Objective To investigate the role and primary mechanism of a novel fusion gene RELCH-RET in driving the malignant transformation of normal human bronchial epithelial(HBE)cells.Methods Based on retrospective clinical data from 456 non-small cell lung cancer(NSCLC)patients admitted in the Second Affiliated Hospital of Army Medical University from January 2019 to June 2022,a fusion gene,RELCH-RET,was identified as a research target.Three cell models were established:negative control(HBE VC,transfected with empty lentiviral vector),RET control(HBE RET,transfected with lentiviral overexpression vector of Flag-RET),and experimental group(HBE RELCH-RET,transfected with lentiviral overexpression vector of Flag-RELCH-RET).MTS assay and Transwell assay were used to detect cell proliferation and migratory and invasive abilities.In vivo tumorigenicity of the 3 cell models was assessed in 15 female non-obese diabetic/severe combined immunodeficiency(NOD/SCID)mice(SPF grade,4 weeks old,weighing 15.1±0.4 g)via subcutaneous xenograft experiments,with 5 animals in each group.Western blotting was employed to detect the autophosphorylation of RET(Y905)and the phosphorylation of downstream signaling proteins ERK1/2,EGFR(Y845)and STAT3(Y705).Dimerization and multimerization status of RELCH-RET were analyzed by chemical cross-linking(DTME treatment)in combination with Western blotting,with the reversibility being confirmed through de-cross-linking experiments.Results There were 3 cases carrying RELCH-RET fusion gene screened out from the 469 NSCLC patients.Compared with the HBE VC and HBE RET groups,the HBE RELCH-RET group exhibited significantly enhanced cell proliferation(P<0.01),and acquired migratory and invasive abilities(P<0.01),while the control groups did not demonstrate the abilities.In the mouse xenograft tumor model,HBE cells stably expressing RELCH-RET developed significant tumor nodules(P<0.001),whereas the control groups(empty vector and wild-type RET)failed to exhibit detectable tumor growth.Western blotting revealed that RELCH-RET could induce the autophosphorylation of the RET tyrosine residue(Y905)and significantly up-regulate the phosphorylation levels of ERK1/2,EGFR(Y845),and STAT3(Y705)proteins.Chemical cross-linking combined with Western blot analysis demonstrated that RELCH-RET formed a dimer(～170 kDa)in HBE cells,which is reversibly dissociated into monomers upon decross-linking treatment.Conclusion The novel fusion gene RELCH-RET,promotes ligand-independent dimerization/oligomerization,thereby mediating RET autophosphorylation,subsequently activates the downstream typical RET signaling pathway and ultimately drives the malignant transformation of normal HBE cells.

Objective To elucidate the effects of miR-616 on the malignant biological processes of osteosarcoma and to preliminarily explore its potential mechanisms.Methods In situ hybridization(ISH)was employed to analyze miR-616 expression in 11 paraffin-embedded osteosarcoma specimens collected in our department during January 2018 to December 2019.Quantitative real-time PCR(qRT-PCR)was used to compare the mRNA expression level of miR-616 in the osteoblast cell line hFOB1.19 and osteosarcoma cell lines 143B and HOS.Stable cell lines with miR-616 knockdown or overexpression were established via lentiviral transfection in 143B and HOS cells.Cell proliferation and apoptosis were detected by flow cytometry,while cell invasion and migration were assessed using Transwell and colony formation assays,respectively.To evaluate the effect of miR-616 on tumor growth in vivo,10 female nude mice(4 weeks old,weighing 18～20 g)were randomized into a control group and a miR-616 overexpression group.After the xenograft tumor model was constructed,the growth of subcutaneous tumors was monitored.Finally,next-generation sequencing and a dual-luciferase reporter assay were performed to identify the target genes of miR-616.Results ISH results showed that miR-616 expression was up-regulated in osteosarcoma tissues than adjacent tissues,and primarily localized in the cytoplasm.qRT-PCR confirmed that miR-616 level was significantly higher in 143B and HOS cells than hFOB1.19 cells(P<0.05).In vitro experiments revealed that miR-616 overexpression enhanced the proliferation,migration and invasion,while suppressing apoptosis in 143B and HOS cells(P<0.01).Conversely,miR-616 knockdown weakened these malignant phenotypes(P<0.05),with miR-616-3p showing a stronger effect on apoptosis than miR-616-5p.Animal experiments demonstrated that the tumor weight in the miR-616 overexpression group was significantly greater than that of the control group(98.00±17.22 vs 33.60±8.08 mg,P<0.01).Furthermore,KLF2 was identified and confirmed as a direct target of miR-616.Conclusion MiR-616 promotes malignant biological behaviors in osteosarcoma,and its expression level indicates that it may serve as a potential therapeutic target.

Objective:To apply CBL combined with PBL based on SMART (specific, measurable, attainable, relevant and time-based) principle in nursing practice teaching in radiotherapy.Methods:A total of 100 nurses who performed nursing practice in the Department of Radiotherapy in Guangdong Provincial Hospital of Traditional Chinese Medicine from May 2016 to May 2020 were selected as the research objects. They were divided into a control group and a study group according to their admissions, with 50 people in each group. The study group used CBL combined with PBL teaching based on SMART principle, and the control group used traditional practice teaching. After the clinical practice, the two groups were assessed on theoretical and clinical practice skills, and the two groups' teaching satisfaction and teaching effects were evaluated through seminars and questionnaire surveys. SPSS 22.0 was used for t test and chi-square test. Results:The theoretical and clinical practice performance assessment scores of the practical nurses in the study group were higher than those in the control group, with statistically significant differences ( P<0.001). The teaching satisfaction rate of the practice nurses in the control group was 62.00% (31/50), and that of the practice nurses in the study group was 96.00% (48/50), with significant differences ( P<0.001). In terms of improving independent learning ability, information acquisition and problem analysis ability, improving clinical thinking ability, mobilizing learning enthusiasm, enhancing teamwork ability, nurse-patient communication ability, and recognition of innovation ability, the teaching satisfaction of the research group was higher than that of the control group. Conclusion:The application of SMART principle in teaching has the advantages of clear goals and quantifiable evaluation. The combination of CBL and PBL based on SMART principle can help to improve the mastery of theoretical and practical skills of radiotherapy practice nurses, and achieve satisfactory teaching results.

Agrobacterium tumefaciens-mediated transformation system has been widely applied. However, the function of target gene is affected by multiple factors. With this system, we obtained a transgenic rice line CX8621 carrying the bacterial blight resistance gene Xa21. In previous work, we have confirmed that it was selectable maker-free and vector backbone-free. And after 16 generations of breeding, it still maintained perfect resistance to bacterial blight disease. On this basis, we analyzed the integration and expression of Xa21 in CX8621 at the present study. First, based on the border sequences of plasmid pBXa21 and Xa21, we designed nested primers and assured the integrity of Xa21 in CX8621. Second, we cloned the flanking sequences and located Xa21 on chromosome 2 using improved Tail-PCR. Then we analyzed the expression pattern of Xa21 in several tissues and at different developmental stages by RT-PCR. The results show that Xa21 can be stably expressed in CX8621, agreeing well with the disease resistance response as reported previously. In addition, we detected the protein levels of XA21 in CX8621 with antibody of natural XA21 protein. Surprisingly, no XA21 protein was detected in the seeds of CX8621. Thus, the integration and expression analysis of Xa21 in CX8621 provided a part of scientific evidences for the safety assessment of genetically modified rice.
Animals ; DNA Primers ; Disease Resistance ; genetics ; Oryza ; genetics ; Plant Proteins ; genetics ; Plants, Genetically Modified ; genetics ; Polymerase Chain Reaction ; Protein-Serine-Threonine Kinases ; genetics ; Seeds

Objective To explore the difference between T cells in the synovial fluid and peripheral blood in patients with rheumatoid arthritis(RA). Method Samples from 22 patients were studied. The differentiation and activation markers expressed on T cell surface were detected by immunofluorscence using flow cytometer. The specific proliferation of collagen Ⅱ and heat shock protein 70 was analyzed using standard 3H-TdR incorporation method. Restricted V beta usage of these T cell was analyzed by semi-quantitied RT-PCR. Results The majority of the T cell subsets in the synovial fluid were demonstrated to be CD4 and CD8 positive cells in which (40?10)% were CD4 positive and (36?16)% were CD8 T cells respectively. The ratio between CD4 and CD8 was much lower than that found in the PBL of RA patients. The percentage of CD3+/CD25+ T cells was (16?6)%. The specific proliferation of collagen Ⅱ and HSP70 to CD3+/CD25+ T cell was higher than that of CD3+/CD25+ negative T cells. The T cell receptor expressed on the T cells from both peripheral blood and synovial fluid were tested for ?? TCR (70?26)%. However, the T cells in the synovial fluid showed V?14,16 and 17 restriction. Conclusion The data here reported indicates that T cell subsets in the synovial fluid and peripheral blood circulation in patients with rheumatoid arthritis are different. The T cells in the synovial fluid demonstrates more activation and higher reactivation to collagen Ⅱ and HSP70. The TCR of T cells showes V?14,16 and 17 restriction.