LINC00626通过JAK1/STAT3信号通路对肺鳞状细胞癌增殖、侵袭和转移的影响

LINC00626 promotes the proliferation,invasion and metastasis of lung squamous cell carcinoma via the JAK1/STAT3 signaling pathway

目的探究LINC00626在肺鳞状细胞癌恶性进程中的作用及其分子机制。方法采用聚合酶链式反应(PCR)和实时荧光定量聚合酶链式反应(RT-qPCR)检测144对癌组织和癌旁组织、正常肺上皮细胞株和肺癌细胞株中LINC00626的表达量与肺鳞状细胞癌病理指标间的相关性,受试者工作特征(ROC)曲线分析LINC00626对肺鳞癌的诊断价值。将敲降的LINC00626及其敲降对照慢病毒转入H1437细胞中,命名为sh-NC组和sh-LINC00626组。将过表达LINC00626慢病毒及其过表达对照慢病毒转入A549细胞中,命名为Vetor组和LINC00626组。采用细胞计数试剂盒-8(CCK-8)实验、菌落形成实验、细胞划痕实验和Transwell小室实验检测LINC00626体外细胞学功能。采用裸鼠皮下荷瘤和尾静脉注射肺转移模型检测LINC00626在活体动物体内对细胞增殖、侵袭和转移能力的影响。采用qPCR和蛋白免疫印迹(Western blot)实验检测LINC00626表达水平与JAK/STAT信号通路的相关性。结果PCR和RT-qPCR结果显示,与癌旁组织相比,LINC00626在肺鳞癌组织中显著上调(P<0.001),且与患者的TNM分期和淋巴结转移密切相关(P<0.05)。与16HBE细胞相比,LINC00626在H1437和H1975细胞中相对表达较高(P<0.01),在A549和H1299细胞中相对表达较低(P<0.001)。与sh-NC组相比,sh-LINC00626组中p-JAK1/JAK1、STAT3的mRNA水平显著降低(P<0.01);与Vector组相比,LINC00626组中p-JAK1/JAK1、STAT3的mRNA水平显著增加(P<0.01,P<0.05)。体外细胞功能实验和体内动物实验结果显示,与对照组相比,LINC00626过表达或敲低组对肺鳞癌的增殖、侵袭和转移有明显的促进或抑制作用(P<0.001)。WB实验结果显示,与sh-NC组相比,sh-LINC00626组中p-JAK1/JAK1、STAT3的蛋白水平显著降低(P<0.001);与Vector组相比,LINC00626组中p-JAK1/JAK1、STAT3的蛋白水平显著增加(P<0.001)。结论LINC00626通过JAK1/STAT3信号通路促进肺鳞癌的增殖、侵袭和转移。为肺鳞癌的临床诊断提供了新的生物标志物和潜在的治疗靶点。

Objective To investigate the role of LINC00626 in the malignant process of lung squamous-cell carcinoma and its molecular mechanism.Methods The expression of LINC00626 was assessed by polymerase chain reaction(PCR)and quantitative real-time PCR(RT-qPCR)in 144 paired of fresh cancerous and para-cancerous tissues,normal lung epithelial cell lines and lung cancer cell lines.And the correlations between LINC00626 expression and pathological parameters of lung squamous cell carcinoma was analyzed.The diagnostic value of LINC00626 in lung squamous cell carcinoma was analyzed by ROC curve.The knock-down LINC00626 and the knock-down control lentivirus were transferred into H1437 cells and named as sh-NC group and sh-LINC00626 group.Over expressing LINC00626 lentivirus and control lentivirus were transferred into A549 cells and named as Vetor group and LINC00626 group.Cytological experiment in vitro was assessed by cell counting kit-8(CCK-8)experiment,colony formation assay,cell scratch test,and Trans-well assay.Besides,the proliferation,invasion and metastasis of LINC00626 cells in vivo were tested by subcutaneous tumor bearing and tail vein injection in nude mice.The correlation between LINC00626 and JAK/STAT signal pathway was detected by qPCR and Western blot(WB).Results Compared with the adjacent tissues,LINC00626 was significantly up-regulated in lung squamous cell carcinoma(P<0.001),and was closely correlated to TNM stage and lymph node metastasis(P<0.05).Compared with 16HBE,LINC00626 was highly expressed in H1437 and H1975(P<0.01)and low expressed in A549 and H1299(P<0.001).Compared with sh-NC group,the mRNA expressions of p-JAK1/JAK1 and STAT3 in sh-LINC00626 group were significantly decreased(P<0.01);compared with Vector group,the mRNA expressions of p-JAK1/JAK1 and STAT3 were significantly increased(P<0.01,P<0.05).LINC00626 over-expression or knockdown group significantly promoted or inhibited the proliferation,invasion and metastasis of lung squamous cell carcinoma compared with the control group(P<0.001).Compared with sh-NC group,the protein levels of p-JAK1/JAK1 and STAT3 in sh-LINC00626 group were significantly decreased(P<0.001);compared with Vector group,the protein levels of p-JAK1/JAK1 and STAT3 in LINC00626 group were significantly increased(P<0.001).Conclusion LINC00626 can promote the proliferation,invasion and metastasis of lung squamous cell carcinoma via JAK1/STAT3 signaling pathway.Our findings provide a new biomarker and potential therapeutic targets for clinical diagnosis of lung squamous cell carcinoma.