LncRNA CBR3-AS1调节miR-448/TFAM轴对结直肠癌细胞恶性生物学行为的影响

Impact of LncRNA CBR3-AS1 regulating miR-448/TFAM axis on malignant biological behavior of colorectal cancer cell

目的 探讨长链非编码RNA(LncRNA)CBR3-AS1对结直肠癌(CRC)细胞恶性生物学行为的影响及其机制。方法 收集济南市第五人民医院2021年1月至2022年6月获取的40对CRC组织和邻近癌旁组织标本,采用实时荧光定量聚合酶链反应(qRT-PCR)检测人CRC组织、癌旁组织和CRC细胞系、正常结直肠上皮细胞中LncRNA CBR3-AS1、微小RNA-448(miR-448)及线粒体转录因子A(TFAM)mRNA表达。根据不同CRC细胞系中LncRNA CBR3-AS1的表达水平筛选后续实验使用的细胞系。验证miR-448与LncRNA CBR3-AS1、TFAM的靶向关系。在目标细胞中转染靶向LncRNA CBR3-AS1的小干扰RNA(si-CBR3-AS1)、miR-448抑制物(miR-448 inhibitor),采用qRT-PCR、蛋白免疫印迹法(Western blot)检测LncRNA CBR3-AS1、miR-448及TFAM mRNA和蛋白表达;采用CCK-8法、流式细胞术、划痕愈合实验和Transwell实验分别检测细胞活力、凋亡率、迁移及侵袭能力。结果 CRC组织和细胞中LncRNA CBR3-AS1及TFAM mRNA呈高表达,miR-448呈低表达,且HCT116细胞中LncRNA CBR3-AS1的表达最高,故选择HCT116细胞进行后续靶向验证和转染实验。经验证,HCT116细胞中miR-448与LncRNA CBR3-AS1、TFAM均存在靶向关系。转染si-CBR3-AS1可降低HCT116细胞中LncRNA CBR3-AS1及TFAM mRNA和蛋白表达水平,同时降低细胞活力、划痕愈合率、迁移细胞数、侵袭细胞数,升高miR-448表达水平及凋亡率;转染si-CBR3-AS1基础上转染miR-448 inhibitor可减弱干扰LncRNA CBR3-AS1表达对HCT116细胞的影响。结论 干扰LncRNA CBR3-AS1可抑制CRC细胞的恶性生物学行为,其机制可能与靶向miR-448/TFAM轴有关。

Objective To investigate the impact of long non-coding RNA(LncRNA)CBR3-AS1 on the malignant biological behavior of colorectal cancer(CRC)cells and its mechanism.Methods A total of 40 pairs of CRC tissue samples and adjacent paracancerous tissue samples were collected from the Jinan Municipal Fifth People′s Hospital from January 2021 to June 2022.The real-time fluorescence quantitative polymerase reaction(qRT-PCR)was applied to detect the expressions of LncRNA CBR3-AS1,microRNA-448(miR-448)and mitochondrial transcription factor A(TFAM)mRNA in human CRC tissue,paracancerous tissues,CRC cell line and normal colorectal epithelial cells.According to the expression levels of LncRNA CBR3-AS1 in different CRC cell lines,the cell lines used in subsequent experiments were screened.The targeting relationship between miR-448 with LncRNA CBR3-AS1 and TFAM was verified.Small interfering RNA(si-CBR3-AS1)targeting LncRNA CBR3-AS1 and miR-448 inhibitor were transfected into target cells,qRT-PCR and Western blot were applied to detect the mRNA and protein expression of LncRNA CBR3-AS1,miR-448 and TFAM;the cell viability,apoptosis rate,migration and invasion ability were detected by CCK-8 assay,flow cytometry,scratch healing assay and Transwell assay,respectively.Results LncRNA CBR3-AS1 and TFAM mRNA were highly expressed in CRC tissues and cells,while miR-448 was lowly expressed.In addition,the expression of LncRNA CBR3-AS1 was the highest in HCT116 cells,so HCT116 cells were selected for subsequent targeting validation and transfection experiments.After verification,miR-448 had a targeted relationship with LncRNA CBR3-AS1 and TFAM in HCT116 cells.Transfecting si-CBR3-AS1 could reduce the expression levels of LncRNA CBR3-AS1,and TFAM mRNA and protein in HCT116 cells,meanwhile reduce the cell viability,scratch healing rate,number of migrated and invasive cells,and increase the miR-448 expression level and apoptosis rate;transfecting miR-448 inhibitor on the basis of trassfecting si-CBR3-AS1 could weaken the effect of interfering LncRNA CBR3-AS1 expression on HCT116 cells.Conclusion Interfering LncRNA CBR3-AS1 could inhibit the malignant biological behavior of CRC cells,and its mechanism may be related to targeting the miR-448/TFAM axis.