Journal: World Journal of Gastroenterology

Article Title: Long noncoding RNA ZNFX1-AS1 promotes the invasion and proliferation of gastric cancer cells by regulating LIN28 and CAPR1N1

doi: 10.3748/wjg.v28.i34.4973

Figure Lengend Snippet: List of primers used to detect mRNA expression of long noncoding RNA binding proteins

Article Snippet: LIN28, CAPRIN1, CEA, and CK20 were detected by enzyme-linked immunosorbent assay (ELISA) using a Mouse Protein lin-28 homolog A (Lin28a) ELISA Kit (ELISAGenie, Dublin, Ireland), a Human Caprin 1 (CAPRIN1) ELISA Kit (Abbexa, Cambridge, United Kingdom), a CEA ELISA Kit (Bioss, United Kingdom) and CK20 ELISA kits (FKBIO, Chengdu, China), respectively.

Techniques: Expressing, RNA Binding Assay

Journal: World Journal of Gastroenterology

Article Title: Long noncoding RNA ZNFX1-AS1 promotes the invasion and proliferation of gastric cancer cells by regulating LIN28 and CAPR1N1

doi: 10.3748/wjg.v28.i34.4973

Figure Lengend Snippet: The relative mRNA levels of genes encoding ZNFX1-AS1 binding proteins in transduced BGC823 and SGC7901 cells were determined by quantitative reverse transcription polymerase chain reaction. A: UFP1; B: eIF4AIII; C: IGF2BP1; D: FMRP; E: LIN28; F: IGF2BP2; G: FUS; H: ZC3H7B; I: IGF2BP3; J: CAPRIN1. AFAS1: ZNFX1-AS1. a P < 0.05; b P < 0.01.

Article Snippet: LIN28, CAPRIN1, CEA, and CK20 were detected by enzyme-linked immunosorbent assay (ELISA) using a Mouse Protein lin-28 homolog A (Lin28a) ELISA Kit (ELISAGenie, Dublin, Ireland), a Human Caprin 1 (CAPRIN1) ELISA Kit (Abbexa, Cambridge, United Kingdom), a CEA ELISA Kit (Bioss, United Kingdom) and CK20 ELISA kits (FKBIO, Chengdu, China), respectively.

Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction

Journal: World Journal of Gastroenterology

Article Title: Long noncoding RNA ZNFX1-AS1 promotes the invasion and proliferation of gastric cancer cells by regulating LIN28 and CAPR1N1

doi: 10.3748/wjg.v28.i34.4973

Figure Lengend Snippet: The relative protein levels of LIN28 and CAPRIN1 in transduced BGC823 and SGC7901 cells were determined by enzyme-linked immunosorbent assay. A: The levels of CAPRIN1 in transduced BGC823 cells; B: The levels of CAPRIN1 in transduced SGC7901 cells; C: The levels of LIN28 in transduced BGC823 cells; D: The levels of LIN28 in transduced SGC7901 cells. a P < 0.05; b P < 0.01.

Article Snippet: LIN28, CAPRIN1, CEA, and CK20 were detected by enzyme-linked immunosorbent assay (ELISA) using a Mouse Protein lin-28 homolog A (Lin28a) ELISA Kit (ELISAGenie, Dublin, Ireland), a Human Caprin 1 (CAPRIN1) ELISA Kit (Abbexa, Cambridge, United Kingdom), a CEA ELISA Kit (Bioss, United Kingdom) and CK20 ELISA kits (FKBIO, Chengdu, China), respectively.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: World Journal of Gastroenterology

Article Title: Long noncoding RNA ZNFX1-AS1 promotes the invasion and proliferation of gastric cancer cells by regulating LIN28 and CAPR1N1

doi: 10.3748/wjg.v28.i34.4973

Figure Lengend Snippet: Effect of ZNFX1-AS1 on gastric cancer cell tumorigenesis in vivo . A: The expression of long noncoding RNA ZNFX1-AS1 in BALB/c nude mice was detected weekly by quantitative reverse transcription polymerase chain reaction; B and C: Images of tumors from BALB/c nude mice; D and E: Tumor size and weight were measured weekly; F: Expression of LIN28 and CAPRIN1 in tumors of BALB/c nude mice; G: Expression of carcinoembryonic antigen and CK20 in lymph nodes of BALB/c nude mice. CEA: Carcinoembryonic antigen; ZFAS1: ZNFX1-AS1. a P < 0.05; b P < 0.01; c P < 0.001.

Article Snippet: LIN28, CAPRIN1, CEA, and CK20 were detected by enzyme-linked immunosorbent assay (ELISA) using a Mouse Protein lin-28 homolog A (Lin28a) ELISA Kit (ELISAGenie, Dublin, Ireland), a Human Caprin 1 (CAPRIN1) ELISA Kit (Abbexa, Cambridge, United Kingdom), a CEA ELISA Kit (Bioss, United Kingdom) and CK20 ELISA kits (FKBIO, Chengdu, China), respectively.

Techniques: In Vivo, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: iScience

Article Title: LIN28 and histone H3K4 methylase induce TLR4 to generate tumor-initiating stem-like cells

doi: 10.1016/j.isci.2023.106254

Figure Lengend Snippet: Conditional expression of constitutively active Tlr4 ( caTLR4 ) in hepatocytes/hepatoblasts promotes liver tumor and shRNA screening identified H3K4 methylase SETD7 as TLR4 inducer (A) Strategy to conditionally express caTLR4 in liver progenitor cells of transgenic mice. Schematic of the Tet -ca Tlr4 mice. Gene diagrams of pAlb-tTA , pLAP-tTA and pTet-o-CMV-CD4hTLR4 are shown to generate compound mice Alb:tTA;Tet-caTLR4 mice and LAP:tTA;Tet-caTLR4 mice. (B) Immunoblot of LAP in spleen and hepatoblasts versus adult liver. (C) Representative images of spontaneously developed liver tumors in pLAP-tTA / Tet-caTlr4 mice after DOX withdrawal. (D) Liver tumor incidence 12 months after doxycycline withdrawal (Vehicle). Hepatoblasts are predisposed to TLR4-mediated transformation in Tet-caTLR4 Tg mice ( Alb-Tlr4 versus LAP-Tlr4 ). Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (E) The effect of caTLR4 targeted to liver progenitor cells. pLAP-tTA ; Tet-caTlr4 mice were subjected to DCC diet feeding to drive LPC induction. These mice developed aggressive nodular HCC at the incidence rate of 40%. In contrast DDC feeding alone of these mice resulted in only 10% tumor incidence. These results demonstrated that caTLR4 signaling was oncogenic in hepatoblasts but not in mature hepatocytes in vitro . Data are represented as mean ±SEM. Asterisks (∗) indicate statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. Scale bars represent 50 μm. (E, insets) EpCAM+ cell proliferation in non-tumor ductular areas of DDC-diet-fed mice. (F) TIC surface makers were detected by FACS in Huh7 cells and TICs. TICs are CD133(+)/EpCAM double-positive cells. (G) Hepatocytes versus progenitors for genesis of TICs in soft agar assay. Transformation induced by ca Tlr4 expression in hepatoblasts but not in primary hepatocytes. Overexpression of LIN28 and NANOG transformed primary liver progenitor cells (LPCs). Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (H) Nanog mRNA expression in the cells shown in (G) correlated with transformation.CaTLR4 overexpression promoted NANOG, HMGB1, and P65 phosphorylation and reduced phospho-IκB (H, right). Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (I) Hypothetical model of TLR4-mediated oncogenesis in hepatocytes/LPCs. (J) Scheme of forward genetic screen using lentiviral shRNA library in an orthotopic transplant mouse model for identification of metastasis-promoting genes. (K) List of shRNA-mediated tumor regression gene candidates. The shRNA screen followed by validation in orthotopic injection mouse model identified SETD7, NOTCH1 and HIF2 as key shRNA-mediated tumor regression genes.

Article Snippet: ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (F) (Left) The 25 nM dose of LIN28 pharmacological inhibitor (LIN28 1632: R&D Systems) with 1 μM of HIF inhibitor (HIF-2α inhibitor PT2385). (Right) CD133+ TICs were killed by LIN28TALEN delivery via CD133 Ab-NPs in the presence of HIF pharmacological inhibitor.

Techniques: Expressing, shRNA, Transgenic Assay, Western Blot, Transformation Assay, In Vitro, Soft Agar Assay, Over Expression, Phospho-proteomics, Biomarker Discovery, Injection

Journal: iScience

Article Title: LIN28 and histone H3K4 methylase induce TLR4 to generate tumor-initiating stem-like cells

doi: 10.1016/j.isci.2023.106254

Figure Lengend Snippet: DNA hypomethylation promotes TLR4 and NANOG activation in liver cancer through SETD7 expression (A) Evasion of LPS tolerance in TICs. Test of Nanog+/CD133+/CD49f+ TICs avoidance of LPS tolerance, known for attenuated expression of pro-inflammatory genes after repetitive LPS exposure. Cancer stem cells and Nanog-/CD133-/CD49f+ control cells were untreated (C) or repetitively stimulated with 100 ng/mL LPS at 0, 24 and/or 48 h (L1, L2 and/or L3). (B) TICs evade LPS tolerance for Nanog and Tlr4 in TICs. The mRNA levels for NANOG and TLR4 were indeed attenuated after second and third LPS treatment in the control cells but not in the TICs. The TICs evaded LPS-tolerance for Nanog and TLR4 expression under repetitive LPS stimulation. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (C) H3K4 methyltransferase inhibition restored TLR4 expression in non-TICs. Epigenetic and transcriptional regulation of NANOG by LPS/TLR4 signaling in TICs isolated from HCC model. DzNEP inhibits EZH2. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (D) Epigenetic and transcriptional regulation of NANOG by LPS/TLR4 signal in TICs isolated from HCC model. SETD7 pharmacological inhibition or SED7 silencing reduced TLR4 expression. (Right) Inhibition of SETD7 reduced TLR4 expression. To test if SETD7 downregulation reduces TLR4 mRNA levels, shRNA lentivirus targeting SETD7 was transduced. Silencing effect was confirmed by immunoblot analyses. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (E) Comparison of NANOG methylation status in embryonic stem cells (ESC), TICs and primary hepatocytes. (F) E2F1 (orange oval) regulated Nanog enhancer for LPS-induced activation. Truncated promoter luciferase constructs were used to map the region responsive to TLR4 signaling. LPS-mediated Nanog promoter activity in mESCs (Top) and TICs (Bottom) compared to PBS (Vehicle)-treated cells demonstrated Nanog promoter activity upon TLR4 stimulation (n = 3). Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (G) E2F1-binding sites were required for efficient Nanog transactivation (n = 4). Four mutant-luciferase plasmids were constructed by in vitro mutagenesis. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (H) Nanog promoter ChIP assay using anti-E2F1 antibody followed by qPCR in TICs. E2F1 showed increased occupancy in Nanog promoter because TLR4 signaling transactivates Nanog promoter through E2F1-binding sites. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001. (I) TLR4 mRNA upregulation in CD133+ cells through hypoxia via HIF2 and SETD7. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.001. (J) Inhibition of LIN28 or HIF reduced TIC spheroid formation. (K) Epigenetic regulation of TLR4. Demethylation of TLR4 promoter. DNA methylation of CpG islands in TLR4 loci among TICs, hepatocytes, and mESC. Epigenetic regulation of TLR4. Demethylation of TLR4 promoter. TLR4 epigenetic regulation (bisulfite sequencing analysis of TLR4 promoter region). (L) DNA methylation of CpG islands of TLR4 in TICs derived from different mouse tumor model (NS5A Tg fed high-cholesterol high-fat diet: HCFD).

Article Snippet: ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (F) (Left) The 25 nM dose of LIN28 pharmacological inhibitor (LIN28 1632: R&D Systems) with 1 μM of HIF inhibitor (HIF-2α inhibitor PT2385). (Right) CD133+ TICs were killed by LIN28TALEN delivery via CD133 Ab-NPs in the presence of HIF pharmacological inhibitor.

Techniques: Activation Assay, Expressing, Control, Inhibition, Isolation, shRNA, Western Blot, Comparison, Methylation, Luciferase, Construct, Activity Assay, Binding Assay, Mutagenesis, In Vitro, DNA Methylation Assay, Methylation Sequencing, Derivative Assay

Journal: iScience

Article Title: LIN28 and histone H3K4 methylase induce TLR4 to generate tumor-initiating stem-like cells

doi: 10.1016/j.isci.2023.106254

Figure Lengend Snippet: LIN28-mediated let-7 inhibition maintains TLR4 expression and self-renewal ability of TICs (A) Lin28 is one of several induced genes in tumors. (B) Mechanism of TLR4 regulation following LIN28 signal. (C) (Top) LIN28 inhibits Drosha-mediated processing of pri- let-7 and Dicer-mediated cleavage of pre- let-7 . (Bottom) Using TLR4 promoter-3′UTR reporter construct for analysis of post-transcriptional regulation of Tlr4 through the let-7 binding site in CD133-cells. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (D) LIN28 expression reduced mature let-7i (Bottom: Northern blots) and increased pri- let-7i and TLR4; (Top: Immunoblots in hepatoblasts. Plasmid amounts (μg) are indicated in designated lanes. (E) Lentivirus sh-LIN28 transduction increased mature let-7i level, (Bottom: Northern blots) and reduced pri- let-7i (Bottom: Northern blots) and TLR4 (Top: Immunoblots) in hepatoblasts. (F) Inhibition of LIN28, TLR4 or NANOG reduced TIC colony formation. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (G) Spheroid formation assays. TLR4 (−) Huh7 cells had high let-7, but low LIN28. Transduction of LIN28 lentiviral vector induced TLR4 mRNA expression in TLR4(−) Huh7 cells. Data are represented as mean ±SEM. (H) NANOG mRNA induction. LIN28 and TLR4 expression was significantly higher in mouse and human TICs compared to CD133-/CD49f+ cells. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (I) Silencing Hmgb1 reduced TIC proliferation in vitro . Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (J) Silencing Hmgb1 reduced TIC proliferation in immunocompromised NSG mice. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (K) LPS-mediated TLR4 stimulation induced HMGB1 release in culture supernatants that was accentuated by hypoxia treatment. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (L) 3 H-Uridine incorporation enhanced by LPS stimulation in the presence of hypoxia. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (M) LIN28-mediated let-7 inhibition maintained TLR4 expression and self-renewal ability of TICs.

Article Snippet: ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (F) (Left) The 25 nM dose of LIN28 pharmacological inhibitor (LIN28 1632: R&D Systems) with 1 μM of HIF inhibitor (HIF-2α inhibitor PT2385). (Right) CD133+ TICs were killed by LIN28TALEN delivery via CD133 Ab-NPs in the presence of HIF pharmacological inhibitor.

Techniques: Inhibition, Expressing, Construct, Binding Assay, Northern Blot, Western Blot, Plasmid Preparation, Transduction, In Vitro

Journal: iScience

Article Title: LIN28 and histone H3K4 methylase induce TLR4 to generate tumor-initiating stem-like cells

doi: 10.1016/j.isci.2023.106254

Figure Lengend Snippet: Inhibition of TLR4 pathway and chemotherapeutic agents induced cell death of CD133+/CD49f+ cells isolated from the HCC models (A) CD133+/CD49f+ TICs from the Core/Ns5a HCC mouse model, proliferated in vitro in a TLR4 dependent manner. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (B) TLR4 antagonism or chemotherapeutic drugs rapamycin (Rapa) and sorafenib (Sora) effects on TICs. TLR4 antagonist caused 30% growth arrest and complete arrest was achieved when combined with chemotherapeutic drugs. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (C) Effect of TLR antagonist and chemotherapeutic drugs (see (B)) on TIC viability. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (D and E) TLR4 antagonism or chemotherapeutic agent LIN28 inhibitor caused 40% growth reduction, and complete arrest in drug combination. (E middle) Drug effects correlated with cell death of TICs. (E right) TNF-α mRNA levels were quantified by RT-qPCR after testing whether LIN28-TLR4 signaling promoted TNF-α-mediated cell death. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (F) (Top) Experimental mouse models employed and treatments. (Bottom) Tumor volume kinetics of NSG mice injected with TICs. TLR4 antagonism for therapy conjunctive with LIN28 pharmacological inhibitor treatment effectively eliminated implanted tumors transduced for TLR4 antagonism or treatment by chemotherapeutic agents Sorafenib (Sora) + Rapamycin (Rapa) caused 75% growth retardation, and partial arrest by triple combination, which correlated with cell death of TICs (Bottom). Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (G) Tumor volume kinetics of NSG mice injected with TICs. TLR4 antagonism for therapy conjunctive with LIN28 pharmacological inhibitor treatment effectively eliminated injected tumors. TLR4 antagonism or LIN28 pharmacological inhibition resulted in 98% growth retardation, and complete arrest in triple combination. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001.

Article Snippet: ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (F) (Left) The 25 nM dose of LIN28 pharmacological inhibitor (LIN28 1632: R&D Systems) with 1 μM of HIF inhibitor (HIF-2α inhibitor PT2385). (Right) CD133+ TICs were killed by LIN28TALEN delivery via CD133 Ab-NPs in the presence of HIF pharmacological inhibitor.

Techniques: Inhibition, Isolation, In Vitro, Quantitative RT-PCR, Injection

Journal: iScience

Article Title: LIN28 and histone H3K4 methylase induce TLR4 to generate tumor-initiating stem-like cells

doi: 10.1016/j.isci.2023.106254

Figure Lengend Snippet: LIN28-dependent tumorigenicity of CD133+ Huh7.5.1 cells with or without SETD7, HIF inhibitors or TLR4 antagonist (A) Schematic of TALEN-based gene ablation. Specific amino acid repeats in TAL domain recognize respective nucleotides (e.g., Red a.a. repeat recognizes T in LIN28 gene). (Top). In vitro translated LIN28TALEN effectively cleaves LIN28 DNA in vitro (lower). (B) Lin28TALEN suppressed proliferation of CD133+/CD49f+ TICs. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (C) Lin28TALEN suppressed TIC proliferation and killed CD133+/CD49f+ TICs from human HCC. Latter were cultured for 24–120 h and transfected with LIN28TALEN or ControlTALEN mRNAs at 72 h. DNA synthesis assessed by 3 H-uridine incorporation showed significant inhibition by LIN28TALEN. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (D) A PCR product of the LIN28 target region amplified from LIN28TALEN was resistant to Tse I digestion. (E) 43% of TICs were killed by LIN28TALEN. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (F) (Left) The 25 nM dose of LIN28 pharmacological inhibitor (LIN28 1632: R&D Systems) with 1 μM of HIF inhibitor (HIF-2α inhibitor PT2385). (Right) CD133+ TICs were killed by LIN28TALEN delivery via CD133 Ab-NPs in the presence of HIF pharmacological inhibitor. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (G–I) LIN28-dependent tumorigenicity of CD133+ Huh7.5.1 cells with or without NANOG. Lin28 knockout reduced tumor growth from TICs in NOG mice in the presence or absence of conjunctive treatment of SETD7 inhibitor (G), HIF inhibitor (H) or TLR4 antagonists (I). CD133+/CD49f+ but not CD133-/CD49f+ Huh7 cells gave rise to viable tumors in NOG mice in a manner dependent on LIN28 and NANOG (red curve, Right). SCR: Scrambled shRNA-transduced cells. Silencing Lin28 and/or Nanog inhibited tumorigenesis (G, Right).Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (J) Hypothetical model for LIN28-mediated TLR4-NANOG activation. Post-transcriptional regulation: The expression of LIN28 increases TLR4 expression with reduction of functionally mature let-7 microRNA in CD133-cells but not in TICs, suggesting LIN28-mediated let-7 reduction drives TLR4 induction.

Article Snippet: ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (F) (Left) The 25 nM dose of LIN28 pharmacological inhibitor (LIN28 1632: R&D Systems) with 1 μM of HIF inhibitor (HIF-2α inhibitor PT2385). (Right) CD133+ TICs were killed by LIN28TALEN delivery via CD133 Ab-NPs in the presence of HIF pharmacological inhibitor.

Techniques: In Vitro, Cell Culture, Transfection, DNA Synthesis, Inhibition, Amplification, Knock-Out, shRNA, Activation Assay, Expressing

Journal: iScience

Article Title: LIN28 and histone H3K4 methylase induce TLR4 to generate tumor-initiating stem-like cells

doi: 10.1016/j.isci.2023.106254

Figure Lengend Snippet: Pharmacologic TLR4 antagonism and LIN28 inhibitor reduced liver tumor incidence in liver tumor mouse model induced by alcohol Western-diet (A) Non-invasive in vivo imaging (micro-CT) detection of tumors in mice fed alcohol Western diet. We tested tumor development by utilizing ethanol western diet-fed Core/Ns5a double Tg mice which spontaneously develop liver tumors at >60% incidence in males after 12 months of alcohol western diet feeding (lesser incidence in females). We tested the therapeutic but not preventive effect of the TLR4 antagonist. For this, it was critical to know when the animals began tumor development to begin treatment, including SETD7 inhibitor treatment (R)-PFI-2. We performed micro-CT imaging with contrast enhancement with enhancement reagent to allow non-invasive, sensitive early detection of small HCC and subsequent tumor monitoring in Tg mice. (B) Liver tumors begin developing at 12 months of alcohol feeding. , Thus, we imaged the livers from 9 months every two weeks for 3 months at the USC Biomedical Imaging Facility. First, we tested three doses of E5564 which was initiated immediately after the detection of tumors. An inhibitory dose (ID 50 ) of E5564 on TNFα production resulting from by co-injection of galactosamine and LPS in mice is shown to be 0.20 mg/kg. The effective dose was decided from sufficient in vivo efficacy in xenograft experiments. The dosage started with 0.6 mg/kg decreasing incrementally. Based on this, we first tested the dose of 0.6 mg/kg/day in alcohol-fed male Core/Ns5a Tg mice to determine the most optimal dose for suppression of liver tumor progression. The H & E staining of liver tissues of mice is shown in the presence or absence of TLR4 antagonist plus LIN28 TALEN. Data are represented as mean ±SEM. Asterisk (∗) indicates statistical significance. ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (C) Immunofluorescence staining of liver tissues from alcohol associated HCCs. Confocal fluorescent images of liver tissues with alcohol associated HCCs showing LIN28 (green) and NANOG (red). Scale bars represent 50 μm. (D) Hypothetical model of tumor development. Hypoxic tumor microenvironment and LIN28-mediated tumor maturation results from absence of inhibitory mature miR let-7 allowing TLR4 expression. Epigenetically, SETD7-mediated H3K4me1 enrichment and DNA hypomethylation on TLR4 promoter is permissive for constitutive TLR4 expression.

Article Snippet: ∗: p<0.05, ∗∗: p<0.01, ∗∗∗: p<0.001. (F) (Left) The 25 nM dose of LIN28 pharmacological inhibitor (LIN28 1632: R&D Systems) with 1 μM of HIF inhibitor (HIF-2α inhibitor PT2385). (Right) CD133+ TICs were killed by LIN28TALEN delivery via CD133 Ab-NPs in the presence of HIF pharmacological inhibitor.

Techniques: Western Blot, In Vivo Imaging, Micro-CT, Imaging, Biomarker Discovery, Injection, In Vivo, Staining, Immunofluorescence, Expressing

Journal: Advanced Science

Article Title: CHB‐Induced Immune Zonation Chaos Elicited LXRα‐mediated Lipid Metabolism Disorders in Kupffer Cells to Induce Cancer Stem Cell Formation

doi: 10.1002/advs.202510275

Figure Lengend Snippet: Induction of HCSC from HBV‐expressing HepAD38 cells by LMD macrophages. a) GSEA analysis showing dysregulated pathway of regulating pluripotency of stem cells in the hepatocytes of the CHB mice at 12 mpi. b) Induction of Yamanaka factors ( Nanog , Oct4 , Klf4 , c‐Myc , Lin28A ) in HBV‐expressing HepAD38 cells treated by macrophages with or without LMD. THP‐derived macrophages were treated with HepAD38 (tet ‐ or tet + ) cells for 48 h, and then co‐cultured with another well of HBV‐expressing HepAD38 in a Transwell plate for another 48 h. The experiments were conducted in triplicate ( n = 3). c) The representative images of Nanog‐positive HCSC (red) surrounded by LXRα‐negative (low or absent of green) Kupffer cells (F4/80, grey) by mIF analysis of liver sections from the control and AAV‐HBV mice at 12 months and 27 months. d) LXR agonist GW3965 significantly reduced the increased mRNA level of pluripontency inducers in HBV‐expressing HepAD38 co‐cultured with LMD macrophages. The experiments were conducted in triplicate ( n = 3). e) The representative bright field images of spheroids formed from HBV‐expressing HepAD38 cells treated by the normal and LMD macrophages with or without GW3965. f) The average of Feret's diameter of the spheroids and the percentage of spheroids with a diameter larger than 50 µm were analyzed. The experiments were performed in triplicate ( n = 3). g) Western blot analysis of Yamanaka factors and the activation of STAT3, Akt, and Wnt3a signaling in HBV‐expressing HepAD38 cells treated by normal and LMD macrophages with or without GW3965. h) The induction of Nanog and Oct4 in LMD macrophage‐co‐cultured HBV + HepAD38 cells was reduced by knockdown of Stat3 . i) Knockdown of AKT had no effect on the expression of Nanog and Oct4 in LMD macrophage‐co‐cultured HBV + HepAD38 cells. j) The Venn diagram showed the overlapping differentially expressed proteins (DEPs) exhibiting opposite variation patterns before and after GW3965 addition within the macrophage‐HepAD38 crosstalk. The supernatants of the co‐culture system involving HepAD38/tet‐ and normal or LMD macrophages with or without GW3965 were subjected to Olink analysis and 7 DEPs who had opposite changing patterns before and after GW3965 treatment were selected. k) The inhibitory effect of neutralization of CCL19, CXCL10, or CXCL11 on the expression of Nanog and Oct4 in HepAD38/tet ‐ cells within the co‐culture system ( n = 3–6). Data are shown as mean ± SEM; * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001.

Article Snippet: Primary antibodies against LXRα (#14352‐1‐AP), ASGR1 (#11739‐1‐AP), BRCA1 (#22362‐1‐AP), BARD1 (#22964‐1‐AP), NANOG (#14295‐1‐AP), LIN28 (#11724‐1‐AP), OCT4 (#11263‐1‐AP), STAT3 (#10253‐1‐AP), WNT3A (#26744‐1‐AP), and CXCL9 (#22355‐1‐AP), were purchased from Proteintech (Wuhan, China).

Techniques: Expressing, Derivative Assay, Cell Culture, Control, Western Blot, Activation Assay, Knockdown, Co-Culture Assay, Neutralization

Journal: Scientific Reports

Article Title: Catalpol improved energy metabolism and inflammation through the SIRT5-mediated signaling pathway to ameliorate myocardial injury

doi: 10.1038/s41598-024-80505-z

Figure Lengend Snippet: The regulation of CAT in CoCl 2 -induced H9c2 cells through SIRT5-mediated Lin28a. ( A-B ) The H9c2 cells were pretreated with CAT (10, 20, and 40 µmol/L) for 24 h before stimulating with CoCl 2 (400 µmol/L) for 24 h. The expressions of Lin28a mRNA ( A ) and protein ( B ) were determined by qRT-PCR and western blotting, respectively. Original blots are presented in Supplementary Fig. 6B and the samples derive from the same experiment and that blots were processed in parallel. ( C-D ) The H9c2 cells were stimulated with CoCl 2 (400 µmol/L) for 24 h after transfecting with SIRT5 siRNA or NC siRNA and pretreating with CAT (20 µmol/L). The expressions of Lin28a mRNA ( C ) and protein ( D ) were determined by qRT-PCR and western blotting, respectively. Original blots are presented in Supplementary Fig. 6D and the samples derive from the same experiment and that blots were processed in parallel. Data indicated the mean of three independent experiments ± SEM, n = 3. *p < 0.05, **p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. CoCl 2 group; $ p < 0.05, $$ p < 0.01 vs. CoCl 2 + CAT group.

Article Snippet: The antibody against Lin28a was obtained from Proteintech (Wuhan, China).

Techniques: Quantitative RT-PCR, Western Blot, Control

Journal: Scientific Reports

Article Title: Catalpol improved energy metabolism and inflammation through the SIRT5-mediated signaling pathway to ameliorate myocardial injury

doi: 10.1038/s41598-024-80505-z

Figure Lengend Snippet: Primer sequences used for qRT-PCR.

Article Snippet: The antibody against Lin28a was obtained from Proteintech (Wuhan, China).

Techniques: