微小RNA-224对乳腺癌锁骨上淋巴结转移的影响及其作用机制

Effect of microRNA-224 on supraclavicular lymph node metastasis in breast cancer and mechanism

目的探讨微小RNA(miR)-224对乳腺癌锁骨上淋巴结转移的影响及其作用机制。方法选择2018年6月至2019年6月南阳市中心医院收治的45例锁骨上淋巴结转移的乳腺癌和45例无淋巴结转移的乳腺癌作为研究对象。采用转录组织化学方法分析淋巴结转移和无淋巴结转移癌组织中差异表达的miRNA。采用荧光定量聚合酶链反应(PCR)分析差异表达的miRNA(miR-224);采用慢病毒介导miRNA阴性对照和miR-224过表达在人乳腺癌细胞(MDA-MB-231)乳腺癌细胞建立对照细胞系(对照组)和miR-224过表达细胞系(miR-224组)。将对照组和miR-224组细胞接种至裸鼠腋下,并于30 d后处死,解剖分析锁骨上淋巴结转移;生物信息学和双荧光素酶报告基因分析miR-224靶基因,蛋白质印迹法(Western blot)分析临床样本和细胞系miRNA靶蛋白(PHLPP)1表达水平,组间比较采用t检验。结果转录组化学结果显示,淋巴结转移乳腺癌组织miR-224表达水平(5.08±0.69)明显高于未淋巴结转移乳腺癌组织(2.10±0.69),差异有统计学意义(t=3.109,P<0.05)。荧光定量PCR结果验证显示,淋巴结转移乳腺癌组织miR-224表达水平(2.98±0.41)高于无淋巴结转移乳腺癌组织miR-224表达水平(1.20±0.31),差异有统计学意义(t=2.839,P<0.05)。miR-224组细胞侵袭数量[(119.49±9.72)个]高于对照组细胞侵袭数量[(69.39±6.09)个],差异有统计学意义(t=4.091,P<0.05)。miR-224组细胞接种移植瘤30 d后淋巴结转移病灶数[(25.98±3.12)个]高于对照组细胞接种移植瘤30 d后淋巴结转移病灶数[(10.54±2.39)个],差异有统计学意义(t=3.001,P<0.05)。PHLPP1是miR-224靶基因。淋巴结转移乳腺癌组织中PHLPP1蛋白表达水平(0.36±0.12)低于未淋巴结转移乳腺癌组织中PHLPP1蛋白表达水平(0.94±0.16),差异有统计学意义(t=2.816,P<0.05)。miR-224组细胞PHLPP1蛋白表达水平(0.50±0.12)低于对照组细胞PHLPP1蛋白表达水平(1.04±0.18),差异有统计学意义(t=2.212,P<0.05)。结论miR-224在淋巴结转移的乳腺癌中呈高表达,其可能机制为通过调节PHLPP1蛋白表达参与了乳腺癌的侵袭和转移。

Objective To investigate the effect of microRNA(miRNA,miR)-224 on supraclavicular lymph node metastasis in breast cancer and the underlying mechanism.Methods A total of 45 cases of breast cancer with supraclavicular lymph node metastasis and 45 cases of breast cancer without lymph node metastasis admitted to our hospital from June 2018 to June 2019 were selected as the research objects.Transcriptomics was used to analyze the differentially expressed miRNAs in lymph node metastasis and non-lymph node metastasis cancer tissues.The differentially expressed miRNA(miR-224)was analyzed by fluorescence quantitative polymerase chain reaction(PCR).Lentivirus-mediated miRNA negative control and miR-224 overexpression were used to establish a control cell line(control group)and miR-224 overexpression cell line(miR-224 group)in MD anderson-metastatic breast-231 cell(MDA-MB-231)breast cancer cells respectively.The control group and miR-224 group cell lines were inoculated into the armpits of nude mice,and the animals were sacrificed 30 days later.The supraclavicular lymph node metastasis was dissected and analyzed.The miR-224 target genes were analyzed by bioinformatics and dual luciferase reporter genes,and Western blotting analysis was done to detect the expression level of miRNA target protein pleckstrin homology domain and leucine rich repeat protein phosphatase 1(PHLPP1)in clinical samples and cell lines.Results Transcriptomics showed that the expression level of miR-224(5.08±0.69)was significantly higher than that in breast cancer without lymph node metastasis(2.10±0.69)(t=3.109,P<0.05).The real-time PCR showed that the expression level of miR-224 in breast cancer with lymph node metastasis was significantly higher than that in breast cancer without lymph node metastasis(1.20±0.31)(t=2.839,P<0.05).Compared with the control group(69.39±6.09),the number of cell invasion in miR-224 group(119.49±9.72)was significantly increased(t=4.091,P<0.05).Compared with the control group(10.54±2.39),the number of lymph node metastases in miR-224 group(25.98±3.12)significantly increased at 30 days after transplantation(t=3.001,P<0.05).PHLP1 is a target gene of miR-224.Compared with the expression level of PHLP1 protein in breast cancer without lymph node metastasis(0.94±0.16),the expression level of PHLP1 protein in breast cancer with lymph node metastasis(0.36±0.12)was significantly decreased(t=2.816,P<0.05).Compared with the control group(1.04±0.18),the expression level of PHLP1 protein in miR-224 group(0.50±0.12)decreased significantly(t=2.212,P<0.05).Conclusion miR-224 is highly expressed in breast cancer with lymph node metastasis probably participating in the invasion and metastasis of breast cancer by regulating the expression of PHLPP1 protein.