摘要
目的 探讨LncRNA BCYRN1调节miR-363-3p/PAX6轴对非小细胞肺癌(NSCLC)细胞恶性生物学行为的影响。方法 qRT-PCR检测正常肺上皮细胞BEAS-2B和NSCLC细胞(H460、HCC-827、A549)中BCYRN1、miR-363-3p和PAX6 mRNA的表达水平,筛选最佳干预细胞;然后将A549细胞分为control组、sh-NC组、sh-BCYRN1组、oe-NC组、oe-BCYRN1组、sh-BCYRN1+inhibitor NC组、sh-BCYRN1+miR-363-3p inhibitor组;qRT-PCR检测各组细胞BCYRN1、miR-363-3p和PAX6 mRNA表达水平;MTT检测细胞增殖;流式细胞术检测细胞凋亡率;Transwell实验检测细胞迁移和侵袭;Western blot检测细胞中PAX6、Ki67、MMP-2、caspase3的蛋白表达量。双荧光素酶报告基因实验检测LncRNA BCYRN1和miR-363-3p、miR-363-3p和PAX6的靶向关系。结果 与正常肺上皮细胞BEAS-2B相比,NSCLC细胞(H460、HCC-827、A549)中BCYRN1和PAX6 mRNA表达水平显著升高,miR-363-3p表达水平显著降低(P<0.05)。与control组和sh-NC组相比,sh-BCYRN1组细胞活力、细胞增殖率、细胞迁移和侵袭数量、BCYRN1和PAX6 mRNA表达水平、PAX6、Ki67和MMP-2蛋白表达水平显著降低,细胞凋亡率、miR-363-3p、caspase3表达显著升高(P<0.05);与oe-NC组比较,oe-BCYRN1组细胞活力、细胞增殖率、细胞迁移和侵袭数量、BCYRN1和PAX6 mRNA表达水平、PAX6、Ki67和MMP-2蛋白表达水平显著升高,细胞凋亡率、miR-363-3p、caspase3表达显著降低(P<0.05);与sh-BCYRN1+inhibitor NC组比较,sh-BCYRN1+miR-363-3p inhibitor组细胞活力、细胞增殖率、细胞迁移和侵袭数量、PAX6 mRNA表达、PAX6、Ki67和MMP-2蛋白表达水平显著升高,细胞凋亡率、miR-363-3p、caspase3表达显著降低(P<0.05),BCYRN1表达无显著性变化(P>0.05);双荧光素酶报告基因实验验证miR-652-5p和BCYRN1、PAX6存在靶向调控关系。结论 干扰BCYRN1可通过调控miR-363-3p/PAX6轴抑制NSCLC恶性发展。
Objective To investigate the regulatory effect of long noncoding RNA(lncRNA)brain cytoplasmic RNA 1(BCYRN1)on the malignant biological behaviors of non-small cell lung cancer(NSCLC)cells by regulating the microRNA-363-3p(miR-363-3p)/paired-box 6(PAX6)axis.Methods The mRNA expressions of BCYRN1,miR-363-3p and PAX6 in the lung epithelial cell line BEAS-2B and NSCLC cell lines H460,HCC-827,and A549 were detected by quantitative real-time polymerase chain reaction(qRT-PCR)to screen the optimal intervention cell line.A549 cells were grouped into control group,sh-NC group,sh-BCYRN1 group,oe-NC group,oe-BCYRN1 group,sh-BCYRN1+inhibitor NC group,and sh-BCYRN1+miR-363-3p inhibitor group.The mRNA expressions of BCYRN1,miR-363-3p and PAX6 in each group were detected by qRT-PCR.Cell proliferation and apoptosis were detected by MTT assay,and flow cytometry,respectively.Transwell assay was applied to detect cell migration and invasion.Western blot was applied to detect the protein expressions of PAX6,Ki67,matrix metalloprotease-2(MMP-2)and caspase3.The binding of lncRNA BCYRN1 to miR-363-3p,and the binding of miR-363-3p to PAX6 were detected by dual-luciferase reporter assay.Results Compared with those of the normal lung epithelial cell line BEAS-2B,BCYRN1 and PAX6 were upregulated,and miR-363-3p was downregulated in NSCLC cell lines H460,HCC-827,and A549)(P<0.05).Compared with those of control group and sh-NC group,we detected significantly lower viability,proliferative rate,migratory and invasive cell numbers,mRNA levels of BCYRN1 and PAX6,and protein levels of PAX6,Ki67 and MMP-2,and higher apoptotic rate and expression levels of miR-363-3p and caspase3 in sh-BCYRN1 group(P<0.05).Compared with those of oe-NC group,we detected significantly higher viability,proliferative rate,migratory and invasive cell numbers,mRNA levels of BCYRN1 and PAX6,and protein levels of PAX6,Ki67 and MMP-2,and lower apoptotic rate and expression levels of miR-363-3p and caspase3 in oe-BCYRN1 group(P<0.05).Compared with those of sh-BCYRN1+inhibitor NC group,we detected significantly higher viability,proliferative rate,migratory and invasive cell numbers,mRNA levels of PAX6,and protein levels of PAX6,Ki67 and MMP2,and lower apoptotic rate and expression levels of miR-363-3p and caspase3 in sh-BCYRN1+miR-363-3p inhibitor group(P<0.05);while the expression level of BCYRN1 was comparable(P>0.05).Dual-luciferase reporter assay verified that miR-652-5p had a targeted regulatory relationship with BCYRN1 and PAX6.Conclusion Knockdown of BCYRN1 can inhibit the malignant development of NSCLC by regulating the miR-363-3p/PAX6 axis.
作者
曲梦媛
张丽君
姜楠
范伟
QU Mengyuan;ZHANG Lijun;JIANG Nan(Department of Oncology,Qingdao Central Hospital Affiliated to Qingdao University,Shandong,Qingdao 266013,China;不详)
出处
《河北医药》
CAS
2023年第21期3205-3210,3216,共7页
Hebei Medical Journal
基金
吴阶平医学基金会临床科研专项资助基金(编号:320.6750.2021-2-66)。
关键词
LncRNA
BCYRN1
miR-363-3p
PAX6
非小细胞肺癌
long noncoding RNA(lncRNA)brain cytoplasmic RNA 1(BCYRN1)
microRNA-363-3p(miR-363-3p)
paired-box 6(PAX6)
non-small cell lung cancer
