论文部分内容阅读
目的:探讨长链非编码RNA(lncRNA) HNF1A-AS1对乳腺癌细胞增殖、侵袭和转移的影响及其分子机制。方法:选取新乡医学院附属中心医院2017年6月至2019年6月手术切除的236例乳腺癌组织和癌旁组织作为研究对象，采用荧光定量聚合酶链反应(PCR)分析表达水平；将乳腺癌MDA-MB-231细胞系按照随机数字表法分为短发卡RNA(shRNA)对照组、shRNA HNF1A-AS1组、miRNA对照组和miR-32-5p组。采用细胞计数试剂盒(CCK-8)、Transwell和体内移植瘤实验分析lncRNA HNF1A-AS1和miR-32-5p对乳腺癌细胞增殖、侵袭和转移的影响。荧光素酶基因报告实验检测lncRNA HNF1A-AS1和miR-32-5p的关系与miR-32-5p与FBXW7的关系。免疫印迹法(Western blot)分析靶蛋白表达水平。应用SPSS 17.0统计学软件分析，符合正态分布的计量资料以均数±标准差(n ±n s)表示。n 结果:癌旁组织lncRNA HNF1A-AS1表达水平(1.09±0.21)低于乳腺癌组织lncRNA HNF1A-AS1表达水平(3.01±0.32)，差异有统计学意义(n t＝3.819，n P<0.05)；癌旁组织miR-32-5p表达水平(0.94±0.18)高于乳腺癌组织miR-32-5p表达水平(0.47±0.13)，差异有统计学意义(n t＝2.209，n P<0.05)。shRNA对照组细胞48 h吸光度(n A)值(1.87±0.24)高于shRNA HNF1A-AS1组细胞48 h n A值(1.31±0.18)，差异有统计学意义(n t＝2.781，n P<0.05)。miRNA对照组细胞48 hn A值(1.94±0.20)低于miR-32-5p组细胞48 h n A值(1.19±0.12)，差异有统计学意义(n t＝2.799，n P<0.05)。shRNA对照组细胞侵袭数量[(84.39±8.01)个]高于shRNA HNF1A-AS1组细胞侵袭数量[(45.19±4.11)个]，差异有统计学意义(n t＝6.192，n P<0.05)。miRNA对照组细胞侵袭数量[(80.15±7.69)个]低于miR-32-5p组细胞侵袭数量[(38.63±5.71)个]，差异有统计学意义(n t＝7.109，n P<0.05)。shRNA对照组细胞淋巴结转移数量(15.90±3.10)高于shRNA HNF1A-AS1组细胞淋巴结转移数量(8.12±2.09)，差异有统计学意义(n t＝4.163，n P<0.05)。miRNA对照组细胞淋巴结转移数量(14.03±3.54)低于miR-32-5p组细胞淋巴结转移数量(7.19±3.74)，差异有统计学意义(n t＝3.853，n P<0.05)。生物信息学显示miR-32-5p是lncRNA HNF1A-AS1的靶基因。FBXW7是miR-32-5p的靶基因。shRNA对照组和miRNA对照组细胞FBXW7蛋白表达水平(1.48±0.24、1.38±0.19)高于shRNA HNF1A-AS1组和miR-32-5p组FBXW7蛋白表达水平(0.79±0.15、0.85±0.25)，差异有统计学意义(n t＝3.163、2.833，n P<0.05)。n 结论:lncRNA HNF1A-AS1在乳腺癌中高表达，通过吸附结合miR-32-5p，调节癌蛋白FBXW7表达，进而调控乳腺癌细胞增殖、侵袭和转移等生物学过程。“,”Objective:To investigate the effect of long noncoding RNA (lncRNA) HNF1A-AS1 on proliferation, invasion and metastasis of breast cancer cells and its molecular mechanism.Methods:Breast cancer tissues and adjacent tissues (236 cases) from June 2017 to June 2019 were selected as the research objects. The MDA-MB-231 cells were divided into short hairpin RNA (shRNA) control group, shRNA HNF1A-AS1 group, miRNA control group and miR-32-5p group by random digits table. The effects of lncRNA HNF1A-AS1 and miR-32-5p on the proliferation, invasion and metastasis of breast cancer cells were analyzed by cell counting kit-8 (CCK-8), transwell and n in vivo tumor transplantation experiments. Luciferase gene reporter assay was used to detect the relationship between lncRNA HNF1A-AS1 and miR-32-5p, and the relationship between miR-32-5p and FBXW7. The expression of target protein was analyzed by Western blotting. SPSS 17.0 statistical software was used to analyze the data. The data in accordance with normal distribution were expressed as mean±standard deviation.n Results:The expression level of lncRNA HNF1A-AS1 in adjacent tissues tissue (1.09±0.21) was significantly lower than that in breast cancer tissue (3.01±0.32, n t=3.819, n P<0.05). The expression level of miR-32-5p in adjacent tissues (0.94±0.18) was significantly lower than that in breast cancer tissue (0.47±0.13,n t=2.209, n P<0.05). The 48-h absorbance (n A) value of shRNA control group (1.87±0.24) was significantly higher than that of shRNA HNF1A-AS1 group n A value (1.31±0.18, n t=2.781, n P<0.05). The 48-hn A value of miRNA control group (1.94±0.20) was significantly lower than that of miR-32-5p group (1.19±0.12, n t=2.799, n P<0.05). The number of invasive cells in shRNA control group [(84.39±8.01) cells] was significantly higher than that in shRNA HNF1A-AS1 group [(45.19 4.11) cells,n t=6.192, n P<0.05]. The number of invasive cells in miRNA control group [(80.15±7.69) cells] was significantly higher than that in miR-32-5p group [(38.63±5.71) cells,n t=7.109, n P<0.05]. The number of lymph node metastases in shRNA control group (15.90±3.10) was significantly higher than that in shRNA HNF1A-AS1 group (8.12±2.09,n t=4.163, n P<0.05). The number of lymph node metastases in miRNA control group (14.03±3.54) was significantly higher than that in miR-32-5p group (7.19±3.74,n t=3.853, n P<0.05). Bioinformatics showed that miR-32-5p was the target gene of lncRNA HNF1A-AS1. FBXW7 was the target gene of miR-32-5p. The expression level of FBXW7 protein in shRNA control group and miRNA control group (1.48±0.24, 1.38±0.19) was significantly higher than that in shRNA HNF1A-AS1 group and miR-32-5p group (0.79±0.15, 0.85±0.25,n t=3.163, 2.833, n P<0.05).n Conclusion:lncRNA HNF1A-AS1 is highly expressed in breast cancer, which can regulate the expression of oncoprotein FBXW7 by binding miR-32-5p, and then regulate the biological process of breast cancer cell proliferation, invasion and metastasis.