1.江西省吉安市妇幼保健院病理科,江西 吉安 343000;2.井冈山大学医学部,江西 吉安 343000;3.江西省吉安市妇幼保健院检验科,江西 吉安 343000
1. Department of Pathology, Maternal and Child Health Hospital of Ji’an, Ji’an 343000, China. 2. Medical Science Center of Jinggangshan University, Ji’an 343000. 3. Clinical Laboratory, Maternal and Child Health Hospital of Ji’an, Ji’an 343000
目的 探究miR-34a-5p 通过靶向胰岛素样生长因子Ⅱ mRNA 结合蛋白3(IMP3)、程序性死亡配体-1(PD-L1)表达对乳腺癌细胞生物学行为的影响。 方法 利用Human Protein Atlas 和GEPIA 在线分析TCGA 数据库和GTEx 项目中乳腺癌及正常组织IMP3、PD-L1 的表达水平、患者的预后生存期;qRT-PCR、Western blot 检测160例患者乳腺癌组织及癌旁正常组织中miR-34a-5p、IMP3 、PD-L1 mRNA 与蛋白表达;Pearson 检验分析miR-34a-5p分别与IMP3 mRNA、PD-L1 mRNA 的相关性;免疫组化检测患者乳腺癌及正常组织IMP3、PD-L1、白细胞分化抗原44 变异体6(CD44v6)的表达,分析IMP3、PD-L1 表达水平与患者临床病理参数的关系;靶基因预测、双荧光素酶报告实验验证miR-34a-5p 对IMP3、PD-L1 的靶向调控作用。MCF7 细胞分为control 组、miR-NC 组、miR-34a-5p 组、miR-NC+pcDNA-NC 组、miR-NC+IMP3 组、miR-NC+PD-L1 组、miR-34a-5p+pcDNA-NC 组、miR-34a-5p+IMP3 组和miR-34a-5p+PD-L1 组,采用MTT 法、克隆形成、划痕、Transwell 小室实验测定细胞增殖活力、迁移、侵袭能力;Western blot 检测IMP3、PD-L1、CD44v6、基质金属蛋白酶2(MMP-2)、MMP-9、E-钙黏蛋白(E-cadherin)表达水平。 结果 生物数据库显示乳腺癌组织IMP3、PD-L1 表达水平越高,患者生存期越短(P<0. 05);qRT-PCR、Western blot结果显示,乳腺癌组织miR-34a-5p 表达水平明显低于正常组织,IMP3、PD-L1 mRNA 与蛋白表达水平明显高于正常组织,miR-34a-5p 表达分别与IMP3 mRNA、PD-L1 mRNA 呈明显负相关(P<0. 05);免疫组化结果显示,乳腺癌组织IMP3、PD-L1、CD44v6 呈阳性表达,且IMP3、PD-L1 表达越高,TNM 分期越晚,肿瘤分化程度越低、越容易发生淋巴结和远处转移(P<0. 05);双荧光素酶实验验证了miR-34a-5p 对IMP3、PD-L1 的靶向调控作用;与control 组和miRNC组相比,miR-34a-5p 组的细胞活力、单克隆形成数目、划痕愈合率、细胞侵袭数目、IMP3、PD-L1、CD44v6、MMP-2、MMP-9 蛋白表达明显降低,E-cadherin 蛋白表达明显升高,与miR-NC+pcDNA-NC 组相比,miR-NC+IMP3 组和miR-NC+PD-L1 组细胞活力、单克隆形成数目、划痕愈合率、细胞侵袭数目、IMP3、PD-L1、CD44v6、MMP-2、MMP-9 蛋白表达明显升高,E-cadherin 蛋白表达明显降低,与miR-34a-5p+pcDNA-NC 组相比,miR-34a-5p+IMP3 组和miR-34a-5p+PD-L1 组细胞活力、单克隆形成数目、划痕愈合率、细胞侵袭数目、IMP3、PD-L1、CD44v6、MMP-2、MMP-9 水平升高,E-cadherin 水平降低(P<0. 05)。 结论 miR-34a-5p 能够通过靶向调控IMP3、PD-L1 表达抑制乳腺癌细胞的增殖、迁移、侵袭和上皮间质化(EMT)。
Objective To explore how miR-34a-5p regulates insulin-like growth factor Ⅱ mRNA-binding protein 3 (IMP3) and programmed death ligand-1 (PD-L1) expression and its influence on the biological behavior of breast cancer cells. Methods We analyzed IMP3 and PD-L1 expression in breast cancer and normal tissues using the Human Protein Atlas and GEPIA, and patient prognosis and survival using The Cancer Genome Atlas and GTEx project. miR-34a-5p, IMP3, and PD-L1 mRNA levels, IMP3 and PD-L1 protein levels were detected in breast cancer and adjacent normal tissues by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Correlations between miR-34a-5p and IMP3 and PD-L1 mRNAs were analyzed by Pearson’ s test. Immunohistochemistry was used to detect IMP3, PD-L1, and leukocyte differentiation antigen 44 variant 6 (CD44v6) expression in breast cancer and normal tissues, and the relationships between IMP3 and PD-L1 expression and clinicopathological parameters were analyzed. Target gene prediction and dual luciferase reporter assay were used to verify the targeted regulation of miR-34a-5p on IMP3 and PD-L1. MCF7 cells were divided into control group, miR-NC group, miR-34a-5p group, miR-NC+pcDNA-NC group, miR-NC+IMP3 group, miR-NC+PD-L1 group, miR-34a-5p+pcDNA-NC group, miR-34a-5p+IMP3 group, and miR-34a-5p+PD-L1 group. Cell proliferation, migration, and invasion were determined by MTT, colony-formation, scratch, and Transwell chamber tests, respectively. Expression levels of IMP3, PD-L1, CD44v6, matrix metalloproteinase-2 (MMP-2), MMP-9, and E-cadherin were detected by Western blot. Results The biological databases showed that higher expression levels of IMP3 and PD-L1 in breast cancer tissues were associated with shorter survival (P<0. 05). Expression levels of miR-34a-5p were significantly lower while expression levels of IMP3 and PD-L1 mRNAs and proteins were significantly higher in breast cancer tissues than in normal tissues. miR-34a-5p expression was negatively correlated with IMP3 and PD-L1 mRNAs, respectively (P<0. 05). Immunohistochemistry showed positive expression of IMP3, PD-L1, and CD44v6 in breast cancer, with higher IMP3 and PD-L1 expression associated with later TNM stage, lower degree of tumor differentiation, and increased risks of lymph node and distant metastases (P<0. 05). Dual luciferase assay verified the targeted regulation of miR-34a-5p on IMP3 and PD-L1. The cell viability, number of clones formed, scratch-healing rate, cell invasion, and expression levels of IMP3, PD-L1, CD44v6, MMP-2, and MMP-9 protein were all significantly lower and E-cadherin expression was significantly higher in the miR-34a-5p group compared with the control group and miR-NC group. The cell viability, number of clones formed, scratch-healing rate, cell invasion, and expression levels of IMP3, PD-L1, CD44v6, MMP-2, and MMP-9 proteins were all significantly increased while expression of E-cadherin protein was significantly decreased in the miR-NC+IMP3 and miR-NC+PD-L1 groups compared with the miR-NC+pcDNANC group. The cell viability, number of clones formed, rate of scratch healing, cell invasion, and expression levels of IMP3, PD-L1, CD44v6, MMP-2, and MMP-9 protein were all significantly increased while E-cadherin expression was significantly decreased in the miR-34a-5p+IMP3 and miR-34a-5p+PD-L1 groups compared with the miR-34a-5p+pcDNANC group (P< 0. 05). Conclusions miR-34a-5p can inhibit the proliferation, migration, invasion, and epithelialmesenchymal transition of breast cancer cells by targeting the expression of IMP3 and PD-L1.
JIANG Liping, WANG Xia, PENG Xinhua. miR-34a-5p 通过靶向IMP3、PD-L1 对乳腺癌细胞生物学行为的影响[J].中国比较医学杂志,2023,33(7):67~77.复制