摘要
目的探究长链非编码RNA 626(long intergenic non-protein coding RNA 626,LINC00626)通过JAK1/STAT3/KHSRP信号轴调控肺腺癌恶性进展及分子机制。方法采用实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,q RT-PCR)检测非小细胞肺癌细胞系(A549、H1299、H1975、H1437)和人正常气管上皮细胞系(16HBE),以及144例肺腺癌组织标本及其正常组织标本中LINC00626、KH型剪接调控蛋白(KH-type splicing regulatory protein,KHSRP)m RNA的表达量。将敲降LINC00626慢病毒及其对照慢病毒转入H1299、H1437细胞中,分别命名为sh-LINC00626组(转染短发卡RNA慢病毒载体敲降LINC00626)和sh-NC组(转染短发卡RNA慢病毒空白载体)。将过表达LINC00626慢病毒及其过表达对照慢病毒转入A549、H1975细胞中,分别命名为LINC00626组和Vector组。在H1437细胞中加入敲降LINC00626慢病毒基础上的KHSRP载体和敲降LINC00626慢病毒基础上的空白载体,分别命名为sh-LINC00626+KHSRP组和shLINC00626+Vector组。采用细胞计数试剂盒-8、Transwell迁移/侵袭实验检测细胞增殖、迁移和侵袭等生物学特征,采用蛋白质印迹法检测稳定转染细胞中JAK/STAT、KHSRP蛋白的表达水平。裸鼠实验分析LINC00626在体内的作用。核质分离和RNA荧光原位杂交实验分析LINC00626和KHSRP的亚细胞定位。RNA下拉和质谱分析用于鉴定LINC00626的结合蛋白。结果与人正常支气管上皮细胞相比,LINC00626和KHSRP在非小细胞肺癌细胞系中表达水平均显著增加。与正常组织比较,LINC00626、KHSRP在肺腺癌患者组织中高表达。与sh-NC组相比,sh-LINC00626组细胞增殖率、细胞迁移、侵袭数明显降低,过表达组相应结果则反之。与sh-LINC00626+Vector组相比,sh-LINC00626+KHSRP组H1437细胞在转染敲降LINC00626和KHSRP后细胞增殖率、细胞迁移、侵袭数均明显增加。裸鼠实验中,与对照组相比,敲降组中肿瘤体积和重量、细胞增殖率和增殖指数、肺转移病灶数目明显下降;过表达组呈相反的效果,各组间差异均有统计学意义(P<0.01)。LINC00626和KHSRP位于细胞核内,LINC00626与KHSRP蛋白直接结合。与对照组相比,敲降组JAK1、STAT3 m RNA和蛋白的表达降低(P<0.05);与对照组相比,过表达组JAK1、STAT3 m RNA和蛋白的表达增加(P<0.05)。结论LINC00626通过JAK1/STAT3/KHSRP信号轴,促进肺腺癌的恶性进程。
Objective To explore the molecular mechanism of LINC00626 regulating malignant progression of lung adenocarcinoma metastasis through JAK1/STAT3/KHSRP axis.Methods Quantitative real-time polymerase chain polymerase chain reaction was used to detect the expression of LINC00626 and KHSRP mRNA in human non-small-cell lung carcinoma cell lines(A549,H1299,H1975,H1437),human normal bronchial epithelial cell line(16HBE)and 144 lung adenocarcinoma tissues.The knockdown LINC00626 lentivirus and the control lentivirus were transferred into H1299 and H1437 cells,and named as sh-LINC00626 group(silencing of LINC00626 by transfecting short hairpin RNA lentiviral vector and sh-NC Group negative control by transfecting short hairpin RNA lentiviral).The overexpressed LINC00626 lentivirus and the control lentivirus were transferred into A549 and H1975 cells and named as LINC00626 group and Vector group.KHSRP vector on the basis of silencing LINC00626 and blank vector on the basis of silencing LINC00626 were added in H1437 cells.Cell counting kit-8 assay and Transwell migration/invasion assay were used to detect cell proliferation,migration and invasion.The expression levels of JAK/STAT and KHSRP in stably transfected cells were detected by Western blot.The effect of LINC00626 in vivo was studied in nude mice.Nuclear-cytoplasmic separation and RNA fluorescence in situ hybridization assay are used to predict the subcellular localization of LINC00626 and KHSRP.RNA pull down and mass spectrometry analysis were used to identify LINC00626 binding proteins.Results The expression levels of LINC00626 and KHSRP in non-small-cell lung carcinoma cell lines were significantly higher than those in normal human bronchial epithelial cells.LINC00626 and KHSRP were highly expressed in lung adenocarcinoma.Compared with the control group,the cell proliferation rate,colony formation,cell migration and invasion of H1437 cells were significantly decreased in knockdown group,while the reverse was true for over-expression.LINC00626 and KHSRP were located in the nucleus.LINC00626 directly binded to the KHSRP protein.Compared with the control group,H1437 cells transfected with knockdown LINC00626 and KHSRP significantly increased cell proliferation rate,cell migration,number of invasions.Compared with the control group,knockdown group showed a significant decrease in tumor volume and weight,cell proliferation rate and proliferation index,and the number of lung metastases.While the overexpression group showed an opposite effect,there were significant differences among the groups(P<0.01).The expression of JAK1 and STAT3 mRNA and protein in sh-LINC00626 group was lower than that in sh-NC Group(P<0.05),and the expression of JAK1 and STAT3 mRNA and protein in sh-LINC00626 group was higher than that in Vector group(P<0.05).Conclusion LINC00626 promotes malignant progression of lung adenocarcinoma metastasis through JAK1/STAT3/KHSRP signaling axis.
作者
徐锋
范林林
康霞
刘洋洋
韦海涛
李丽
XU Feng;FAN Linlin;KANG Xia;LIU Yangyang;WEI Haitao;LI Li(Department of Respiratory Medicine,Huaihe Hospital,Henan University,Kaifeng,Henan 475001,P.R.China;School of Nursing and Health,Henan University,Kaifeng,Henan 475001,P.R.China;Department of Thoracic Surgery,Huaihe Hospital,Henan University,Kaifeng,Henan 475001,P.R.China)
出处
《中国呼吸与危重监护杂志》
CAS
CSCD
北大核心
2023年第9期646-656,共11页
Chinese Journal of Respiratory and Critical Care Medicine
基金
河南省教育厅资助项目(23A320012)
河南省科技厅指导项目(222102310422)
河南省科技厅指导项目(232102310007)。
作者简介
共同第一作者:徐锋;共同第一作者:范林林;通信作者:李丽,Email:10210051@vip.henu.edu.cn;通信作者:韦海涛,Email:taoge9885@163.com。
