Objective To explore the role of ClC-7 in lamellar body(LB)biogenesis and to investigate the possible pathogenesis of some ClC-7 mutants from patients. Methods siRNA knockdown was employed to decrease the expression of ClC-7 in A549 cells,followed by observing LB morphological changes using laser confocal microscopy and transmission electron microscopy.Wild-type and mutant ClC-7 plasmids were constructed,and the co-localization of ClC-7 with LB and other organelles was observed using immunofluorescence method. Results ClC-7 was localized to LBs and lysosomes.The size of LBs in the ClC-7 knockdown cells was enlarged.Mutations of p.G203D,p.G215R,p.G292E,p.R403Q,and p.L766P,led to mislocalization of ClC-7 to the endoplasmic reticulum.Mutation of p.P470L lead to partial retention of ClC-7 on early endosomes but mostly localizes on LBs. Conclusion ClC-7 in A549 cells is localized to both LBs and lysosomes.Knockdown of ClC-7 in cultured cells leads to enlarged LBs.Most of the mutated ClC-7 cannot be correctly localized on lamellar bodies and lysosomes.

Objective To investigate how long non-coding RNA(lncRNA)GAS5 regulates Th17/Treg imbalance through targeting miR-155 in celiac disease(CeD). Methods Peripheral blood was collected from 20 CeD patients(CeD group)and 20 healthy individuals without CeD or other autoimmune diseases(healthy group),CD4⁺T cells were isolated from both groups.the expression levels of lncRNA GAS5 and miR-155 in CD4⁺T cells from CeD patients and the control group were dected by qRT-PCR.Flow cytometry was used to analyze the Th17/Treg ratio.Pearson statistical analysis was performed to evaluate the correlation between the expression levels of lncRNA GAS5 and miR-155 with the Th17/Treg ratio in CeD patients.In vitro,CD4⁺T cells from the CeD patients were divided into 7 groups:control group,pcDNA3.1-NC group,pcDNA3.1-GAS5 group,miR-NC group,miR-155-mimics group,pcDNA3.1-GAS5+miR-NC group,and pcDNA3.1-GAS5+miR-155-mimics group.qRT-PCR was used to detect the expression levels of lncRNA GAS5 and miR-155 in each group.Edu staining and Hoest33342 staining was used to detect the proliferation and apoptosis of CD4⁺T cells,respectively.Immunofluorescence staining was used to measure the expression of IL-17 and Foxp3.Flow cytometry was used to detect the proportion of IL-17⁺ and Foxp3⁺ cells in each group. Results Compared to those in the healthy group,the expression level of lncRNA GAS5 in the CeD group was decreased,while the expression level of miR-155 was increased.lncRNA GAS5 was negatively correlated with the Th17/Treg ratio(r=-0.65,P<0.05),and miR-155 was positively correlated with the Th17/Treg ratio(r=0.70,P<0.05).In CD4⁺T cells from the CeD group,the pcDNA3.1-GAS5 group showed increased expression levels of lncRNA GAS5 and Foxp3,increased proliferation rate of CD4⁺T cells,and increased proportion of Foxp3⁺ cells(allP<0.05),while the expression levels of miR-155 and IL-17,the apoptosis rate of CD4⁺T cells,and the proportion of IL-17⁺ cells were decreased(allP<0.05)when compared with those in the pcDNA3.1-NC group.When compared with the miR-NC group,the miR-155-mimics group showed decreased Foxp3 expression level,decreased proliferation rate of CD4⁺T cells,and decreased proportion of Foxp3⁺ cells(allP<0.05),while the expression levels of miR-155 and IL-17,the apoptosis rate of CD4⁺T cells,and the proportion of IL-17⁺ cells were increased(allP<0.05).Compared with the pcDNA3.1-GAS5+miR-NC group,the pcDNA3.1-GAS5 ⁺ miR-155-mimics group showed decreased Foxp3 expression level,decreased proliferation rate of CD4⁺T cells,and decreased proportion of Foxp3⁺ cells(allP<0.05),while the expression levels of miR-155 and IL-17,the apoptosis rate of CD4⁺T cells,and the proportion of IL-17⁺ cells were increased(allP<0.05). Conclusion lncRNA GAS5 regulates Th17/Treg imbalance by targeting miR-155,affecting the proliferation and apoptosis of CD4⁺T cells in CeD patients,thereby influencing IL-17 and Foxp3 expression.This suggests that lncRNA GAS5 may serve as a potential therapeutic target for CeD.

Objective To investigate the effect of Wnt 5a/Smad 2/3 activation and sclerostin inhibition in subchondral bone cells on trabecular sclerosis in osteoarthritis. Methods The co-cultured system of chondrocytes and osteoblasts was established.Cells were divided into 5 groups:Control group,OE-vector group,LIF OE group,shRNA-NC group and shRNA-LIF group.RNA immunoprecipitation(RIP)was used to detect the direct interaction between LIFR and sclerostin encoding gene SOST mRNA.The relative expression levels of SOST mRNA was detected by qRT-PCR.LIF expression in chondrocytes was overexpressed or knocked-down,and the relative expression levels of LIF and LIFR was detected by Western blotting.fourty male C57BL 6J mice aged 7-8 weeks were randomly divided into 7 groups:Control group,Model group,Sham group with 4-week or 8-week induction,and Model+LIFR antagonist group with 8-week induction.Micro-CT was used to measure the bone volume fraction(BV/TV),trabecular thickness(Tb.Th)and trabecular number(Tb.N).HE staining and safranin O-fast green staining were used to analyze the histopathological changes in mice joint tissues.The relative expression levels of Wnt 5a,Smad 2/3,Sclerostin,leukemia inhibitory factor(LIF)/LIF receptor(LIFR),tartrate-resistant acid phosphatase(TRAP)and matrix metalloproteinases 13(MMP-13)were detected by Western blotting. Results RIP results showed that LIFR and SOST mRNA had direct interaction.Overexpression of LIF increased LIFR expression levels(P<0.05)and decreased Sclerostin expression levels(P<0.05);knocking down LIF produced the opposite results.After 8 weeks of induction,the BV/TV and Tb.Th were increased(P<0.05),and the Tb.N was decreased(P>0.05)in the Model group when compared with those in the Sham group.Compared with those in the Model group,the BV/TV and Tb.Th were decreased(P<0.05)in the Model+LIFR antagonist group,and the Tb.N was increased(P>0.05).HE staining and Safranin O-fast green cartilage staining results showed that in the Model group,cartilages was destroyed,the intercellular matrix degraded,cell arrangement was disrupted,the cartilage layer disintegrated,and the subchondral bone was affected.In the Model+LIFR antagonist group,the cartilage surfaces area and proliferation area were destroyed,but the hypertrophy zone remained relatively intact,suggesting that cartilage zone destruction was relatively alleviated following LIFR antagonist treatment.After 4 weeks or 8 weeks of induction,the expression levels of Wnt 5a,p-Smad 2/3 in the Model group were increased(P<0.05),the expression levels of sclerostin was decreased(P<0.05),and the expression levels of LIF,LIFR,TRAP and MMP-13 were up-regulated(P<0.05)when compared with those in the Sham group. Conclusion Wnt 5a/Smad 2/3 activation and sclerostin inhibition in subchondral bone cells promote trabecular sclerosis.

Objective To explore the effect and mechanism of microRNA-214-5p(miR-214-5p)on DNA damage and chemotherapy sensitivity in neuroblastoma(NB). Methods The relationship between Fanconi anemia complementary group A (FACNA) protein and clinicopathological characteristics of NB was analyzed by TARGET database.NB cell lines with low expression of miR-214-5p and overexpression/low expression of FANCA were constructed,and their effects on the proliferation of NB in vivo and in vitro were detected by cell counting kit-8(CCK-8)and transplanted tumor model.The targeted relationship between miR-214-5p and FANCA was verified. Results FANCA was screened out as the intersection gene correlated with NB in GEO and TARGET databases,and its expression level was related to cells proliferation pathways,and the prognosis and clinicopathological characteristics of NB.Compared with those in the empty vector(Vector)group,the cells viability,and the volume and weight of transplanted tumors were increased in the FANCA group(P<0.05).Compared with those in the shNC group,the cells viability was decreased in the shFANCA1/2 groups(P<0.05).Compared with those in the control group,the phosphorylated histone γ-H2AX was increased after treated with 1 mmol/L H₂O₂(P<0.05),but γ-H2AX was decreased after FANCA overexpression(P<0.05).With the increase of doxorubicin concentration,cells viability was decreased,but it was higher in the FANCA group than in the Vector group(P<0.05).There was a targeted relationship between miR-214-5p and FANCA. Conclusion miR-214-5p can inhibit the proliferation of NB cells by reducing FANCA expression level,and regulate the DNA damage and chemotherapy sensitivity in cells.

Objective To explore the immunological mechanisms of sodium butyrate on improving Type 2 diabetic mouse model’s insulin sensitivity. Methods Type 2 diabetic mouse model was established by feeding with a high-fat/high-sucrose diet combined with streptozotocin induction.Thirty male C57BL/6 mice were divided into three groups:control group,model group,model+sodium butyrate group,10 mice in each group.The values of glucose tolerance test and insulin tolerance test were measured.The proportion of infiltrated regulatory T cells(Treg)(%)and T helper type 17(Th17)(%)in mice peripheral blood and adipose tissues were measured by flow cytometry.Western blotting was used to determine the relative expression levels of p-mTOR,mTOR,p-p70S6K and p70S6K in infiltrated Treg and Th17 cells in adipose tissues. Results Glucose tolerance test results have shown that compared with control group,in model+sodium butyrate group blood glucose levels increased at 20 min(P<0.05);there was no statistically significant difference in blood glucose at subsequent time points(P>0.05).Insulin tolerance test results have shown that there was no significant difference in blood glucose levels in model+sodium butyrate group at any time points(P>0.05).Compared with those in the control group,the percentage(%)of infiltrating Th17 cells in peripheral blood and adipose tissues was increased in the model group(P<0.05),and the percentage(%)of infiltrating Treg cells was decreased(P<0.05),and the relative expression levels of p-mTOR and p-p70S6K in infiltrated Treg cells and Th17 cells in adipose tissues were increased(P<0.05).Compared with the model group,the model+sodium butyrate group significantly reversed the above indicator values(P<0.05). Conclusion Sodium butyrate treatment could inhibit the activation levels of mTOR/p70S6K signaling pathway,regulate the numbers of Treg cells and Th17 cells in peripheral blood and adipose tissues,and improve insulin sensitivity in type 2 diabetes mice model.

Objective To investigate the effect of oxysophocarpine on breast cancer(BC)cell proliferation,apoptosis,and oxidative stress by modulating the nuclear factor erythroid 2-related factor 2,(Nrf2)/ heme oxygenase-1(HO-1)/ NAD(P)H:quinone oxidoreductase 1(NQO1)pathway. Methods MDA-MB-231 and MCF-7 cell lines were used.Cell viability was detected by CCK-8 assay.Quantitative real-time polymerase chain reaction(qRT-PCR)or Western blotting were used to measure the expression level of proliferation-related(Survivin,Ki67,P21),apoptosis-related(cleaved caspase-3/9,Bax/Bcl-2 ratio),and Nrf2/HO-1/NQO1 pathway-related moleculars.Apoptosis and mitochondrial membrane potential were analyzed by flow cytometry.Oxidative stress markers superoxide dismutase(SOD),glutathione(GSH),malondialdehyde(MDA))were assessed via enzyme-linked immunosorbent assay(ELISA).Nrf2 was overexpressed in MDA-MB-231 cells to validate the Nrf2/HO-1/NQO1 pathway’s effect.A nude mouse xenograft model was established to evaluate tumor growth,Ki67 expression,caspase-3 positivity,apoptosis rate(TUNEL),and Nrf2/HO-1/NQO1 pathway protein expression levels. Results Oxysophocarpine inhibited BC cell viability,induced apoptosis,and reduced mitochondrial membrane potential in a dose-dependent manner.It significantly suppressed the expression of proliferation-related molecules(Survivin,Ki67),and promoted the expression of apoptosis-related molecules(P21,cleaved caspase-3/9,Bax/Bcl-2 ratio).Additionally,it inhibited Nrf2/HO-1/NQO1 pathway activation and exacerbated oxidative stress(reduced SOD,GSH;increased MDA).Nrf2 overexpression partially reversed these effects of oxysophocarpine.Animal experiments confirmed that oxysophocarpine inhibited tumor growth,reduced proliferation(Ki67),promoted apoptosis(caspase-3,TUNEL),and suppressed Nrf2 pathway activation in vivo. Conclusion Oxysophocarpine effectively inhibits breast cancer cell proliferation,induces apoptosis,and enhances oxidative stress by suppressing the Nrf2/HO-1/NQO1 signaling pathway.

Objective To investigate the role of circular RNA vesicle-associated membrane protein-associated protein A(circRNA VAPA)on Zinc-finger E-box binding homeobox 2(ZEB2)in paclitaxel resistance of breast cancer by targeting miR-377-3p. Methods Human breast cancer cell line MCF7 and paclitaxel-resistant strain MCF7/Taxol were cultured,and the expression level of circVAPA,miR-377-3p and ZEB2 were detected and compared.MCF7/Taxol cells were divided into 9 groups.Dual luciferase reporter gene assay was used to detect the targeting relationship between circVAPA and miR-377-3p,and that between miR-377-3p and ZEB2.Cell proliferation inhibition rate was measured by CCK-8 assay and the sensitivity to paclitaxel was assessed. Results The expression levels of circVAPA and ZEB2 in the MCF7/Taxol cells were higher than those in the MCF7 cells,and the expression levels of miR-377-3p were lower than those in the MCF7 cells(P<0.05).The activities of circVAPA and ZEB2 wild-type reporter genes in the miR-377-3p mimic group were both lower than those in the NC group(P<0.05),while there was no significant difference in the activities of circVAPA and ZEB2 mutant reporter genes(P>0.05).Compared with those in the NC group,the paclitaxel sensitivity of the si-circVAPA group and the miR-377-3p mimic group were increased(P<0.05),and the expression level of ZEB2 were decreased(P<0.05).Compared with those in the control plasmid group,the paclitaxel sensitivity in the circVAPA group was decreased,and the expression level of ZEB2 was increased.Compared with those in the circVAPA group,the paclitaxel sensitivity in the circVAPA+miR-377-3p group was increased,and the expression level of ZEB2 was decreased(P<0.05).The paclitaxel sensitivity of MCF7/Taxol cells in the si-circVAPA+miR-377-3p inhibitor group was lower than that in the si-circVAPA group(P<0.05),and the expression level of ZEB2 was higher than that in the si-circVAPA group(P<0.05). Conclusion The regulation of ZEB2 by circular RNA VAPA/miR-377-3p is related to paclitaxel resistance in breast cancer cells.

Objective To explore the expression patterns of migrasome-related genes(MRGs)in prostate cancer and elucidate their underlying mechanisms using bioinformatics tools. Methods Transcriptomic data of 498 cases of prostate cancer were obtained from The Cancer Genome Atlas(TCGA)database.The limma package was used to identify differentially expressed genes in prostate cancer tissues,and the results were validated using GSE70770 and GSE88808 datasets.Migrasome-related differentially expressed genes(MRDEGs)were screened using protein annotation databases.Gene Ontology(GO)was applied to investigate the functional enrichment of MRDEGs.The LASSO regression and SVM-RFE model were used to screen key diagnostic MRDEGs for prostate cancer,and a predictive Nomogram was constructed.The correlation between 22 types of infiltrating immune cells and MRDEGs was analyzed. Results Six MRDEGs were identified in prostate cancer tissues.PKD1,ITGB1 and ITGA5 were significantly expressed in the GSE70770 and GSE88808 datasets.GO enrichment analysis showed that these genes were primarily enriched in biological processes or molecular functions such as the positive regulation of cyclin-dependent serine/threonine protein kinase activity,calcium channel activity,and transmembrane transporter binding.Machine learning identified ITGB1 and ITGA5 as the key MRDEGs for diagnosing prostate cancer.The prediction model constructed based on the key MRDEGs for prostate cancer in the training set TCGA database had an AUC value of 0.833,and the AUC value in the external validation set GSE70770 was 0.868.Resting dendritic cells and neutrophils showed a positive correlation with the expression levels of ITGB1 and ITGA5,while regulatory T cells(Tregs)and M0 macrophages showed a negative correlation. Conclusion Our study employed bioinformatics methods to systematically elucidate the significant role of the migration-related genes ITGB1 and ITGA5 in the pathogenesis of prostate cancer,potentially providing new insights into molecular targets for the treatment of prostate cancer.

Objective To investigate the effect of renal cancer cell exosome on CD8⁺T cell tumor killing fuction. Methods HK-2 exo,786-O exo,SN12-PM6 exo,and an equal volume of PBS were co-cultured with CD8⁺T cells,cytotoxicity assay was used to detect the killing function of CD8⁺T cells.ELISA was used to detect the secretion of IFN-γ,IL-2 and TNF-α.Western blotting was used to detect the expression level of PD-1 in CD8⁺T cells.CD8⁺T cell transfected PD-1 siRNA(si-PD-1)and siRNA Negative Control(si-NC)were co-incubated with 786-O exo and SN12-PM6 exo for 24 hours,respectively.Cytotoxicity experiments were conducted to detect the cytotoxicity of CD8⁺T cells against 786-O and SN12-PM6 cells after transfection. Results Compared with those in the PBS group,the secretion of IFN-γ,IL-2 and TNF-α in the 786-O exo group and SN12-PM6 exo group were significantly downregulated(P<0.05).The ability to kill 786-O and SN12-PM6 was significantly reduced(P<0.05),and PD-1 protein expression level was significantly upregulated(P<0.05).After co-incubation with 786-O exo and SN12-PM6 exo,the the secretion of IFN-γ,IL-2 and TNF-α,as well as the ability to kill 786-O and SN12-PM6 were significantly increased when compared with those in the CD8⁺T cells transfected with si-NC(P<0.05). Conclusion Renal cancer exosome can induce the expression of PD-1 of CD8⁺T cells to inhibit the tumor killing function.

Objective To investigate the effect of long non-coding RNA(lncRNA)MIR100HG on epithelial-mesenchymal transition(EMT)of triple-negative breast cancer MDA-MB-231 cells. Methods MIR100HG was overexpressed or inhibited in the MDA-MB-231 cells,cell proliferation was measured by MTS assay,cell apoptosis rate was analyzed by flow cytometry,and cell migration and invasion were determined by Transwell assay.Western blotting was used to detect the regulatory effect of MIR100HG on epithelial-mesenchymal transition related markers. Results Overexpression of MIR100HG promoted the proliferation,migration and invasion of MDA-MB-231 cells,reduced cell apoptosis,downregulated the expression of epithelial markers and upregulated the expression of mesenchymal markers; Inhibition of MIR100HG produced opposite results. Conclusion Overexpression of LncRNA MIR100HG can promote the proliferation,migration and invasion of MDA-MB-231 cells,reduce the apoptosis of MDA-MB-231 cells,and induce the epithelial-mesenchymal transition of MDA-MB-231 cells.

Objective To study the correlation of serum defensin 5 level and severe acute pancreatitis(SAP)complicated with peripancreatic necrosis. Methods A retrospective study was conducted on 224 SAP patients admitted to Kunshan Hospital of Traditional Chinese Medicine from May 2020 to December 2023,and they were divided into peripancreatic necrosis group(n=96)and non-peripancreatic necrosis group(n=128)according to whether peripancreatic necrosis infection occurred.Compare the differences in general characteristics,and serum defensin 5 levels and laboratory indicators within 24 hours of admission between the two groups.Logistic regression was used to analyze the influencing factors of SAP combined with peripancreatic necrosis,and receiver operating characteristics(ROC)curve was used to analyze the diagnostic indicators of peripancreatic necrosisn. Results The levels of serum defensin 5 and serum calcium in the peripancreatic necrosis group were lower than those in the non-peripancreatic necrosis group,and the time from onset to hospital admission,the levels of C-reactive protein(CRP)and procalcitonin(PCT)were higher than those in the non-peripancreatic necrosis group(P<0.05).Logistic regression analysis showed that the level of serum defencin 5,the time from onset to admission,and the levles of serum calcium,CRP and PCT were the influencing factors of peripancreatic necrosis(P<0.05).ROC curve analysis demonstrated that serum defensin 5 level,time from symptom onset to admission,serum calcium levels,CRP,and PCT levels—both individually and in combination—possess diagnostic value for SAP complicated by peripancreatic necrosis infection.The combined diagnostic approach achieved a sensitivity of 82.81 % and a specificity of 96.99 %. Conclusion The decrease of serum defensin 5 level is related to SAP complicated with peripancreatic necrosis.The combination of defensin 5 and time from onset to hospital admission,serum calcium,CRP and PCT have better diagnostic efficacy for SAP complicated with peripancreatic necrosis.

Objective To explore the problems of immunohistochemistry and fluorescence in situ hybridization in HER2 detection of breast cancer. Methods A total of 309 HER2 IHC 2+ breast cancer samples underwent FISH analysis,including 159 specimens submitted at our hospital in 2018,54 specimens between 2010 and 2012,and 96 specimens from external hospitals between 2016 and 2019. Results The positive rate of FISH test results for breast cancer HER2 IHC 2+ cases in 2018 was significantly lower than that of specimens from other hospitals and the hospital from 2010 to 2012. Conclusion Patients with breast cancer expected to undergo HER2-targeted therapy,particularly those from subordinate hospitals or diagnosed at an early stage,should be further tested using immunohistochemical analysis or FISH to guide precision medicine.

Retinoic acid-induced gene Ⅰ(RIG-Ⅰ),served as a cytoplasmic pattern recognition receptor,is capable of recognizing RNA viruses,and can activate mitochondrial antiviral signaling protein(MAVS)through conformational changes and trigger the TBK1-IRF3/IKK-NF-κB pathway to induce type I interferon(IFN-Ⅰ)production.Mutations in the RIG-I encoding gene DDX58 or excessive activation of RIG-I can lead to the overproduction of inflammatory mediators such as IFN-I,promoting apoptosis and autoantibody formation,resulting in damage to multiple organs including the heart,brain,and kidneys.The cross-disease role of RIG-I in both infectious and non-infectious diseases has gained significant attention in recent years.This article reviews RIG-Ⅰand its roles in systemic lupus erythematosus,tumors,and cardiovascular diseases.

Amyotrophic lateral sclerosis(ALS)is a fatal neurodegenerative disease,and the selective death of motor neurons is its prominent pathological feature.The mechanisms triggering the death of ALS neurons are intricate.Currently,there is no effective treatment plan.It is urgent to deeply explore the potential disease mechanisms and search for neuroprotective agents that may delay disease progression,extend survival,and ultimately reduce the disease burden.Ferroptosis is a programmed cell death mode mediated by iron ions,which causes neuronal damage through lipid peroxidation and oxidative stress.Recent studies have revealed that ferroptosis is closely related to the pathological mechanism of ALS and may become a potential therapeutic target.This article reviews the molecular mechanisms of ferroptosis,pathological characteristics of ALS,and potential connections between the two.It also explores the latest advances in clinical trials targeting ferroptosis for the treatment of ALS,with the aim of opening up new avenues for ALS treatment.

With the rapid development of artificial intelligence technology,it provides new pathways for cultivation of precision nursing professionals.This paper reviews the goals of precision nursing talent development,the advantages and current applications of artificial intelligence in precision nursing education,analyzes the challenges,and proposes development recommendations.It aims to provide reference for the application of artificial intelligence in precision nursing talent training.