LETM1对骨肉瘤细胞恶性生物学行为的作用及其机制  

Effect and mechanism of LETM1 on malignant behavior of osteosarcoma cells

作　　者：石雨鹭 康权[3] 岳小涵 罗庆[1] Shi Yu-Lu;Kang Quan;Yue Xiao-Han;Luo Qing(The Children's Hospital of Chongqing Medical University/National Clinical Research Center for Child Health and Disorders/Ministry of Education Key Laboratory of Child Development Disease Research/Chongqing Key Laboratory of Pediatrics,Chongqing 400014,China;Department of Pediatrics,Affiliated Hospital of North Sichuan Medical College,Nanchong,Sichuan 637000,China;Department of General and Trauma Surgery,the Children's Hospital of Chongqing Medical University,Chongqing 400014,China)

机构地区：[1]重庆医科大学附属儿童医院/国家儿童健康与疾病临床医学研究中心/儿童发育疾病研究教育部重点实验室/儿科学重庆市重点实验室,重庆400014 [2]川北医学院附属医院儿科,四川南充637000 [3]重庆医科大学附属儿童医院普通外科创伤外科,重庆400014

出　　处：《解放军医学杂志》2024年第10期1163-1173,共11页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81172545);重庆市自然科学基金(cstc2020jcyj-msxmX0113)。

摘　　要：目的 探讨亮氨酸拉链-EF-hand跨膜蛋白1(LETM1)对人骨肉瘤MG63及143B细胞增殖、迁移、凋亡、成骨分化及体内成瘤的作用及其机制。方法 将骨肉瘤MG63及143B细胞分为空白对照组(未加入腺病毒感染)、阴性对照组(sh-NC组,用RNAi阴性对照病毒感染)及LETM1敲低组(sh-LETM1组,用sh-LETM1腺病毒感染)。采用Western blotting检测各组LETM1蛋白在健康人成骨细胞hFOB1.19及骨肉瘤细胞中的表达水平,以验证腺病毒敲低效果;细胞克隆形成实验及CCK-8法检测细胞增殖情况;划痕愈合实验及Transwell实验检测细胞迁移情况;DAPI染色法及Annexin V-APC/7-AAD流式细胞术双染法检测细胞凋亡情况;碱性磷酸酶(ALP)染色及茜素红S染色检测细胞的早期及晚期成骨分化情况。将10只裸鼠分为sh-NC组(n=5,以RNAi阴性对照病毒感染的143B细胞注入裸鼠皮下)与sh-LETM1组(n=5,以sh-LETM1腺病毒感染的143B细胞注入裸鼠皮下),并采用裸鼠皮下成瘤实验检测各组细胞的体内成瘤能力。结果 Western blotting检测结果显示,LETM1蛋白在骨肉瘤MG63及143B细胞中的表达明显高于健康人成骨细胞hFOB1.19(P<0.05),sh-LETM1腺病毒感染MG63及143B细胞后明显降低了LETM1蛋白的表达。细胞克隆形成实验及CCK-8法检测结果显示,在MG63及143B骨肉瘤细胞中,sh-LETM1组较sh-NC组及空白对照组的克隆形成能力及增殖能力明显降低(P<0.01)。Wound-healing实验及Transwell实验结果显示,在MG63及143B骨肉瘤细胞中,sh-LETM1组较sh-NC组及空白对照组的细胞迁移率明显降低(P<0.01)。DAPI染色及流式细胞术结果显示,在MG63及143B骨肉瘤细胞中,sh-LETM1组较sh-NC组的细胞凋亡率明显增高(P<0.01)。ALP染色及茜素红S染色实验结果显示,在MG63及143B骨肉瘤细胞中,sh-LETM1组较sh-NC组及空白对照组染色区域及钙盐结节增多。裸鼠皮下成瘤实验结果显示,143B sh-LETM1组较143B sh-NC组细胞的皮下成瘤能力降低。结论 LETMObjective To investigate the effect and mechanism of leucine zipper/EF-hand-containing transmembrane protein 1(LETM1)on proliferation,migration,apoptosis,osteogenic differentiation,and tumorigenesis in vivo of human osteosarcoma MG63 and 143B cells.Methods The osteosarcoma MG63 and 143B cells were divided into blank control group(without adenovirus infection),negative control group(sh-NC group,infected with RNAi negative control virus),and LETM1 knockdown group(sh-LETM1 group,infected with sh-LETM1 adenovirus).Western blotting was performed to detect LETM1 expression in normal human osteoblasts hFOB1.19 and osteosarcoma cells,and to verify the knockdown effect of adenovirus;cell clone formation assays and CCK-8 method were used to detect the proliferation of MG63 and 143B cells;wound-healing assay and Transwell assay were used to test cell migration;DAPI staining and Annexin V-APC/7-AAD flow cytometry double staining were used to detect the apoptosis of MG63 and 143B cells;alkaline phosphatase(ALP)staining and Alizarin Red S staining were used to evaluate early and late osteogenic differentiation of MG63 and 143B cells.Ten nude mice were divided into sh-NC group(n=5,injected subcutaneously into nude mice with 143B cells infected with RNAi negative control virus)and sh-LETM1 group(n=5,injected subcutaneously into nude mice with 143B cells infected with sh-LETM1 adenovirus),and nude mice subcutaneous tumor formation assay was used to examine the in vivo tumor-forming ability of 143B cells in each group.Results Western blotting showed that the expression of LETM1 protein in osteosarcoma MG63 and 143B cells was significantly higher than that in human normal osteoblasts hFOB1.19(P<0.05),and that the expression of LETM1 protein was markedly reduced after injection with sh-LETM1 adenovirus in MG63 and 143B cells.The results of cell clone formation assay and CCK-8 assay indicated that in MG63 and 143B osteosarcoma cells,the clone formation ability and proliferation ability were significantly reduced in sh-LETM1 group com

关 键 词：骨肉瘤细胞 亮氨酸拉链-EF-hand跨膜蛋白1 细胞增殖 细胞迁移 凋亡 细胞分化 

分 类 号：R738.1[医药卫生—肿瘤]