摘要
目的 基于单细胞测序技术解析微管相关蛋白1轻链3β(LC3B)在胃癌中的表达及其与患者预后的关系.方法 选用3对胃癌及配对的癌旁组织样本行单细胞转录组测序(scRNA-seq).CellTypist对过滤后的单细胞表达数据进行亚群分类,使用Wilcoxon rank-sum对测序数据进行拟时序分析,探究不同细胞簇中LC3B的差异表达.通过癌症基因组图谱(TCGA)及免疫组织化学技术(IHC)分析LC3B在胃癌组织中的表达.采用单因素和多因素Cox比例风险回归模型确定影响胃癌患者预后的独立因素.实时定量反转录聚合酶链反应(RT-qPCR)技术分析胃癌细胞中LC3B的表达水平,采用细胞计数试剂盒(CCK-8)、克隆形成、Transwell检测LC3B表达改变后,胃癌细胞增殖、迁移、侵袭能力的变化.两组间比较采用两独立样本t检验.结果 scRNA-seq分析结果显示,LC3B在胃癌组织中的表达量高于癌旁组织(2.001±2.706比1.010±1.634,t=44.094,P＜0.01)且LC3B在内皮细胞、单核细胞中表达量高于其余细胞亚群(3.610±3.294,3.214±3.889,F=1 039.267,P＜0.01).通过TCGA和IHC分析结果显示,LC3B在原发性胃癌组织中的表达高于正常组织[67.983(53.874,83.827)比 48.114(39.147,78.174),P＜0.01;60.3％比 19.1％,x2=9.299,P＜0.01].生存分析显示,LC3B高表达的胃癌患者总体生存时间短于LC3B低表达的患者[风险比(HR)=4.143,95％可信区间(CI):2.352～7.298,P＜0.05].多因素Cox回归分析显示,LC3B 为总生存期相关的危险因素(HR=4.966,95％CI:2.241～11.005,P＜0.01).CCK-8、Transwell迁移及侵袭实验、克隆形成实验检测分析发现,过表达LC3B后胃癌细胞的活力、迁移、侵袭、增殖能力高于空载体组[(1.837±0.057)比(1.287±0.091)、(338.000±39.950)个 比(152.333±4.509)个、(287.667±44.230)个 比(148.667±27.465)个、(202.333±12.503)个 比(104.667±11.240)个,t=8.883、7.999、4.624、10.062,P＜0.05],抑制 LC3B 表达后,胃癌细胞的活力、迁移、侵袭、增殖能力低于空载体组[(0.810±0.064)比(1.460±0.058)、(129.667±37.859)个 比(389.667±43.822)个、(126.000±60.531)个 比(290.000±70.682)个、(54.667±16.166)个 比(109.667±3.055)个,t=12.963、7.776、3.052、5.790,P＜0.05].结论 LC3B 在胃癌中高表达,并且促进胃癌细胞的增殖和转移.
Abstract
Objective To analyze the expression of microtubule associated protein 1 light chain 3 beta(LC3B)in gastric cancer and its correlation with patients'prognosis based on single cell RNA sequen-cing technology.Methods Three pairs of gastric cancer and paired adjacent tissue samples were selected for single-cell RNA sequencing(scRNA-seq).CellTypist classified the filtered single cell expression data into subgroups,and Wilcoxon rank sum was used to perform quasi temporal analysis on the sequencing data to explore the differential expression of LC3B in different cell clusters.The expression of LC3B in gastric cancer tissue was analyzed using the cancer genome atlas(TCGA)and immunohistochemistry(IHC)tech-niques.Univariate and multivariate Cox proportional risk regression models were used to determine inde-pendent factors affecting the prognosis of gastric cancer patients.Real-time quantitative reverse tran-scriptase-polymerase chain reaction(RT-qPCR)was used to analyze the expression level of LC3B in gastric cancer cells,cell counting kit-8(CCK-8)assay,clone formation assay,and Transwell assay were used to detect changes in the proliferation,migration,and invasion ability of gastric cancer cells after changes in LC3B expression.Student's t-test was used to compare two groups.Results ScRNA-seq analysis revealed a significant increase in the expression of LC3B in gastric cancer tissue(2.001±2.706 vs.1.010± 1.634,t=44.094,P＜0.01).The expression of LC3B in endothelial cells and monocytes were higher than that in other subgroups(3.610±3.294,3.214±3.889,F=1 039.267,P＜0.01).Through TCGA and IHC analysis,it was found that the expression of LC3B in primary gastric cancer tissue was significant-ly higher than that in normal tissue[67.983(53.874,83.827)vs.48.114(39.147,78.174),P＜0.01;60.3％vs.19.1％,x2=9.299,P＜0.01].Patients with high LC3B expression tended to have poorer overall survival[hazard ratio(HR)=4.143,95％confidence interval(CI):2.352-7.298,P＜0.05].Multivariate Cox regression analysis showed that the expression of LC3B was a risk factor related to o-verall survival(HR=4.966,95％CI:2.241-11.005,P＜0.001).CCK-8,Transwell and clone formation analysis showed that compared with the empty body group,overexpression of LC3B significantly increased the vitality,migration,invasion and proliferation ability of gastric cancer cells[(1.837±0.057)vs.(1.287± 0.091),(338.000±39.950)vs.(152.333±4.509),(287.667±44.230)vs.(148.667±27.465),(202.333±12.503)vs.(104.667±11.240),t=8.883,7.999,4.624,10.062,P＜0.05],while inhibi-tion of LC3B expression significantly decreased the vitality,migration,invasion and proliferation ability of gastric cancer cells[(0.810±0.064)vs.(1.460±0.058),(129.667±37.859)vs.(389.667±43.822),(126.000±60.531)vs.(290.000±70.682),(54.667±16.166)vs.(109.667±3.055),t=12.963,7.776,3.052,5.790,P＜0.05].Conclusion LC3B is highly expressed in gastric cancer and promotes the proliferation and metastasis of gastric cancer cells.
基金项目
湖北省科技厅大健康领域重大项目(2022BCE024)
硒与人体健康研究院开放课题(D20200018)
NETL
NSTL
万方数据