Lnc-TMEM132D-AS1 overexpression reduces sensitivity of non-small cell lung cancer cells to osimertinib / 南方医科大学学报

OBJECTIVE@#To screen the differentially expressed long non-coding RNAs (lncRNAs) in non-small cell lung cancer (NSCLC) cells with acquired resistance to osimertinib and explore their roles in drug resistance of the cells.@*METHODS@#The cell lines H1975_OR and HCC827_OR with acquired osimertinib resistance were derived from their osimertinib-sensitive parental NSCLC cell lines H1975 and HCC827, respectively, and their sensitivity to osimertinib was assessed with CCK-8 assay, clone formation assay and flow cytometry. RNA sequencing (RNA-seq) and real-time quantitative PCR (qPCR) were used to screen the differentially expressed lncRNAs in osimertinib-resistant cells. The role of the identified lncRNA in osimertinib resistance was explored using CCK-8, clone formation and Transwell assays, and its subcellular localization and downstream targets were analyzed by nucleoplasmic separation, bioinformatics analysis and qPCR.@*RESULTS@#The resistance index of H1975_OR and HCC827_OR cells to osimertinib was 598.70 and 428.82, respectively (P < 0.001), and the two cell lines showed significantly increased proliferation and colony-forming abilities with decreased apoptosis (P < 0.01). RNA-seq identified 34 differentially expressed lncRNAs in osimertinib-resistant cells, and among them lnc-TMEM132D-AS1 showed the highest increase of expression after acquired osimertinib resistance (P < 0.01). Analysis of the TCGA database suggested that the level of lnc-TMEM132D-AS1 was significantly higher in NSCLC than in adjacent tissues (P < 0.001), and its high expression was associated with a poor prognosis of the patients. In osimertinib-sensitive cells, overexpression of Lnc-TMEM132D-AS1 obviously promoted cell proliferation, colony formation and migration (P < 0.05), while Lnc-TMEM132D-AS1 knockdown partially restored osimertinib sensitivity of the resistant cells (P < 0.01). Lnc-TMEM132D-AS1 was localized mainly in the cytoplasm, and bioinformatics analysis suggested that hsa-miR-766-5p was its candidate target, and their expression levels were inversely correlated. The target mRNAs of hsa-miR-766-5p were mainly enriched in the Ras signaling pathway.@*CONCLUSION@#The expression of lnc-TMEM132D-AS1 is significantly upregulated in NSCLC cells with acquired osimertinib resistance, and may serve as a potential biomarker and therapeutic target for osimertinibresistant NSCLC.

Berberine inhibits autophagy and promotes apoptosis of fibroblast-like synovial cells from rheumatoid arthritis patients through the ROS/mTOR signaling pathway / 南方医科大学学报

OBJECTIVE@#To evaluate the regulatory effect of berberine on autophagy and apoptosis balance of fibroblast-like synoviocytes (FLSs) from patients with in rheumatoid arthritis (RA) and explore the mechanism.@*METHODS@#The inhibitory effect of 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L berberine on RA-FLS proliferation was assessed using CCK-8 method. Annexin V/PI and JC-1 immunofluorescence staining was used to analyze the effect of berberine (30 μmol/L) on apoptosis of 25 ng/mL TNF-α- induced RA-FLSs, and Western blotting was performed to detect the changes in the expression levels of autophagy- and apoptosis-related proteins. The cells were further treated with the autophagy inducer RAPA and the autophagy inhibitor chloroquine to observe the changes in autophagic flow by laser confocal detection of mCherry-EGFP-LC3B. RA-FLSs were treated with the reactive oxygen species (ROS) mimic H2O2 or the ROS inhibitor NAC, and the effects of berberine on ROS, mTOR and p-mTOR levels were observed.@*RESULTS@#The results of CCK-8 assay showed that berberine significantly inhibited the proliferation of RA-FLSs in a time- and concentration-dependent manner. Flow cytometry and JC-1 staining showed that berberine (30 μmol/L) significantly increased apoptosis rate (P < 0.01) and reduced the mitochondrial membrane potential of RA-FLSs (P < 0.05). Berberine treatment obviously decreased the ratios of Bcl-2/Bax (P < 0.05) and LC3B-II/I (P < 0.01) and increased the expression of p62 protein in the cells (P < 0.05). Detection of mCherry-EGFP-LC3B autophagy flow revealed obvious autophagy flow block in berberine-treated RA-FLSs. Berberine significantly reduced the level of ROS in TNF-α-induced RA-FLSs and upregulated the expression level of autophagy-related protein p-mTOR (P < 0.01); this effect was regulated by ROS level, and the combined use of RAPA significantly reduced the pro-apoptotic effect of berberine in RA-FLSs (P < 0.01).@*CONCLUSION@#Berberine can inhibit autophagy and promote apoptosis of RA-FLSs by regulating the ROS-mTOR pathway.

Study on the Biological Function of Abemecilib in Inhibiting the Proliferation, Invasion and Migration of Small Cell Lung Cancer with High c-Myc Expression / 中国肺癌杂志

BACKGROUND@#Small cell lung cancer (SCLC) with high c-Myc expression is prone to relapse and metastasis, leading to extremely low survival rate. Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor Abemaciclib plays a key role in the treatment of tumors, but the effects and mechanisms on SCLC remain unclear. This study was to analyze the effect and molecular mechanism of Abemaciclib in inhibiting proliferation, migration and invasion of SCLC with high c-Myc expression, with a view to expanding a new direction for reducing the recurrence and metastasis.@*METHODS@#Proteins interacting with CDK4/6 were predicted using the STRING database. The expressions of CDK4/6 and c-Myc in 31 cases of SCLC cancer tissues and paired adjacent normal tissues were analyzed by immunohistochemistry. The effects of Abemaciclib on the proliferation, invasion and migration of SCLC were detected by CCK-8, colony formation assay, Transwell and migration assay. Western blot was used to detect the expressions of CDK4/6 and related transcription factors. Flow cytometry was used to analyze the effects of Abemaciclib on the cell cycle and checkpoint of SCLC.@*RESULTS@#The expression of CDK4/6 was associated with c-Myc by STRING protein interaction network. c-Myc can directly modalize achaete-scute complex homolog 1 (ASCL1), neuronal differentiation 1 (NEUROD1) and Yes-associated protein 1 (YAP1). Moreover, CDK4 and c-Myc regulate the expression of programmed cell death ligand 1 (PD-L1). Immunohistochemistry showed that the expressions of CDK4/6 and c-Myc in cancer tissues were higher than those in adjacent tissues(P<0.0001). CCK-8, colony formation assay, Transwell and migration assay verified that Abemaciclib could effectively inhibit the proliferation, invasion and migration of SBC-2 and H446OE(P<0.0001). Western blot analysis further showed that Abemaciclib not only inhibited CDK4 (P<0.05) and CDK6 (P<0.05), but also affected c-Myc (P<0.05), ASCL1 (P<0.05), NEUROD1 (P<0.05) and YAP1 (P<0.05), which are related to SCLC invasion and metastasis. Flow cytometry showed that Abemaciclib not only inhibited the cell cycle progression of SCLC cells (P<0.0001), but also significantly increased PD-L1 expression on SBC-2 (P<0.01) and H446OE (P<0.001).@*CONCLUSIONS@#Abemaciclib significantly inhibits the proliferation, invasion, migration and cell cycle progression of SCLC by inhibiting the expressions of CDK4/6, c-Myc, ASCL1, YAP1 and NEUROD1. Abemaciclib can also increase the expression of PD-L1 in SCLC.

Effect and Mechanism of Atorvastatin on Reversing Drug Resistance in Leukemia by Regulating Glycolysis through PTEN/mTOR Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the influence and mechanism of atorvastatin on glycolysis of adriamycin resistant acute promyelocytic leukemia (APL) cell line HL-60/ADM.@*METHODS@#HL-60/ADM cells in logarithmic growth phase were treated with different concentrations of atorvastatin, then the cell proliferation activity was measured by CCK-8 assay, the apoptosis was detected by flow cytometry, the glycolytic activity was checked by glucose consumption test, and the protein expressions of PTEN, p-mTOR, PKM2, HK2, P-gp and MRP1 were detected by Western blot. After transfection of PTEN-siRNA into HL-60/ADM cells, the effects of low expression of PTEN on atorvastatin regulating the behaviors of apoptosis and glycolytic metabolism in HL-60/ADM cells were further detected.@*RESULTS@#CCK-8 results showed that atorvastatin could inhibit the proliferation of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.872, r=0.936), and the proliferation activity was inhibited most significantly when treated with 10 μmol/L atorvastatin for 24 h, which was decreased to (32.3±2.18)%. Flow cytometry results showed that atorvastatin induced the apoptosis of HL-60/ADM cells in a concentration-dependent manner (r=0.796), and the apoptosis was induced most notably when treated with 10 μmol/L atorvastatin for 24 h, which reached to (48.78±2.95)%. The results of glucose consumption test showed that atorvastatin significantly inhibited the glycolytic activity of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.915, r=0.748), and this inhibition was most strikingly when treated with 10 μmol/L atorvastatin for 24 h, reducing the relative glucose consumption to (46.53±1.71)%. Western blot indicated that the expressions of p-mTOR, PKM2, HK2, P-gp and MRP1 protein were decreased in a concentration-dependent manner (r=0.737, r=0.695, r=0.829, r=0.781, r=0.632), while the expression of PTEN protein was increased in a concentration-dependent manner (r=0.531), when treated with different concentrations of atorvastatin for 24 h. After PTEN-siRNA transfected into HL-60/ADM cells, it showed that low expression of PTEN had weakened the promoting effect of atorvastatin on apoptosis and inhibitory effect on glycolysis and multidrug resistance.@*CONCLUSION@#Atorvastatin can inhibit the proliferation, glycolysis, and induce apoptosis of HL-60/ADM cells. It may be related to the mechanism of increasing the expression of PTEN, inhibiting mTOR activation, and decreasing the expressions of PKM2 and HK2, thus reverse drug resistance.

Influence of antimicrobial peptide biofunctionalized TiO2 nanotubes on the biological behavior of human keratinocytes and its antibacterial effect / 中华口腔医学杂志

Objective: To fabricate TiO2 nanotube material functionalized by antimicrobial peptide LL-37, and to explore its effects on biological behaviors such as adhesion and migration of human keratinocytes (HaCaT) and its antibacterial properties. Methods: The TiO2 nanotube array (NT) was constructed on the surface of polished titanium (PT) by anodization, and the antimicrobial peptide LL-37 was loaded on the surface of TiO2 nanotube (LL-37/NT) by physical adsorption. Three samples were selected by simple random sampling in each group. Surface morphology, roughness, hydrophilicity and release characteristics of LL-37 of the samples were analyzed with a field emission scanning electron microscope, an atomic force microscope, a contact angle measuring device and a microplate absorbance reader. HaCaT cells were respectively cultured on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of cell was observed by field emission scanning electron microscope. The number of cell adhesion was observed by cellular immunofluorescence staining. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation. Wound scratch assay was used to observe the migration of HaCaT. The above experiments were used to evaluate the effect of each group on the biological behavior of HaCaT cells. To evaluate their antibacterial effects, Porphyromonas gingivalis (Pg) was respectively inoculated on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of bacteria was observed by field emission scanning electron microscope. Bacterial viability was determined by live/dead bacterial staining. Results: A uniform array of nanotubes could be seen on the surface of titanium samples in LL-37/NT group, and the top of the tube was covered with granular LL-37. Compared with PT group [the roughness was (2.30±0.18) nm, the contact angle was 71.8°±1.7°], the roughness [(20.40±3.10) and (19.10±4.11) nm] and hydrophilicity (the contact angles were 22.4°±3.1° and 25.3°±2.2°, respectively) of titanium samples increased in NT and LL-37/NT group (P<0.001). The results of in vitro release test showed that the release of antimicrobial peptide LL-37 was characterized by early sudden release (1-4 h) and long-term (1-7 d) slow release. With the immunofluorescence, more cell attachment was found on NT and LL-37/NT than that on PT at the first 0.5 and 2.0 h of culture (P<0.05). The results of CCK-8 showed that there was no significant difference in the proliferation of cells among groups at 1, 3 and 5 days after culture. Wound scratch assay showed that compared with PT and NT group, the cell moved fastest on the surface of titanium samples in LL-37/NT group at 24 h of culture [(96.4±4.9)%] (F=35.55, P<0.001). A monolayer cells could be formed and filled with the scratch in 24 h at LL-37/NT group. The results of bacterial test in vitro showed that compared with the PT group, the bacterial morphology in the NT and LL-37/NT groups was significantly wrinkled, and obvious bacterial rupture could be seen on the surface of titanium samples in LL-37/NT group. The results of bacteria staining showed that the green fluorescence intensity of titanium samples in LL-37/NT group was the lowest in all groups (F=66.54,P<0.001). Conclusions: LL-37/NT is beneficial to the adhesion and migration of HaCaT cells and has excellent antibacterial properties, this provides a new strategy for the optimal design of implant neck materials.

Echinacoside hampers malignant progression of breast cancer MCF-7 cells by modulating AKR1B10/ERK signal transduction / 中国中药杂志

This study analyzes the impact of echinacoside(ECH) in the proliferation, metastasis and adriamycin(ADR) resistance of breast cancer(BC) MCF-7 cells via the modulation of aldo-keto reductase family 1 member 10(AKR1B10)/extracellular signal-regulated kinase(ERK) pathway. The chemical structure of ECH was firstly confirmed. MCF-7 cells were treated with different concentration(0, 10, 20, 40 μg·mL~(-1)) of ECH for 48 h. Western blot was used to analyze expression of AKR1B10/ERK pathway-associated proteins and cell counting kit-8(CCK-8) assay to determine cell viability. MCF-7 cells were collected and classified into control group, ECH group, ECH + Ov-NC group, and ECH + Ov-AKR1B10 group. Then Western blot was employed to analyze the expression of AKR1B10/ERK pathway-associated proteins. CCK-8 and 5-ethynyl-2'-deoxyuridine(EdU) assay were used to examine cell proliferation. Cell migration was appraised with scratch assay, Transwell assay, and Western blot. Eventually, MCF-7 cells were treated with ADR for 48 h to induce ADR resistance. Cell viability was tested by CCK-8 assay and cell apoptosis was estimated based on terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) assay and Western blot. Based on Protein Data Bank(PDB) and molecular docking, the binding affinity of ECH to AKR1B10 was assessed. Various doses of ECH decreased the expression of AKR1B10/ERK pathway-associated proteins in a dose-dependent manner and declined cell viability compared with the control group. Compared with the control group, 40 μg·mL~(-1) ECH blocked the AKR1B10/ERK pathway in MCF-7 cells and inhibited the proliferation, metastasis and ADR resistance of the cells. Compared with the ECH + Ov-NC group, ECH + Ov-AKR1B10 group showed the recovery of some biological behaviors of MCF-7 cells. ECH also targeted AKR1B10. ECH can inhibit the proliferation, metastasis, and ADR resistance of BC cells by blocking AKR1B10/ERK pathway.

Exosomes secreted from human umbilical cord mesenchymal stem cells promote pancreatic ductal adenocarcinoma growth by transferring miRNAs / 中华肿瘤杂志

Objective: To observe the effects of exosomes derived from human umbilical cord mesenchymal stem cells on the proliferation and invasion of pancreatic cancer cells, and to analyze the contents of exosomes and explore the mechanisms affecting pancreatic cancer cells. Methods: Exosomes extracted from human umbilical cord mesenchymal stem cells were added to pancreatic cancer cells BxPC3, Panc-1 and mouse models of pancreatic cancer, respectively. The proliferative activity and invasion abilities of BxPC3 and Panc-1 cells were measured by cell counting kit-8 (CCK-8) and Transwell assays. The expressions of miRNAs in exosomes were detected by high-throughput sequencing. GO and KEGG were used to analyze the related functions and the main metabolic pathways of target genes with high expressions of miRNAs. Results: The results of CCK-8 cell proliferation assay showed that the absorbance of BxPC3 and Panc-1 cells in the hucMSCs-exo group was significantly higher than that in the control group [(4.68±0.09) vs. (3.68±0.01), P<0.05; (5.20±0.20) vs. (3.45±0.17), P<0.05]. Transwell test results showed that the number of invasion cells of BxPC3 and Panc-1 in hucMSCs-exo group was significantly higher than that in the control group (129.40±6.02) vs. (89.40±4.39), P<0.05; (134.40±7.02) vs. (97.00±6.08), P<0.05. In vivo experimental results showed that the tumor volume and weight in the exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exo) group were significantly greater than that in the control group [(884.57±59.70) mm(3) vs. (695.09±57.81) mm(3), P<0.05; (0.94±0.21) g vs. (0.60±0.13) g, P<0.05]. High-throughput sequencing results showed that miR-148a-3p, miR-100-5p, miR-143-3p, miR-21-5p and miR-92a-3p were highly expressed. GO and KEGG analysis showed that the target genes of these miRNAs were mainly involved in the regulation of glucosaldehylation, and the main metabolic pathways were ascorbic acid and aldehyde acid metabolism, which were closely related to the development of pancreatic cancer. Conclusion: Exosomes derived from human umbilical cord mesenchymal stem cells can promote the growth of pancreatic cancer cells and the mechanism is related to miRNAs that are highly expressed in exosomes.

Hydrogen-rich water reduces cell damage by reducing excessive autophagy in mouse neuronal cells after oxygen glucose deprivation/reoxygenation / 中华危重病急救医学

OBJECTIVE@#To investigate whether hydrogen-rich water exerts a protective effect against cellular injury by affecting the level of autophagy after oxygen glucose deprivation/reoxygenation (OGD/R) in a mouse hippocampal neuronal cell line (HT22 cells).@*METHODS@#HT22 cells in logarithmic growth phase were cultured in vitro. Cell viability was detected by cell counting kit-8 (CCK-8) assay to find the optimal concentration of Na2S2O4. HT22 cells were divided into control group (NC group), OGD/R group (sugar-free medium+10 mmol/L Na2S2O4 treated for 90 minutes and then changed to normal medium for 4 hours) and hydrogen-rich water treatment group (HW group, sugar-free medium+10 mmol/L Na2S2O4 treated for 90 minutes and then changed to medium containing hydrogen-rich water for 4 hours). The morphology of HT22 cells was observed by inverted microscopy; cell activity was detected by CCK-8 method; cell ultrastructure was observed by transmission electron microscopy; the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 was detected by immunofluorescence; the protein expression of LC3II/I and Beclin-1, markers of cellular autophagy, was detected by Western blotting.@*RESULTS@#Inverted microscopy showed that compared with the NC group, the OGD/R group had poor cell status, swollen cytosol, visible cell lysis fragments and significantly lower cell activity [(49.1±2.7)% vs. (100.0±9.7)%, P < 0.01]; compared with the OGD/R group, the HW group had improved cell status and remarkably higher cell activity [(63.3±1.8)% vs. (49.1±2.7)%, P < 0.01]. Transmission electron microscopy showed that the neuronal nuclear membrane of cells in the OGD/R group was lysed and a higher number of autophagic lysosomes were visible compared with the NC group; compared with the OGD/R group, the neuronal damage of cells in the HW group was reduced and the number of autophagic lysosomes was notably decreased. The results of immunofluorescence assay showed that the expressions of LC3 and Beclin-1 were outstandingly enhanced in the OGD/R group compared with the NC group, and the expressions of LC3 and Beclin-1 were markedly weakened in the HW group compared with the OGD/R group. Western blotting assay showed that the expressions were prominently higher in both LC3II/I and Beclin-1 in the OGD/R group compared with the NC group (LC3II/I: 1.44±0.05 vs. 0.37±0.03, Beclin-1/β-actin: 1.00±0.02 vs. 0.64±0.01, both P < 0.01); compared with the OGD/R group, the protein expression of both LC3II/I and Beclin-1 in the HW group cells were notably lower (LC3II/I: 0.54±0.02 vs. 1.44±0.05, Beclin-1/β-actin: 0.83±0.07 vs. 1.00±0.02, both P < 0.01).@*CONCLUSIONS@#Hydrogen-rich water has a significant protective effect on OGD/R-causing HT22 cell injury, and the mechanism may be related to the inhibition of autophagy.

Expression and effect of heterogeneous nuclear ribonucleoprotein A2/B1 in tongue squamous cell carcinoma / 中南大学学报(医学版)

OBJECTIVES@#Tongue squamous cell carcinoma (TSCC) is a common cancer in the oral and maxillofacial region, which seriously endangers people's life and health.Heterogeneous nuclear ribonucleoprotein A2/B1(hnRNP A2/B1) is an RNA-binding protein that regulates the expression of a variety of genes and participates in the occurrence and development of a variety of cancers. This study aims to investigate the role of hnRNP A2/B1 in TSCC progression.@*METHODS@#The differential expression of hnRNP A2/B1 in oral squamous cell carcinoma (OSCC) and normal oral mucosa cells and tissues was analyzed based on the gene expression profiles of GSE146483 and GSE85195 in the Gene Expression Omnibus (GEO) database. The correlation between hnRNP A2/B1 expression and disease-free survival of TSCC patients was analyzed based on TSCC related chip of GSE4676. TSCC cancer and paracancerous tissue samples of 30 patients were collected in Hunan Cancer Hospital from July to December 2021. Real-time RT-PCR and Western blotting were used to verify the mRNA and protein expression of hnRNP A2/B1 in TSCC patients'samples, respectively. Human TSCC Tca-8113 cells were transfected with hnRNP A2/B1 empty vector (a sh-NC group), knockdown plasmid (a sh-hnRNP A2/B1 group), empty vector overexpression plasmid (an OE-NC group) and overexpression plasmid (an OE-hnRNP A2/B1 group), respectively. The knockdown or overexpression efficiency of hnRNP A2/B1 was detected by Western blotting. The proliferation activity of Tca-8113 cells was detected by cell counting kit-8 (CCK-8), and the apoptosis rate of Tca-8113 cells was detected by flow cytometry.@*RESULTS@#Based on the analysis of OSCC-related chips of GSE146483 and GSE85195 in the GEO database, it was found that hnRNP A2/B1 was differentially expressed in the OSCC and normal oral mucosa cells and tissues (all P<0.01). Meanwhile, the analysis of TSCC related chip GSE4676 confirmed that the expression of hnRNP A2/B1 was negatively correlated with the disease-free survival of TSCC patients (P=0.006). The results of real-time RT-PCR and Western blotting showed that the relative expression levels of hnRNP A2/B1 mRNA and protein in TSCC tissues were significantly up-regulated compared with those in adjacent tissues (all P<0.01). The results of Western blotting showed that the expression level of hnRNP A2/B1 in Tca-8113 cells was significantly inhibited or promoted after knockdown or overexpression of hnRNP A2/B1 (all P<0.01). The results of CCK-8 and flow cytometry showed that inhibition of hnRNP A2/B1 expression in Tca-8113 cells reduced cell proliferation activity (P<0.05) and increased cell apoptic rate (P<0.01). Overexpression of hnRNP A2/B1 in Tca-8113 cells significantly increased cell proliferation (P<0.05) and decreased cell apoptosis (P<0.01).@*CONCLUSIONS@#HnRNP A2/B1 is a key factor regulating the proliferation and apoptosis of TSCC cells. Inhibition of hnRNP A2/B1 expression can reduce the proliferation activity of TSCC cells and promote the apoptosis of TSCC cells.

Naringin Inhibits Colorectal Carcinogenesis by Inhibiting Viability of Colorectal Cancer Cells / 中国结合医学杂志

OBJECTIVE@#To explore the therapeutic effect of naringin on colorectal cancer (CRC) and the related mechanism.@*METHODS@#Cell counting kit-8 (CCK-8) assay and annexin V-FITC/PI assay were used to detect the effect of naringin (50-400 µg/mL) on cell proliferation and apoptosis of CRC cells, respectively. The scratch wound assay and transwell migration assay were used to assess the effect of naringin on CRC cell migration. Four-week-old male nude mice were injected with HCT116 cells subcutaneously to establish the tumor xenograft model. Naringin was injected intraperitoneally at 50 mg/(kg·d), with solvent and 5-fluorouracil treatment as control. The width and length of the tumors were measured and recorded every 6 days, and tumor tissues were photographed and weighed on the last day of the 24-d observation period. Immunohistochemical staining for caspase-3, proliferating cell nuclear antigen and TUNEL assay were used to evaluate the effect of naringin on cell proliferation and apoptosis in tumor tissues. The body weight, food and water intake of mice were recorded, and the major organs in different treatment groups were weighed on the last day and stained with hematoxylin and eosin for histological analysis. Meanwhile, the routine blood indicators were recorded.@*RESULTS@#CCK-8 and annexin V-FITC/PI results confirmed that naringin (100, 200, and 400 µg/mL) could inhibit proliferation and promote apoptosis. The scratch wound assay and transwell migration assay results confirmed the inhibitory activity of naringin against CRC cells migration. In vivo results demonstrated the inhibitory effect of naringin on tumor growth with good bio-compatibility.@*CONCLUSION@#Naringin inhibited colorectal carcinogenesis by inhibiting viability of CRC cells.

Cigarette Smoke Induces Gefitinib Resistance in NSCLC Cells via ROS/Sirt3/SOD2 Pathway / 中国肺癌杂志

BACKGROUND@#Epidermal growth factor receptor (EGFR) gene mutations are the most common driver mutations in non-small cell lung cancer (NSCLC). To prolong the survival of the patients, EGFR tyrosine kinase inhibitors (TKIs) resistance in NSCLC is a major challenge that needs to be addressed urgently, and this study focuses on investigating the mechanism of cigarette smoke (CS) induced Gefitinib resistance in NSCLC.@*METHODS@#PC-9 and A549 cells were cultured in vitro and treated with 1 µmol/L Gefitinib for 4 h and 10% cigarette smoke extract (CSE) for 48 h. Western blot was used to detect Sirtuin 3 (Sirt3) and superoxide dismutase 2 (SOD2) protein expressions; DCFH-DA probe was used to detect intracellular reactive oxygen species (ROS); CCK-8 kit was used to detect cell activity, and EdU was used to detect cell proliferation ability. Sirt3 overexpression plasmid (OV-Sirt3) was transfected in PC-9 and A549 cells and treated with 1 µmol/L Gefitinib for 4 h and 10% CSE for 48 h after N-acetylcysteine (NAC) action. The expressions of Sirt3 and SOD2 were detected by Western blot; the ROS level in the cells was detected by DCFH-DA probe, and the cell activity was detected by CCK-8.@*RESULTS@#CSE induced an increase in the 50% inhibitory concentration (IC50) of both PC-9 and A549 cells to Gefitinib (P<0.01) and enhanced the proliferation of PC-9 and A549 cells, suggesting that CS induced Gefitinib resistance in NSCLC. ROS was involved in CSE-induced Gefitinib resistance (P<0.05). CSE induced low expressions of Sirt3 and SOD2 (P<0.01), and Sirt3/SOD2 was associated with poor prognosis in lung cancer patients (P<0.05). OV-Sirt3 in PC-9 and A549 cells reversed CSE-induced Gefitinib resistance (P<0.05) and significantly reduced ROS production. NAC reversed CSE-induced Gefitinib resistance in PC-9 and A549 cells (P<0.05).@*CONCLUSIONS@#The ROS/Sirt3/SOD2 pathway is involved in CS-induced Gefitinib resistance in NSCLC.

Cytotoxicity of 4 Wild Mushrooms in a Case of Yunnan Sudden Unexplained Death / 法医学杂志

OBJECTIVES@#To explore the cytotoxicity of four wild mushrooms involved in a case of Yunnan sudden unexplained death (YNSUD), to provide the experimental basis for prevention and treatment of YNSUD.@*METHODS@#Four kinds of wild mushrooms that were eaten by family members in this YNSUD incident were collected and identified by expert identification and gene sequencing. Raw extracts from four wild mushrooms were extracted by ultrasonic extraction to intervene HEK293 cells, and the mushrooms with obvious cytotoxicity were screened by Cell Counting Kit-8 (CCK-8). The selected wild mushrooms were prepared into three kinds of extracts, which were raw, boiled, and boiled followed by enzymolysis. HEK293 cells were intervened with these three extracts at different concentrations. The cytotoxicity was detected by CCK-8 combined with lactate dehydrogenase (LDH) Assay Kit, and the morphological changes of HEK293 cells were observed under an inverted phase contrast microscope.@*RESULTS@#Species identification indicated that the four wild mushrooms were Butyriboletus roseoflavus, Boletus edulis, Russula virescens and Amanita manginiana. Cytotoxicity was found only in Amanita manginiana. The raw extracts showed cytotoxicity at the mass concentration of 0.1 mg/mL, while the boiled extracts and the boiled followed by enzymolysis extracts showed obvious cytotoxicity at the mass concentration of 0.4 mg/mL and 0.7 mg/mL, respectively. In addition to the obvious decrease in the number of HEK293 cells, the number of synapses increased and the refraction of HEK293 cells was poor after the intervention of Amanita manginiana extracts.@*CONCLUSIONS@#The extracts of Amanita manginiana involved in this YNSUD case has obvious cytotoxicity, and some of its toxicity can be reduced by boiled and enzymolysis, but cannot be completely detoxicated. Therefore, the consumption of Amanita manginiana is potentially dangerous, and it may be one of the causes of the YNSUD.

Leonurine inhibits ferroptosis in renal tubular epithelial cells by activating p62/Nrf2/HO-1 signaling pathway / 中国中药杂志

To investigate the protective effect and the potential mechanism of leonurine(Leo) against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells), an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins. HK-2 cells were cultured in vitro, and the effects of Leo on the viability of HK-2 cells at 10, 20, 40, 60, 80 and 100 μmol·L~(-1) were examined by CCK-8 assay to determine the safe dose range of Leo administration. A ferroptosis cell model was induced by erastin, a common ferroptosis inducer, and the appropriate concentrations were screened. CCK-8 assay was used to detect the effects of Leo(20, 40, 80 μmol·L~(-1)) and positive drug ferrostatin-1(Fer-1, 1, 2 μmol·L~(-1)) on the viability of ferroptosis model cells, and the changes of cell morphology were observed by phase contrast microscopy. Then, the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2) activation, and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis. Flow cytometry was performed to detect reactive oxygen species(ROS), and the level of glutathione(GSH) was measured using a GSH assay kit. The expressions of glutathione peroxidase 4(GPX4), p62, and heme oxygenase 1(HO-1) in each group were quantified by Western blot. RESULTS:: showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100 μmol·L~(-1). The viability of HK-2 cells decreased as the concentration of erastin increased, and 5 μmol·L~(-1) erastin significantly induced ferroptosis in the cells. Compared with the model group, Leo dose-dependently increased cell via-bility and improved cell morphology, and 80 μmol·L~(-1) Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus. Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin, inhibited the release of intracellular ROS, elevated GSH and GPX4, promoted the nuclear translocation of Nrf2, and significantly upregulated the expression of p62 and HO-1 proteins. In conclusion, Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells, which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.

Single-copy Loss of Rho Guanine Nucleotide Exchange Factor 10 ( arhgef10) Causes Locomotor Abnormalities in Zebrafish Larvae / 生物医学与环境科学（英文）

OBJECTIVE@#To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation.@*METHODS@#Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 -/-, arhgef10 +/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively.@*RESULTS@#WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 -/- zebrafish had more severe symptoms than arhgef10 +/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.@*CONCLUSION@#Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.

JAG1 promotes migration, invasion, and adhesion of triple-negative breast cancer cells by promoting angiogenesis / 南方医科大学学报

OBJECTIVE@#To investigate the effect of JAG1 on the malignant phenotype of triple-negative breast cancer (TNBC) and its role in angiogenesis in breast cancer microenvironment.@*METHODS@#The expressions of Notch molecules were detected in human TNBC 231 and 231B cells using RT-qPCR. Five female nude mice were inoculated with 231 cells and another 5 with 231B cells into the mammary fat pads, and 4-6 weeks later, the tumors were collected for immunohistochemical and immunofluorescence tests. 231 cells and 231B cells were treated with recombinant JAG (rJAG) protein and DAPT, respectively, and changes in their malignant phenotypes were assessed using CCK-8 assay, Hoechst 33258 staining, wound healing assay, Transwell chamber assay and endothelial cell adhesion assay. Western blotting was used to detect the changes in the expressions of proteins related with the malignant phenotypes of 231 and 231B cells. The effects of conditioned medium (CM) derived from untreated 231 and 231 B cells, rJAG1-treated 231 cells and DAPT-treated 231B cells on proliferation and tube formation ability of cultured human umbilical vein endothelial cells (HUVECs) were evaluated using CCK-8 assay and tube-forming assay.@*RESULTS@#The expression of JAG1 was higher in 231B cells than in 231 cells (P < 0.05). Tumor 231B showed higher expression of VEGFA and CD31. Compared with 231-Blank group, the migration, invasion and adhesion of 231 cells in 231-rJAG1 were significantly enhanced (P < 0.05). Protein levels of Twist1 and Snail increased (P < 0.01), anti-apoptotic protein Bcl-2 increased (P < 0.05), while DAPT inhibited the related phenomena and indicators of 231B. The 231-rJAG1-CM increased the cell number and tubule number of HUVEC (P < 0.05).@*CONCLUSION@#JAG1 may affect the malignant phenotype of TNBC and promote angiogenesis in the tumor microenvironment.

Function of AXL and molecular mechanisms in regulation of nasopharyngeal carcinoma / 中南大学学报(医学版)

OBJECTIVES@#Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignant tumor with unique geographical and ethnic distribution characteristics. NPC is mostly found in south China and Southeast Asia, and its treatment mainly depends on radiotherapy and chemotherapy. However, NPC is usually found in the late stage, and local recurrence and distant metastasis are common, leading to poor prognosis. The receptor tyrosine kinase AXL is up-regulated in various tumors and it is involved in tumor proliferation, migration, invasion, and other processes, which are associated with poor prognosis of tumors. This study aims to detect the expression of AXL in NPC cell lines and tissues, and to investigate its biological function of AXL and the underlying molecular mechanisms in regulation of NPC.@*METHODS@#The expression levels of AXL in normal nasopharyngeal epithelial tissues and NPC tissues were analyzed by GSE68799, GSE12452, and GSE53819 data sets based on Gene Expression Omnibus (GEO) database. The Cancer Genome Atlas (TCGA) database was used to analyze the relationship between AXL and prognosis of head and neck squamous cell carcinoma (HNSC). The indicators of prognosis included overall survival (OS), disease-free interval (DFI), disease-specific survival (DSS), and progression-free interval (PFI). Western blotting assay was used to detect the AXL protein expression levels in normal nasopharyngeal epithelial cell line and NPC cell lines. Immunohistochemical method was used to detect AXL expression levels in normal nasopharyngeal epithelial tissues and NPC tissues. Cell lines with stable AXL knockdown were established by infecting 5-8F and Fadu cells with lentivirus interference vector, and cell lines with stable AXL overexpression were established by infecting C666-1 and HK-1 cells with lentivirus expression vector. Real-time PCR and Western blotting were used to detect the efficiency of knockdown and overexpression in stable cell lines. The effects of AXL knockdown or overexpression on proliferation, migration, and invasion of NPC cells were detected by CCK-8, plate colony formation, and Transwell assays, and the effect of AXL knockdown on tumor growth in nude mice was detected by subcutaneous tumor formation assay. The sequence of AXL upstream 2.0 kb promoter region was obtained by UCSC online database. The PROMO online database was used to predict AXL transcription factors with 0% fault tolerance, and the JASPAR online database was used to predict the binding sites of ETS1 to AXL. Real-time PCR and Western blotting were used to detect the effect of ETS1 on AXL protein and mRNA expression. The AXL upstream 2.0 kb promoter region was divided into 8 fragments, each of which was 250 bp in length. Primers were designed for 8 fragments. The binding of ETS1 to AXL promoter region was detected by chromatin immuno-precipitation (ChIP) assay to determine the direct regulatory relationship between ETS1 and AXL. Rescue assay was used to determine whether ETS1 affected the proliferation, migration, and invasion of NPC cells through AXL.@*RESULTS@#Bioinformatics analysis showed that AXL was highly expressed in NPC tissues (P<0.05), and AXL expression was positively correlated with OS, DFI, DSS, and PFI in HNSC patients. Western blotting and immunohistochemical results showed that AXL was highly expressed in NPC cell lines and tissues compared with the normal nasopharyngeal epithelial cell line and tissues. Real-time PCR and Western blotting results showed that knockdown and overexpression efficiency in the stable cell lines met the requirements of subsequent experiments. The results of CCK-8, plate colony formation, Transwell assays and subcutaneous tumor formation in nude mice showed that down-regulation of AXL significantly inhibited the proliferation, migration, invasion of NPC cells and tumor growth (all P<0.05), and the up-regulation of AXL significantly promoted the proliferation, migration, and invasion of NPC cells (all P<0.05).As predicted by PROMO and JASPAR online databases, ETS1 was a transcription factor of AXL and had multiple binding sites in the AXL promoter region. Real-time PCR and Western blotting results showed that knockdown or overexpression of ETS1 down-regulated or up-regulated AXL protein and mRNA expression levels. ChIP assay result showed that ETS1 bound to AXL promoter region and directly regulate AXL expression. Rescue assay showed that AXL rescued the effects of ETS1 on proliferation, migration and invasion of NPC cells (P<0.05).@*CONCLUSIONS@#AXL is highly expressed in NPC cell lines and tissues, which can promote the malignant progression of NPC, and its expression is regulated by transcription factor ETS1.

miR-20a-5p inhibits proliferation of lung cancer A549 cells by down-regulating HOXB13 / 南方医科大学学报

OBJECTIVE@#To investigate the molecular mechanism by which miR-20a-5p regulates HOXB13 gene expression and inhibits lung cancer cell proliferation.@*METHODS@#The expression levels of HOXB13 mRNA and protein in lung cancer A549 cells transfected with HOXB13 overexpression plasmid or HOXB13 siRNA were detected with real-time fluorescence quantitative PCR (qRT-PCR) and Western blotting. CCK-8 and EdU assays were used to examine the effect of modulation of HOXB13 expression on cell proliferation. We screened possible binding miRNAs of HOXB13 by bioinformatics analysis. In A549 cells transfected with miR-20a-5p mimic or miR-20a-5p inhibitor, the expression level of miR-20a-5p was detected by qRT-PCR and the protein expression of HOXB13 was determined with Western blotting. CCK-8 and EdU assays were used to assess the effect of miR-20a-5p overexpression on the proliferation of A549 cells. miR-20a-5p mimic and HOXB13 overexpression plasmids were co-transfected into A549 cells, and the changes in cell proliferation were evaluated with CCK-8 and EdU assays.@*RESULTS@#HOXB13 overexpression obviously promoted the proliferation of A549 cells (P < 0.05). miR-20a-5p was identified as the potential binding miRNA of HOXB13. Overexpression of miR-20a-5p in A549 cells significantly decreased the expression of HOXB13 protein (P < 0.05), while interference of miR-20a-5p obviously increased HOXB13 expression (P < 0.05). The results of cell proliferation experiment showed that miR-20a-5p and HOXB13 had opposite effects on cell proliferation, and the cells overexpressing both miR-20a-5p and HOXB13 showed a lower proliferation activity than the cells overexpressing HOXB13 but higher than the cells overexpressing miR-20a-5p alone (P < 0.05).@*CONCLUSION@#miR-20a-5p inhibits proliferation of lung cancer cells by down-regulating the expression of HOXB13.

Inhibitory Effect of CCK-8 on Methamphetamine-Induced Apoptosis / 法医学杂志

OBJECTIVES@#To investigate the inhibitory effect of cholecystokinin octapeptide (CCK-8) binding to cholecystokinin 2 receptor (CCK2R) on methamphetamine (METH)-induced neuronal apoptosis, and to explore the signal transduction mechanism of β-arrestin 2 in CCK-8 inhibiting METH-induced neuronal apoptosis.@*METHODS@#SH-SY5Y cell line was cultured, and HEK293-CCK1R and HEK293-CCK2R cell line were constructed by lentivirus transfection. Small interfering RNA (siRNA) was used to knockdown the expression of β-arrestin 2. Annexin Ⅴ-FITC/PI staining and flow cytometry were used to detect the apoptotic rate of cells, and Western blotting was used to detect the expression of apoptosis-related proteins.@*RESULTS@#The apoptosis of SH-SY5Y cells was induced by 1 mmol/L and 2 mmol/L METH treatment, the number of nuclear fragmentation and pyknotic cells was significantly increased, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were increased. CCK-8 pre-treatment at the dose of 0.1 mmol/L and 1 mmol/L significantly reversed METH-induced apoptosis in SH-SY5Y cells, and inhibited cell nuclear fragmentation, pyknosis and the changes of apoptosis-related proteins induced by METH. In lentivirus transfected HEK293-CCK1R and HEK293-CCK2R cells, the results revealed that CCK-8 had no significant effect on METH-induced changes of apoptosis-related proteins in HEK293-CCK1R cells, but it could inhibit the expression level of apoptosis-related proteins in HEK293-CCK2R cells induced by METH. The inhibitory effect of CCK-8 on METH-induced apoptosis was blocked by the knockdown of β-arrestin 2 expression in SH-SY5Y cells.@*CONCLUSIONS@#CCK-8 can bind to CCK2R and exert an inhibitory effect on METH-induced apoptosis by activating the β-arrestin 2 signal.

Effects of Pigment Epithelium-derived Factor and Its Peptides on Proliferation, Apoptosis and Migration of Non-small Cell Lung Cancer / 中国肺癌杂志

BACKGROUND@#The anti-tumor effect of pigment epithelium-derived factor (PEDF) has been widely confirmed. However, the anti-tumor effect of its peptides is rarely reported. This study aims to investigate the effects of PEDF and its peptides on the apoptosis and migration of non-small cell lung cancer (NSCLC).@*METHODS@#In this study, A549 cells and H1299 cells were selected as the research object, and the cells were divided into normal group, PEDF treatment group, 34 peptide treatment group, 44 peptide treatment group and 34+44 peptide treatment group by administering different drugs at the same concentration to the cells. The proliferation activity of cells in each group was detected by CCK-8 method; the migration ability of cells was detected by scratch test; the expression levels of apoptosis related proteins such as protein kinase 3 (RIP3) and cleaved-caspase-3 were detected by Western blot; the expression levels of epithelial mesenchymal transition (EMT) markers in each group, such as cadherin (E-cadherin) and α-smooth muscle actin (α-SMA) were detected by Western blot; the apoptosis rate of each group was detected by flow cytometry.@*RESULTS@#The results of CCK-8 showed that PEDF and its peptides could inhibit cell proliferation, and the inhibitory effect of 34+44 peptide was the strongest (P<0.05); Observation under the microscope found that PEDF and its peptides can inhibit the proliferation and mesenchymal transformation of A549 cells and H1299 cells, and the inhibitory effect of the 34+44 peptide group is the most obvious; Western blot indicated that compared with other groups, the expressions of cleaved-caspase-3 and RIP3 in 34+44 peptide group were significantly higher (P<0.05), and the expressions of EMT protein E-cadherin were higher, the expression of α-SMA decreased (P<0.05); The results of flow cytometry showed that the apoptosis rate of 34+44 peptide group was significantly higher than those of other groups (P<0.05); The scratch test showed that compared with all the other groups, the healing rate of 34+44 peptide group was the lowest (P<0.05).@*CONCLUSIONS@#34+44 combination peptide can better promote the apoptosis of NSCLC, inhibit the migration of NSCLC, and thereby inhibit the growth of NSCLC.

The MicroRNA hsa-let-7g Promotes Proliferation and Inhibits Apoptosis in Lung Cancer by Targeting HOXB1

PURPOSE: The goal of this study was to explore the effects of hsa-let-7g on cell proliferation and apoptosis, and elucidate its role in lung cancer development.MATERIALS AND METHODS: The expression levels of has-let-7g and HOXB1 in tissues and cells were measured by qRT-PCR. An inhibitor of hsa-let-7g or one targeting a control messenger RNA were transfected into A549 and H1944 lung cancer cells, and the effects of hsa-let-7g dysregulation on cell viability and apoptosis were analyzed using CCK-8 and apoptosis detection assays. HOXB1 was confirmed as the target gene of hsa-let-7g, based on luciferase reporter assay results. The relationship between hsa-let-7g and HOXB1 was confirmed by co-transfection of inhibitors of hsa-let-7g and HOXB1 followed by Western blot, CCK-8, and apoptosis detection assays.RESULTS: We observed high expression of hsa-let-7g in lung cancer tissues compared to the corresponding normal tissues, and generally higher expression of hsa-let-7g in patients with advanced tumor classification. The results of CCK-8 and apoptosis detection experiments showed that the inhibition of hsa-let-7g significantly inhibited proliferation of A549 and H1944 cells, but also promoted apoptosis. HOXB1 is a specific target of hsa-let-7g, and downregulation of HOXB1 in lung cancer cells reversed the suppressive effects caused by knocking down hsa-let-7g.CONCLUSION: These data collectively suggest that the expression of hsa-let-7g inhibits lung cancer cells apoptosis and promotes proliferation by down-regulating HOXB1. The results from this study demonstrate the potential of hsa-let-7g/HOXB1 axis as a therapeutic target for the treatment of lung cancer.