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 investigate the pathological feathers,diagnosis,differential diagnosis and prognosis of esophageal granular cell tumor. Methods The data of seven patients of esophageal granular cell tumor diagnosed from 2008 to 2014 in our hospital were analyzed. Seven cases of esophageal granular cell tumor were studied by hematoxylin-eosin and EliVision immunohistochemical staining,the clinical and pathological feathers were analyzed with review of the literatures. Results The pathological feathers of seven cases were very typical. The tumors located in the submucosa,and showed gray to grayish yellow,nodular,with no capsule. The size of these tumors ranged from 0. 4 ~1. 0 cm in diameter. Microscopically,the tumor cells were round,polygonal or spindle with small central nuclei and rich eosinophilic granules cytoplasm,the nucleolus could not be easily seen. Immunohistochemical stain showed that the tumor cells were positive for S-100,CD68 and NSE,but negative for SMA,Desmin,CK,CD34,CD117,DOG1,HMB45. The proportion of Ki-67 positive cells was very low. Histochemical stain showed that the tumor cells were positive for PAS. All 7 cases were benign,and the follow-up time was from 8 months to 6 years without recurrence in patients. Conclusion Granular cell tumor of esophagus is very rare in soft tissue tumors,most of which are benign and located in the middle and lower segment of esophagus. The tumor has a better prognosis. The definite diagnosis should depend on the pathological di-agnosis.