One major strategy to generate genetically modified mouse models is gene targeting in mouse embryonic stem (ES) cells, which is used to produce gene-targeted mice for wide applications in biomedicine. However, a major bottleneck in this approach is that the robustness of germline transmission of gene-targeted ES cells can be significantly reduced by their genetic and epigenetic instability after long-term culturing, which impairs the efficiency and robustness of mouse model generation. Recently, we have established a new type of pluripotent cells termed extended pluripotent stem (EPS) cells, which have superior developmental potency and robust germline competence compared to conventional mouse ES cells. In this study, we demonstrate that mouse EPS cells well maintain developmental potency and genetic stability after long-term passage. Based on gene targeting in mouse EPS cells, we established a new approach to directly and rapidly generate gene-targeted mouse models through tetraploid complementation, which could be accomplished in approximately 2 months. Importantly, using this approach, we successfully constructed mouse models in which the human interleukin 3 (IL3) or interleukin 6 (IL6) gene was knocked into its corresponding locus in the mouse genome. Our study demonstrates the feasibility of using mouse EPS cells to rapidly generate mouse models by gene targeting, which have great application potential in biomedical research.
// Shuo Han 1 , Jinhai Guo 2 , Yinan Liu 1 , Zhi Zhang 3 , Qihua He 1 , Peng Li 1 , Mingzhi Zhang 1 , Haojie Sun 1 , Ruizhi Li 1 , Yang Li 3 , Wotan Zeng 2 , Jinwen Liu 2 , Lejian Lian 2 , Yi Gao 3 , Li Shen 1 1 Department of Cell Biology, Stem Cell Research Center, Department of Basic Medical Sciences, Peking University Health Science Center, Beijing, People's Republic of China 2 Beijing DongFang YaMei Gene Science and Technology Research Institute, Beijing, People's Republic of China 3 State Key Laboratory of Organ Failure Research, Co-Innovation Center for Organ Failure Research, Guangdong Provincial Research Center of Artificial Organ and Tissue Engineering, Second Department of Hepatobiliary Surgery, ZhuJiang Hospital, Southern Medical University, Guangzhou, People's Republic of China Correspondence to: Li Shen, e-mail: shenli@bjmu.edu.cn Yi Gao, e-mail: gaoyi6146@163.com Keywords: induced liver cancer stem cells, CD44, transcriptional regulation, CRISPR/Cas9, C3A Received: July 30, 2015 Accepted: October 09, 2015 Published: October 22, 2015 ABSTRACT CD44 is a widely known cancer stem cells marker in various cancers and validated to function in tumor growth, survival and tumor metastasis. In this study, we first established C3A-derived liver cancer stem cells by OSKM method [OCT4, SOX2, KLF4, and c-MYC], termed C3A-induced cancer stem cells (C3A-iCSCs) which acquired self-renewal and stemness abilities. Then we found CD44 was positive in C3A-iCSCs and mainly located in cell nuclear. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) results showed nuclear CD44 combined promoter regions of c- MYC and SOX2 . These results suggested that CD44 participated in C3A-iCSCs transcriptional regulation. To explore CD44 overall influence in liver cancer stem cells, CD44 was knocked out in C3A-iCSCs using CRISPR/Cas9 technology. Our results showed a dramatic increase in the expression of stem cell markers OCT4, SOX2 and NANOG in CD44 − C3A-iCSCs compared with that in CD44 + C3A-iCSCs. Tumor derived from CD44 − C3A-iCSCs also displayed well-differentiated tumor cells compared to CD44 + C3A-iCSCs, which suggested CD44 − C3A-iCSCs derived tumor cells exhibited lower malignant degree. Our data indicated nuclear CD44 in liver cancer stem cells is responsible for the poorly differentiated highly malignant tumor cells by maintenance of low stemness state.
In order to reveal the mechanisms of photosynthetic regulation of Lavandula angustifolia Mill. under low temperature stress, photosynthesis-related genes were screened and the molecular mechanism were analyzed for this species growing in Harbin, northeast of China. RNA-seq technique and photosynthetic physiology measurement were performed under 20°C, 10°C, and 0°C in this study. The results showed that the observing modified rectangular hyperbola mode could accurately reflect the light-response processes under low temperature stress and the low temperature reduced the light energy utilization of L. angustifolia. The stomatal conductance decreased with the temperature dropping, which was associated with the up-regulation of LaBAM1s, LaMPK4-1 and LaMMK2. The up-regulation of LaMPK4-1 and LaMMK2 was beneficial for ROS scavenging. The improvement of cold resistance in L. angustifolia was related to the up-regulated expression of LaFBA and LaOMTs and down-regulated expression of LaGAPAs, LaGOX, and LaTKL1s with the temperature decreasing. The up-expression of LaPSY at 10°C than it at 20°C could protect the photosynthetic organs from oxidative damage. Moreover, the photosynthetic rates at 10°C and 0°C were close to the measured values, which was related to the interactions of RCA with SBPase and Rubisco with SBPase. These findings could provide a theoretical reference for further exploring the cold tolerance mechanism of L. angustifolia, as an important aromatic plant resource, and promoting its cultivation and distribution in the northeast of China.
N7-methylguanosine (m7G) tRNA modification is closely implicated in tumor occurrence and development. However, the precise function and molecular mechanisms of m7G tRNA modification in gastric cancer (GC) remain unclear. In this study, we evaluated the expression and function of methyltransferase-like 1 (METTL1) and WD repeat domain 4 (WDR4) in GC and elucidated the mechanisms underlying the role of METTL1/WDR4-mediated m7G tRNA modifications in promoting GC progression. Upregulation of m7G methyltransferase complex proteins, METTL1 and WDR4, in GC tissues significantly correlates with poor patient prognosis. Functionally, METTL1 and WDR4 facilitate GC progression in vitro and in vivo. Mechanistically, METTL1 knockdown reduces the expression of m7G-modified tRNAs and attenuates the translation of oncogenes enriched in pathways associated with oxidative phosphorylation. Furthermore, METTL1 strengthens mitochondrial electron transport chain complex II (ETC II) activity by promoting succinate dehydrogenase assembly factor 4 (SDHAF4) translation, thereby accelerating GC metabolism and progression. Forced expression of SDHAF4 and chemical modulators of ETC II could reverse the effects of METTL1 on mouse GC. Collectively, our findings delineate the oncogenic role and molecular mechanisms of METTL1/WDR4-mediated m7G tRNA modifications in GC progression, suggesting METTL1/WDR4 and its downstream signaling axis as potential therapeutic targets for GC.
// Lina Wu 1, * , Bo Hu 2, * , Bingtian Zhao 3, * , Yinan Liu 2 , Yue Yang 2 , Lijian Zhang 2 and Jinfeng Chen 2 1 Central Laboratory, Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, Beijing Cancer Hospital & Institute, Peking University Cancer Hospital, Beijing, China 2 Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, Department of Thoracic Surgery II, Peking University School of Oncology, Beijing Cancer Hospital & Institute, Beijing, China 3 Department of Respiratory & Critical Care Medicine, Tianjin Chest Hospital, Tianjin, China * These authors have contributed equally to this work Correspondence to: Jinfeng Chen, email: chenjinfengdoctor@163.com Keywords: microRNA, miR-422a, lung cancer, lymphatic metastasis, biomarker Received: August 15, 2016 Accepted: January 10, 2017 Published: February 02, 2017 ABSTRACT To identify specific circulating microRNAs that were associated with the lymphatic metastasis in lung cancer, we performed miRNA microarray analysis of lymph node with and without metastasis from five lung cancer patients. Top six differentially expressed miRNAs were selected for further validation. A training cohort of 26 patients with lung cancer was firstly recruited and the selected miRNAs in the plasma samples were investigated. miRNA-422a, with highest diagnostic accuracy in lymphatic metastasis was identified (AUC, area under the receiver operating characteristic curve, 0.744; 95%CI, 0.570-0.918). The diagnostic value of miR-422a was also demonstrated by a validation cohort of 51 lung cancer patients (AUC, 0.880; 95%CI, 0.787-0.972). Moreover, a high diagnostic value was also observed after integrated analysis of training and validation cohorts (AUC, 0.792; 95%CI, 0.688-0.896). The odds ratio of high miR-422a expression for lymphatic metastasis in lung cancer was 13.645 (95%CI, 2.677-69.553) after adjustment of the potential confounding factors. Furthermore, we predicted the target genes of miR-422a by combining the online database, miRcords, and the data from GEO and TCGA. Sixty-one target genes of miR-422a that might be involved in lymphatic metastasis in lung cancer were identified. And GO analysis suggested multiple target genes relatively concentrated in the biological processes of apoptosis, transport, and protein phosphorylation.
To investigate the effects of rapamycin on cardiac differentiation, murine embryonic stem cells (ESCs) were induced into cardiomyocytes by 10−4 M ascorbic acid (AA), 20 nM rapamycin alone or 0.01% solvent DMSO. We found that rapamycin alone was insufficient to initiate cardiomyogenesis. Then, the ESCs were treated with AA and rapamycin (20 nM) or AA and DMSO (0.01%) as a control. Compared with control, mouse ESCs (mESCs) treated with rapamycin (20 nM) and AA yielded a significantly higher percentage of cardiomyocytes, as confirmed by the percentage of beating embryonic bodies (EBs), the immunofluorescence and FACS analysis. Rapamycin significantly increased the expression of a panel of cardiac markers including Gata4, α-Mhc, β-Mhc, and Tnnt2. Additionally, rapamycin enhanced the expression of mesodermal and cardiac transcription factors such as Mesp1, Brachyury T, Eomes, Isl1, Gata4, Nkx2.5, Tbx5, and Mef2c. Mechanistic studies showed that rapamycin inhibits Wnt/β-catenin and Notch signaling but promotes the expression of fibroblast growth factor (Fgf8), Fgf10, and Nodal at early stage, and bone morphogenetic protein 2 (Bmp 2) at later stages. Sequential treatment of rapamycin showed that rapamycin promotes cardiac differentiation at the early and later stages. Interestingly, another mammalian target of rapamycin (mTOR) inhibitor Ku0063794 (1 µM) had similar effects on cardiomyogenesis. In conclusion, our results highlight a practical approach to generate cardiomyocytes from mESCs by rapamycin.
Although patients with non-small cell lung cancer harboring activating mutations in the epidermal growth factor receptor (EGFR) show good clinical response to EGFR tyrosine kinase inhibitors (TKIs), patients eventually develop acquired resistance. Previous studies have shown that several microRNAs (miRNAs) are involved in EGFR TKI resistance. Here, we aimed to investigate whether miR-146b-5p sensitizes the EGFR TKI-resistant lung cancer cells. Clinical analysis showed that miR-146b-5p expression in lung cancer cells isolated from pleural effusions of treatment-naive patients was significantly higher than that after acquiring resistance to EGFR TKI treatment. Ectopic expression of miR-146b-5p in EGFR TKI-resistant cells enhanced EGFR TKI-induced apoptosis. The same results were observed in EGFR-dependent and -independent osimertinib-resistant primary cancer cells (PE3479 and PE2988). Mechanically, miR-146b-5p suppressed nuclear factor κB (NF-κB) activity and NF-κB-related IL-6 and IL-8 production by targeting IRAK1. A negative correlation was observed between miR-146b-5p and IRAK1 in clinical specimens. In rescue experiments, restoration of IRAK1 expression reversed the effects of miR-146b-5p on EGFR TKI sensitivity and recovered NF-κB-regulated IL-6 and IL-8 production. In conclusion, miR-146b-5p/IRAK1/NF-κB signaling is important in promoting EGFR TKI resistance, and miR-146b-5p may be a useful tool for overcoming EGFR TKI resistance.
Methyltransferase 3 (METTL3)-mediated N6-methyladenosine (m6 A) RNA modification has been demonstrated to be a potential factor in promoting gastric cancer (GC). METTL3 regulates a series of signaling pathways by modifying various mRNAs. This study aimed to identify novel METTL3-mediated signaling pathways and explored possible targets for use in the clinical setting of gastric cancer.To investigate the proliferation and metastatic capacity of GC cell lines with METTL3 knockdown, a xenograft, lung metastasis, and popliteal lymph node metastasis model was used. The m6 A-modified RNA immunoprecipitation (Me-RIP) sequence was utilized to explore the target mRNAs of METTL3. Cell counting kit 8 and transwell assays were performed to investigate the promoting function of pre-B cell leukemia homeobox 1 (PBX1) and GTP cyclohydrolase 1 (GCH1). Western blotting and chromatin immunoprecipitation were employed to confirm the involvement of the METTL3-PBX1-GCH1 axis. ELISA and liquid chromatography-mass spectrometry were used to explore the biological function of tetrahydrobiopterin (BH4 ).Knockdown of METTL3 suppressed xenograft tumor growth and lung/lymph node metastasis in vivo. Mechanistically, we found that METTL3 combined with and stabilized PBX1 mRNAs. Chromatin immunoprecipitation (ChIP) and further experiments suggested that PBX1 acted as a transcription factor inducing GCH1 expression. Moreover, the METTL3-PBX1-GCH1 axis increased BH4 levels in GC cells, thereby promoting tumor progression.This study suggested that METTL3 enzymes promote tumor growth and lung/lymph node metastasis via METTL3-PBX1-GCH1 axis increasing BH4 levels in GC.
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