长链非编码RNA TP73-AS1调控miR-181b及其靶基因HMGB1的表达以及对非小细胞肺癌细胞生物学行为的影响

Effects of long non-coding RNA TP73-AS1 regulating the expression of miR-181b and its target gene HMGB1 on the biological behavior of non-small cell lung cancer cells

目的:探讨长链非编码RNA TP73-AS1通过调控微RNA(microRNA,miRNA,miR)-181b及其靶基因高迁移率族蛋白B1(high-mobility group box-1,HMGB1)对非小细胞肺癌(non-small cell lung cancer,NSCLC)增殖、侵袭、迁移和凋亡的影响。方法:采用实时荧光定量PCR法检测42例癌组织及其对应的癌旁组织,以及人正常肺上皮细胞BEAS-2B和人NSCLC细胞(A549和NCI-H1299)中TP73-AS1的表达水平,并分析TP73-AS1表达与患者总生存期的相关性。采用脂质体法将shRNA-TP73-AS1(shTP73-AS1)和shRNA-NC(shNC)转入A549和NCI-H1299细胞,分别采用CCK-8法、Transwell小室实验、划痕愈合实验及FCM法检测敲减TP73-AS1表达对NSCLC细胞增殖、侵袭、迁移和凋亡的影响。采用双荧光素酶报告基因验证TP73-AS1、miR-181b和HMGB1间的靶向关系。A549和NCI-H1299细胞中分别转入shTP73-AS1、shTP73-AS1+miR-181b-抑制子(inhibitor)和shTP73-AS1+pcDNA-HMGB1,再用CCK-8法、Transwell小室实验、划痕愈合实验及FCM法检测敲低TP73-AS表达后再沉默miR-181b表达或使HMGB1过表达对A549和NCI-H1299细胞增殖、侵袭、迁移和凋亡的影响。结果:TP73-AS1 mRNA在癌组织中的相对表达量明显高于癌旁组织(P<0.05),癌组织中TP73-AS1的表达水平与miR-181b的表达水平呈负相关(r=-0.623,P<0.05)。A549和NCI-H1299细胞中TP73-AS1的表达水平均明显高于BEAS-2B细胞(P值均<0.05)。敲低TP73-AS1表达可以明显抑制A549和NCI-H1299细胞的增殖、侵袭和迁移能力,并促进细胞凋亡(P值均<0.05)。双荧光素酶报告基因检测证实,TP73-AS1靶向作用于miR-181b并下调其表达,miR-181b可负调控HMGB1的表达。敲低TP73-AS1表达可以明显抑制A549和NCI-H1299细胞中HMGB1 mRNA和蛋白的表达水平(P值均<0.05)。同时,转染shTP73-AS1+miR-181b-inhibitor或者shTP73-AS1+HMGB1过表达载体后,HMGB1 mRNA和蛋白的表达水平较仅转染shTP73-AS1明显上调(P值均<0.05),细胞增殖、侵袭和迁移能力也明显升高,而凋亡水平明显下降(P值均<0.05)。结论:TP73-AS1在NSCLC组织和细胞中高表达,可通过调控miR-181b/HMGB1轴促进癌细胞增殖、侵袭、迁移,并促进凋亡。

Objective:To investigate the effect of long non-coding RNA TP73-AS1 on the proliferation,invasion,migration and apoptosis of non-small cell lung cancer(NSCLC)by regulating microRNA(miR)-181b/high-mobility group box-1(HMGB1).Methods:The expression level of TP73-AS1 in 42 cases of cancer tissues and their corresponding adjacent tissues and human normal lung epithelial cells BEAS-2B and human NSCLC cells(A549 and NCI-H1299)were detected by real-time quantitative PCR method.Analysis of the correlation between TP73-AS1 expression and the overall survival of patients.A549 and NCI-H1299 cells were transfected with shRNA-TP73-AS1(shTP73-AS1)or shRNA-negative control(shNC),and the effects of knockdown of TP73-AS1 in NSCLC cells proliferation,invasion,migration and apoptosis were analyzed.The dual luciferase reporter gene was used to verify the targeting relationship of TP73-AS1,miR-181b and HMGB1.A549 and NCI-H1299 cells were transfected with shTP73-AS1,shTP73-AS1+miR-181b-inhibitor and shTP73-AS1+pcDNA-HMGB1,CCK-8 method,Transwell experiment,scratch healing experiment and FCM method were used to detect the effects of knockdown of TP73-AS1 and then silencing the expression of miR-181b or overexpression of HMGB1 on the proliferation,invasion,migration and apoptosis of A549 and NCI-H1299 cells.Results:The relative expression of TP73-AS1 in cancer tissues was significantly higher than that in adjacent tissues(P<0.05).The relative expression of TP73-AS1 in cancer tissues was negatively correlated with the relative expression of miR-181b(r=-0.623,P<0.05).The expression level of TP73-AS1 in A549 and NCI-H1299 cells was significantly higher than that in BEAS-2B cells(both P<0.05).Knockdown of TP73-AS1 could obviously inhibit the proliferation,invasion and migration of A549 and NCI-H1299 cells,and promote cell apoptosis(all P<0.05).Double luciferase reporter gene detection confirmed that TP73-AS1 targeted miR-181b and down-regulated its expression,miR-181b could negatively regulate the expression of HMGB1.Knockdown of the expression of TP73-AS1 could significantly inhibit the expression levels of HMGB1 mRNA and protein in A549 and NCI-H1299 cells(all P<0.05).After transfection with shTP73-AS1+miR-181b-inhibitor or shTP73-AS1+HMGB1 overexpression vector,the expression levels of HMGB1 mRNA and protein were significantly increased compared with shTP73-AS1 transfection(all P<0.05),The abilities of cell proliferation,invasion and migration also increased significantly,while the level of apoptosis was decreased significantly(all P<0.05).Conclusion:TP73-AS1 is highly expressed in NSCLC tissues and cells.It can promote cancer cell proliferation,invasion,migration and apoptosis by regulating miR-181b/HMGB1 axis.