Acta Universitatis Medicinalis Anhui

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Anhui Provincial Education Department

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Anhui Medical University

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Weng Jianping

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Effects of LINC02086 on proliferation, migration and invasion of gastric cancer cells by regulating Wnt/β-catenin pathway mediated M2 polarization of macrophages

Li Jun1, Bu Yafei2, Chen Jie2, Ding Bo1, Wang Lei1

DOI: 专辑：医药卫生科技

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abstract:
To investigate the effect and mechanism of long intergenic non-coding RNA02086 (LINC02086) overexpression mediated macrophage polarization on the proliferation, migration and invasion of gastric cancer cells. Methods The expression levels of LINC02086 in the human gastric epithelial cell line GES-1 and human gastric cancer cell lines HCG-27, NCI-N87, and AGS were determined by qRT-PCR. Human acute monocytic leukemia cells (THP-1) were induced to differentiate into M0 macrophages using phorbol 12-myristate 13-acetate (PMA). HGC- 27 cells were infected with either LINC02086 overexpression lentivirus (OE-LINC02086) or its negative control lentivirus (Vector), and the culture supernatants were collected as conditioned medium (CM1). M0 macrophages were co-cultured with the infected HGC-27 cells, and the resulting supernatants were designated as conditioned medium 2 (CM2). M0 macrophages were treated with CM1 alone or in combination with Wnt/β-catenin pathway inhibitor IWR-1, forming the Vector+CM1, OE-LINC02086+CM1, and OE-LINC02086+CM1+IWR-1 groups, respectively. Flow cytometry was used to detect mannose receptor C-type 1 (CD206) expression, and qRT-PCR was employed to measure mRNA levels of interleukin-10 (IL-10), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and chemokine ligand 22 (CCL22). Western blot was performed to evaluate protein expression of CD206, VEGF, and key components of the Wnt/β-catenin pathway—Wnt family member 3a (Wnt3a), glycogen synthase kinase-3β (GSK-3β), and β-catenin. HGC-27 cells were treated with CM2 alone or combined with IWR-1, establishing the Vector+CM2, OE-LINC02086+CM2, and OE-LINC02086+CM2+IWR-1 groups. CCK-8 assay was used to evaluate cell proliferation, and Transwell assays were conducted to assess migration and invasion capabilities. Results Compared with GES-1 cells, the expression levels of LINC02086 were upregulated in HCG-27, NCI-N87, and AGS cells (P < 0.05), with the smallest increase observed in HCG-27 cells. Compared with Vector+CM1 group, the level of CD206 and the expression levels of IL-10, TGF-β, VEGF and CCL22 mRNA in macrophages stimulated by OE- LINC02086+CM1 increased (P<0.05). Meanwhile, the expression levels of Wnt3a and β-catenin proteins in cells increased (P<0.05), and the expression level of GSK-3β protein decreased (P<0.05).However, co-treatment with IWR-1 markedly reversed the promoting effects of LINC02086 overexpression on the expression of M2 polarization markers, including CD206, IL-10, and TGF-β mRNA, in macrophages (P<0.05), as well as its activation ofthe Wnt/β-catenin signaling pathway (P<0.05). Compared with Vector+CM2 group, HGC-27 cells infected with OE-LINC02086+CM2 had increased proliferation activity and increased number of migration and invasion cells (P<0.05). However, the combined intervention ofIWR-1 significantly reversed the promotion of LINC02086 overexpression on the proliferation, migration and invasion of HGC-27 cells (P<0.05). Conclusion LINC02086 overexpression promotes the proliferation, migration and invasion of gastric cancer cells by activating Wnt/β-catenin pathway to mediate M2 polarization of macrophages.

Role and mechanism of Lck/Yes-related novel tyrosine kinases in macrophage M1 polarization

Yu Xin, Gao Zhensheng, Bian Weihua, Liu Xiangyong, Sun Yeying

DOI: 专辑：医药卫生科技

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abstract:
To investigate the role and mechanism of Lck/Yes-related novel protein tyrosine kinase (Lyn) on lipopolysaccharide (LPS)-induced M1-type polarization of macrophage. Methods The LentiCRISPR-V2 plasmid was digested with the restriction endonuclease BSMBI- V2, and the digested DNA fragments were recovered. The digested plasmid was ligated with Lyn- sgRNA using T4 ligase to generate the Lenti-Lyn-gRNA lentivirus. THP-1 cells were infected with the Lenti-Lyn-gRNA lentivirus to obtain a stable cell line with Lyn knockout, and a monoclonal THP-1 cell line with complete Lyn knockout (Lyn⁻/⁻) was established subsequently. Wild-type Lyn (Lynᵂᵀ) and Lyn⁻/⁻ THP-1 cells were induced with 100 ng/mL phorbol myristate acetate (PMA) for 48 h to differentiate into M0 macrophages, which were further polarized into M1 macrophages by stimulation with 100 ng/mL LPS for 24 h. Quantitative real-time polymerase chain reaction (qPCR) was performed to detect the expression of M0 macrophage markers, including integrin αM (CD11b), macrophage antigen (CD68), and monocyte differentiation antigen (CD14). The expression of Lyn in M1 macrophages differentiated from wild-type THP-1 cells (Lynᵂᵀ-M1) was measured by qPCR, and the ratio of phosphorylated Lyn to total Lyn (P-Lyn/Lyn) in Lynᵂᵀ-M1 cells was determined by Western blot. In M1 macrophages differentiated from Lyn-knockout THP-1 cells (Lyn⁻/⁻-M1), qPCR was used to detect the mRNA expression of inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and chemokine (C-X-C motif) ligand 10 (CXCL-10). Western blot was conducted to assess the protein expression of iNOS, as well as the protein levels of molecules related to the JAK1- STAT1 signaling pathway, including Janus kinase 1 (JAK1), phosphorylated JAK1 (P-JAK1), signal transducer and activator of transcription 1 (STAT1), and phosphorylated STAT1 (P-STAT1). Additionally, the expression of the M1 macrophage marker cluster of differentiation 80 (CD80) was analyzed by flow cytometry. Results The Lyn-/- monoclonal cell line was successfully constructed. The expression of CD11b was significantly elevated in Lyn-/- M0 macrophages, and the differentiation of THP-1 macrophages was successful. Knockdown of Lyn inhibited mRNA expression of iNOS, IL-6, CXCL-10, protein expression of iNOS and CD80 expression in M1 macrophages. Western blot assay showed that Lyn knockdown inhibited protein expression of JAK1 and P-STAT1. Conclusion After CRISPR/Cas9-mediated Lyn knockout, the expression levels of JAK1 and P-STAT1, the key molecules in the JAK/STAT signaling pathway of M1 macrophages, are significantly downregulated; concomitantly, the expression of M1 macrophage-specific secretory factors (iNOS, IL-6, CXCL-10) and CD80 is also downregulated, which may be achieved via targeted regulation of the JAK1/P-STAT1-mediated JAK/STAT signaling pathway.

Study on the capture of Helicobacter pylori released from Candida using immunomagnetic bead

Luo Tingting1,2, Sun Jianchao2, Yang Tingxiu2, Xu Xiaoli1,2, Cui Guzhen2, Luo Qing3, Zhuo Shuwei4, Liu Qi1, Chen Zhenghong2

DOI: 专辑：医药卫生科技

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abstract:
To investigate the ability of clinically isolated, Helicobacter pylori (H. pylori)-specific gene polymerase chain reaction (PCR)-positive gastric, vaginal, and fecal Candida to release H. pylori. Methods Resuscitate 4 strains of H. pylori -specific 16S rDNA and ureA gene PCR-positive Candida strains isolated in laboratory from clinical sources, including 1 strain of gastric Candida, 1 strain of fecal Candida, 2 strains of vaginal Candida and the standard Candida albicans strain ATCC10231 (Ca10231). The presence of H. pylori-specific ureA in the 5 strains of Candida isolates was confirmed by PCR. The aforementioned strains of Candida and H.pylori were inoculated into urea medium and cultured in a constant temperature incubator at 37 ℃. The color change of the medium was observed daily. A change in the medium’s color from yellow to red indicated the presence ofurease activity. Then, the five strains of Candida and H. pylori were co-incubated with the magnetic beads coated with H. pylori antibodies respectively. Scanning electron microscopy (SEM) was employed to observe the presence of bacilli adsorbed on the surface of the magnetic beads. PCR was used to detect the presence of H.pylori-specific 16S rDNA and ureA genes on magnetic beads. Results The PCR analysis of the ureA gene in the four Candida isolates was positive, whereas the Ca10231 strain tested negative. Upon culturing the four Candida isolates on urea medium, the medium color changed from yellow to red which was determined to be urease positive, while the medium containing Ca10231 remained unchanged, which was urease negative. SEM revealed that bacilli could be observed on the surface of magnetic beads co-incubated with the 4 strains of Candida of clinical origin and H.pylori isolate. Specifically, PCR testing of the magnetic beads co-incubated with one vaginal Candida, one gastric Candida and H.pylori isolate showed positive results for the 16S rDNA and ureA genes of H. pylori; however, the PCR tests for the two genes were negative for the magnetic beads co-incubated with the other two Candida isolate. Conclusion This study demonstrates that H. pylori-specific genes Candida can release H. pylori.

BMP3 inhibits the inflammatory response in rat adjuvant-induced arthritis

Sun Dantong1,2,3, Li Xiaofeng2,3, Li Jun2,3, Song Biao1,2,3

DOI: 专辑：医药卫生科技

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Investigation of the regulatory effect of overexpressed Ptpn2 on SiO2-mediated mouse alveolar macrophages based on iTRAQ technology

Wei Yi1,2, Li Yaqian1,2,3, Li Xinjie1, Feng Mengfei1,2, Jin Fuyu1,2, Xu Hong1,2,4, Zhu Ying1,2

DOI: 专辑：医药卫生科技

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abstract:
To investigate the regulatory effect of overexpressed protein tyrosine phosphatase non-receptor type 2 (Ptpn2) on the inflammatory response of mouse alveolar macrophages (MH-S) induced by SiO₂. Methods Cells with overexpressed Ptpn2 were constructed and induced by SiO₂ . The experimental groups were divided into four groups: the negative control group with an empty vector (NC), the overexpressed Ptpn2 group (P), the negative control group with an empty vector + SiO₂ induction (NS), and the overexpressed Ptpn2 + SiO₂ induction group (PS). Isobaric tags for relative and absolute quantification (iTRAQ) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen differential proteins, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database analyses. Immunofluorescence staining was used to detect the expressions of Tumor necrosis factor (TNF) α, Gasdermin D (GSDMD), and Transforming growth factor (TGF)-β1. Western blot was used to detect the protein expression levels of PTPN2, Toll-like receptor 4 (TLR4), tumor necrosis factor-α (TNF-α), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), and proteins related to the TGF-β1 signaling pathway in the cells of each group. Results iTRAQ results identified 144 differential proteins among the four groups. GO analysis showed that in biological processes (BP), these differential proteins were mainly enriched in IκB kinase/nuclear factor-κB (NF-κB) signaling, cell activation and signal transduction involved in immune responses, and regulation of receptor signaling pathways by signal transducer and activator of transcription (STAT), etc. KEGG analysis revealed that the differential proteins were mainly enriched in Toll- like receptor signaling pathway, NF-κB signaling pathway, NOD-like receptor signaling pathway, TGF-β signaling pathway, and TNF signaling pathway. The results of immunofluorescence staining showed that compared with the NC group, the expressions ofTNF α, GSDMD, and TGF-β1 in the cells ofthe NS group increased; Compared to the NS group, the expression of the aforementioned proteins in the PS group decreased in cellular proteins. The results of Western blot showed that compared with theNC group, the protein expression levels of PTPN2, p-NF-κB、MyD88、TLR4 、 NLRP3、GSDMD、Caspase-1、IL-1β 、 TGF-βR1、 TGF-βR2、p-Smad2/3 intheNS group were significantly upregulated (P < 0.05); Compared with the NS group, the expression levels of the aforementioned proteins in the PS group were significantly downregulated (P < 0.05). Conclusion Overexpression of Ptpn2 can inhibit the protein expressions of TLR4-TNF-α signaling, NLRP3 signaling, and TGF-β1 signaling closely related to inflammatory response in SiO₂-mediated MH-S macrophages.

Effect of RUNX3 on the activation, proliferation, and migration capabilities of hepatic stellate cells

Ling Hui1, Wang Xianchen2, You Junbo2, Fan Jiahao2, Cui Xiao1, Sha Jiming2, Yu Liquan1

DOI: 专辑：医药卫生科技

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abstract:
To investigate the effects of targeted silencing of Runt-related Transcription Factor 3 (RUNX3) on the proliferation and migration of Mouse Hepatic Stellate Cells (HSCs), as well as subsequent collagen deposition. Methods Mouse hepatic stellate cell line (JS-1) was selected and then morphologically observed and identified under a microscope. After the cells had fully adhered, they were treated with 5 ng/mL of transforming growth factor beta 1 (TGF-β1) for 24 hours to induce hepatic stellate cell activation. Furthermore, a RUNX3 silencing model was established using RUNX3 lentiviral infection. The experiment was divided into four groups: Control group, TGF-β1 group, TGF-β1+siRNA-NC group, and TGF-β1+siRNA-RUNX3 group. Protein expression changes of RUNX3, alpha-smooth muscle actin (α-SMA), and Alpha 1 type I collagen (Collagen I) were detected using Western blot method. Cellular immunofluorescence assays were employed to investigate the deposition changes of α-SMA and RUNX3 in hepatic stellate cells. RT-qPCR was utilized to examine the mRNA expression changes of RUNX3, α-SMA, and Collagen I. The proliferative capacity of hepatic stellate cells was assessed using Edu staining. The migratory ability of hepatic stellate cells was evaluated through wound healing assays and Transwell migration experiments. Results Compared with Control group, a significant elevation in RUNX3 was observed in the TGF-β1-induced activated HSCs (P<0.01). Meanwhile, the protein and mRNA levels of fibrosis-related markers and α-SMA and Collagen I were significantly upregulated (P<0.001). Additionally, the proliferation and migration capabilities of HSCs were significantly enhanced (P<0.001). In contrast, when compared to TGF-β1+siRNA-NC group, TGF-β1+siRNA- RUNX3 group exhibited a notable decrease in RUNX3 and other related indicators, such as the protein and mRNA levels of α-SMA and Collagen I (P<0.001). Concurrently, the proliferation and migration capabilities of HSCs were significantly inhibited in TGF-β1+siRNA-RUNX3 group (P<0.001). Conclusion Silencing RUNX3 can inhibit the deposition of collagen and the proliferation and migration of hepatic stellate cells. Conversely, RUNX3 promotes the proliferation and migration capabilities of HSCs, thereby facilitating the activation of HSC.

α-ketoglutarate ameliorated arsenic-induced hepatic lipid deposition in offspring via PI3K/AKT signaling pathway

Bao Shuangrui1,2,3, Wu Hongyan1,2,3, Sun Ying1,2,3, Zhan Tong1,2,3, Yang Qian1,2,3, Liang Xinru 1,2,3, Wan Zhiyan1,2,3, Chen Wenyi2,3, Zhang Cheng1,2,3

DOI: 专辑：医药卫生科技

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abstract:
To investigate the protective effect of α-ketoglutarate (α-KG) on hepatic lipid deposition in offspring caused by arsenic exposure during pregnancy. Methods 8-week-old institute of cancer research (ICR) mice were mated in a ratio of 2:1 between females and males, and the detection of vaginal plugs confirmed pregnant. A total of 32 pregnant mice were randomly divided into four groups: control group, arsenic group, α-KG group, arsenic+α-KG group. On gestational day 0~16 (GD0~GD16), the arsenic and arsenic+α-KG groups were exposed to sodium arsenite (NaAsO2 ,15 mg/L) in drinking water everyday, and the α-KG and arsenic+α-KG groups were gavaged with α-KG (2 g/kg) everyday. On GD16, pregnant mice were euthanized to collect fetal liver, and fetal body weight and crown-rump length were measured. Gene expression differences between the control group and the arsenic group were analyzed by transcriptome. The total triglycerides (TGs) and subtypes in fetal liver were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS). Oil red O staining was used to observe the histopathological changes in the liver. RT-qPCR was used to detect the expression level of genes related to lipid synthesis, transport, and degradation, and phosphatidylinositol 3' -kinase/ protein kinase B (PI3K/ AKT) in the liver of fetus. Results Transcriptomics analysis showed that 2 144 genes were downregulated and 1 675 genes were upregulated in the arsenic exposed fetal liver; body weight and crown-rump length were reduced (PTuKey<0.05); the level of hepatic TGs was elevated in arsenic group (PTuKey<0.05); oil-red O staining showed a significant increase in lipid droplets in arsenic group (PTuKey<0.01); the expression of lipid synthesis-related genes were significantly upregulated (PTuKey<0.05); the expression of β-oxidation-related genes and lipid degradation-related genes were downregulated (PTuKey<0.05); the expression of PI3K, AKT decreased (PTuKey<0.05). Compared with the arsenic group, the body weight and crown-rump length of fetus increased in the arsenic+α-KG group (PTuKey<0.05); the level of hepatic TGs decreased in the arsenic+α-KG group (PTuKey<0.05); oil red O staining showed lipid droplets significantly decreased (PTuKey<0.01); the expression of lipid synthesis-related genes were downregulated (PTuKey<0.05), the expression of β-oxidation-related genes and lipid degradation-related genes were upregulated (PTuKey<0.05); the expression levels of PI3K and AKT increased (PTuKey<0.05). Conclusion α-KG alleviated hepatic lipid deposition in offspring exposed to arsenic during pregnancy through activating PI3K/AKT signaling pathway.

Effects of oleic acid-induced lipid droplet synthesis on the proliferation, migration, invasion, and epithelial-mesenchymal transition of osteosarcoma cells

Wang Mengting1, 2, 3, Wang Yunlong1, 2, Liang Mengxia1, 2, Liu Jun2, Bian Erbao1, 2

DOI: 专辑：医药卫生科技

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