SET and MYND domain-containing protein 3 inhibits tumor cell sensitivity to cisplatin.

Cisplatin resistance has been a major factor limiting its clinical use as a chemotherapy drug. The present study aimed to investigate whether SET and MYND domain-containing protein 3 (SMYD3), a histone methyltransferase closely associated with tumors can affect the sensitivity of tumors to cisplatin chemotherapy. Real time-qPCR, western blotting, the luciferase reporter, MTT and clonogenic assays were performed to detect the effects of SMYD3 on the chemotherapy capacity of cisplatin. In the present study, SMYD3 exhibited different expression patterns in MCF-7 and T47D breast cancer cells. In addition, this differential expression was associated with tumor cell resistance to cisplatin. Furthermore, SMYD3 knockdown following small interfering RNA transfection increased cisplatin sensitivity, whereas SMYD3 overexpression decreased cisplatin sensitivity. In addition, SMYD3 knockdown synergistically enhanced cisplatin-induced cell apoptosis. SMYD3 expression was downregulated during cisplatin treatment. In addition, transcriptional regulatory activities of SMYD3 3'-untranslated region were also downregulated. These results suggested that SMYD3 may affect cell sensitivity to cisplatin and participate in the development of cisplatin resistance, which is a process that may involve microRNA-124-mediated regulation.

SMYD3-associated pathway is involved in the anti-tumor effects of sulforaphane on gastric carcinoma cells.

Sulforaphane (SFN), a natural compound derived from cruciferous vegetables, has been proved to possess potent anti-cancer activity. SMYD3 is a histone methyltransferase which is closely related to the proliferation and migration of cancer cells. This study showed that SFN could dose-dependently induce cell cycle arrest, stimulate apoptosis, and inhibit proliferation and migration of gastric carcinoma cells. Accompanied with these anti-cancer effects, SMYD3 and its downstream genes, myosin regulatory light chain 9, and cysteine-rich angiogenic inducer 61, was downregulated by SFN. Furthermore, overexpression of SMYD3 via transfection could abolish the effects of SFN, suggesting that SMYD3 might be an important mediator of SFN. To the best of our knowledge, this is the first report describing the role of SMYD3 in the anti-cancer of SFN. These findings might throw light on the development of novel anti-cancer drugs and functional food using SFN-rich cruciferous vegetables.

Stable expression of infliximab in CRISPR/Cas9-mediated BAK1-deficient CHO cells.

OBJECTIVES: To establish stable infliximab-expressing Chinese hamster ovary (CHO) cells with high tolerance to serum-free culture. RESULTS: Bcl-2 antagonist/killer 1 (BAK1), which is a key mediator of the apoptosis pathway, was disrupted, and infliximab, which is a broadly used monoclonal antibody for the treatment of rheumatoid arthritis and other autoimmune diseases, was incorporated into the BAK1 locus of the CHO chromosome using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome-editing technique. The activating effects of serum starvation on BAK1 and cytochrome C (CytC) were suppressed in the genome-edited cells, and the ability of the cells to resist the serum starvation-induced loss of mitochondrial membrane potential and apoptosis was increased, as indicated by the results of polymerase chain reaction (PCR), flow cytometry, enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) analysis. In addition, during subsequent passages, infliximab could be stably produced in the genome-edited CHO cells, and the recombinant antibody could effectively antagonize the cytotoxic effect of tumor necrosis factor α (TNFα). CONCLUSIONS: A CHO cell line capable of stably expressing infliximab and adapting to serum-free culture was constructed. This work lays the foundation for the development of infliximab biosimilars.

LncRNA MALAT1 regulates smooth muscle cell phenotype switch via activation of autophagy.

Vascular smooth muscle cells (VSMCs), switching from a differentiated to a proliferative phenotype, contribute to various vascular diseases. However, the role of long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 MALAT1 in the phenotype switching of VSMCs remains unclear. Here, we report that the knockdown of MALAT1 promotes the transformation of smooth muscle cells from a proliferative phenotype to a differentiated phenotype. MALAT1 knockdown inhibited cellular proliferation and migration, leading to significant cell cycle arrest in the G2 phase. MALAT1 was downregulated in bone morphogenetic protein-7 (BMP-7)-induced cellular differentiation, while MALAT1 was upregulated in platelet-derived growth factor-BB (PDGF-BB)-induced cellular proliferation. PDGF induced the transformation of smooth muscle cells into a proliferative phenotype accompanied by an increase in autophagy. The downregulation of MALAT1 attenuated PDGF-BB-induced proliferation and migration by inhibiting autophagy. MALAT1 could act as a competing endogenous RNA (ceRNA) to regulate autophagy-related 7 (ATG7) gene expression by sponging miR142-3p. The present study reveals a novel mechanism by which MALAT1 promotes the transformation of smooth muscle cells from contraction to synthetic phenotypes.

Knockdown of DNMT1 and DNMT3a Promotes the Angiogenesis of Human Mesenchymal Stem Cells Leading to Arterial Specific Differentiation.

Human mesenchymal stem cells (hMSCs) possess the potential to differentiate into endothelial cells (EC). DNA methylation plays an important role in cell differentiation during development. However, the role of the DNA methyltransferases Dnmt1 and Dnmt3a in specific arterial differentiation of hMSCs is not clear. Here, we show that the CpG islands in the promoter regions of the EC specification and arterial marker genes were highly methylated in hMSCs based on bisulfite genomic sequencing. Treatment with the DNMT inhibitor 5-aza-dc induced the reactivation of EC specification and arterial marker genes by promoting demethylation of these genes as well as stimulating tube-like structure formation. The hMSCs with stable knockdown of Dnmt1/Dnmt3a were highly angiogenic and expressed several arterial specific transcription factors and marker genes. A Matrigel plug assay confirmed that Dnmt1/Dnmt3a stable knockdown hMSCs enhanced blood vessel formation compared with WT MSCs. We also identified that the transcription factor E2F1 could upregulate the transcription of arterial marker genes by binding to the promoters of arterial genes, suggesting its critical role for arterial specification. Moreover, miRNA gain/loss-of-function analyses revealed that miR152 and miR30a were involved in endothelial differentiation of hMSCs by targeting Dnmt1 and Dnmt3a, respectively. Taken together, these data suggest that Dnmt1 and Dnmt3a are critical regulators for epigenetic silencing of EC marker genes and that E2F1 plays an important role in promoting arterial cell determination. Stem Cells 2016;34:1273-1283.

STAT3 regulated ATR via microRNA-383 to control DNA damage to affect apoptosis in A431 cells.

Skin cancer is a major cause of morbidity and mortality worldwide. Mounting evidence shows that exposure of the skin to solar UV radiation results in inflammation, oxidative stress, DNA damage, dysregulation of cellular signaling pathways and immunosuppression thereby resulting in skin cancer. Signal transducer and activator of transcription 3 (STAT3) is well known to function as an anti-apoptotic factor, especially in numerous malignancies, but the relationship between STAT3 activation and DNA damage response in skin cancer is still not fully understood. We now report that STAT3 inhibited DNA damage induced by UV and STAT3 mediated upregulation of GADD45γ and MDC-1 and the phosphorylation of H2AX in UV induced DNA damage. Notably, STAT3 can increase the expression of ATR in A431 cells. Luciferase assay shows that STAT3 activates the transcription of ATR promoter. More importantly, microRNA-383 suppressed ATR expression by targeting 3' (untranslated regions)UTR of ATR in A431 cells, and STAT3 down-regulates the transcription of miR-383 promoter. Thus, these results reveal the new insight that ATR is down-regulated by STAT3-regulated microRNA-383 in A431 cells. Moreover, overexpression of STAT3 enhanced expression of antiapoptosis genes BCL-1 and MCL-1, and depletion of STAT3 sensitized A431 cells to apoptotic cell death following UV. Collectively, these studies suggest that STAT3 may be a potential target for both the prevention and treatment of human skin cancer.

Histone acetyltransferase p300 promotes MRTF-A-mediates transactivation of VE-cadherin gene in human umbilical vein endothelial cells.

Vascular endothelial cadherin (VE-cadherin) is the major determinant of endothelial cell contact integrity and is required in vascular development and angiogenesis. Serum response factor (SRF) plays essential roles in postnatal retinal angiogenesis and adult neovascularization. It is unclear whether transcription of VE-cadherin is mediated by a SRF co-activator, myocardin-related transcription factor-A (MRTF-A). Here we have demonstrated that MRTF-A is a key regulatory factor to activate the transcription of VE-cadherin in human umbilical vein endothelial cells (HUVECs). siRNA-mediated knockdown of MRTF-A decreased the level of VE-cadherin in HUVECs. Vascular endothelial growth factor (VEGF) induced MRTF-A binding to the SRF-binding site (CArG box) within VE-cadherin promoter. Histone acetyltransferase p300 and MRTF-A could synergistically augment the expression of VE-cadherin by enhancing acetylation of histone3K9 (H3K9Ac), histone3K14 (H3K14Ac) and histone4 at the SRF-binding site within VE-cadherin promoter. Taken together, these data identified a detailed regulatory mechanism of VE-cadherin gene expression.

Generation of insulin-producing cells from C3H10T1/2 mesenchymal progenitor cells.

Mesenchymal stem cells (MSCs) have been reported to be an attractive source for the generation of transplantable surrogate ß cells. A murine embryonic mesenchymal progenitor cell line C3H10T1/2 has been recognized as a model for MSCs, because of its multi-lineage differentiation potential. The purpose of this study was to explore whether C3H/10T1/2 cells have the potential to differentiate into insulin-producing cells (IPCs). Here, we investigated and compared the in vitro differentiation of rat MSCs and C3H10T1/2 cells into IPCs. After the cells underwent IPC differentiation, the expression of differentiation markers were detected by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR (qRT-PCR) and Western blotting. The insulin secretion was evaluated by enzyme-linked immunosorbent assay (ELISA). Furthermore, these differentiated cells were transplanted into streptozotocin-induced diabetic mice and their biological functions were tested in vivo. This study reports a 2-stage method to generate IPCs from C3H10T1/2 cells. Under specific induction conditions for 7-8 days, C3H10T1/2 cells formed three-dimensional spheroid bodies (SBs) and secreted insulin, while generation of IPCs derived from rat MSCs required a long time (more than 2 weeks). Furthermore, these IPCs derived from C3H10T1/2 cells were injected into diabetic mice and improves basal glucose, body weight and exhibited normal glucose tolerance test. The present study provided a simple and faithful in vitro model for further investigating the mechanism underlying IPC differentiation of MSCs and cell replacement therapy for diabetes.