group (TXNIP overexpression group) stably transferred INS-1 islet beta cell line. TXNIP protein expression was detected by Western blot, aging-related beta-galactosidase activity was detected by immunochemical staining, the changes of expression of p16, p21 and Rb was determined by Western blot. Results: Compared with normal mice, the fasting blood glucose of db/db group was increased significantly (P＜0. 01), the expression of TXNIP protein was increased significantly in pancreatic tissues(P＜0. 05), positive staining rate of β- galactosidase was increased significantly in pancreatic tissues, p16/p21/Rb protein expression levels were increased significantly (P＜0. 05). Compared with Ad-GFP group, the positive staining rate of β- galactosidase in Ad-TXNIP-GFP group was increased significantly, p16/p21/Rb protein expression levels were increased significantly (P＜0. 01). Compared to the scramble ShRNA group, the positive staining rate of β- galactosidase in TXNIP-ShRNA group was decreased, p16/p21/Rb protein expression levels were decreased significantly (P＜0. 05). Conclusion: Diabetes can induce islet β-cell senescence by up-regulating TXNIP expression.