miR-325-3p和FOXP3在结直肠癌组织中的 表达及临床意义

Expression and Clinical Significance of miR-325-3p and FOXP3 in Colorectal Cancer Tissues

摘要: 目的：探讨微小RNA-325-3p (miR-325-3p)与叉头框蛋白P3 (FOXP3)在结直肠癌(CRC)组织中的表达情况，分析两者表达与临床病理特征的关系，探索两者在CRC发生和发展中的临床意义及潜在内在联系。方法：采用实时荧光定量逆转录PCR (qRT-PCR)技术检测30例CRC患者癌组织与配对癌旁组织中miR-325-3p和FOXP3的表达水平；将HCT116结直肠癌细胞分别转染miR-325-3p mimics (过表达组)和miR-325-3p inhibitor (抑制组)，通过CCK-8实验、Transwell实验检测miR-325-3p对CRC细胞增殖、侵袭及迁移能力的影响；利用生物信息学软件预测miR-325-3p与FOXP3的靶向关系，结合双荧光素酶报告基因检测实验验证两者靶向关联；采用SPSS20.0软件分析miR-325-3p、FOXP3表达与CRC患者临床病理特征的相关性，通过Spearman相关性分析法分析两者表达水平的相关性。结果：① miR-325-3p在CRC癌组织中的相对表达量(0.630 ± 0.119)显著低于癌旁组织(1.109 ± 0.206) (P < 0.05)，其表达与患者淋巴结转移、分化程度及TNM分期呈负相关(P < 0.05)，与性别、年龄、肿瘤大小无显著相关性(P > 0.05)；② FOXP3在CRC癌组织中的相对表达量(4.893 ± 1.753)显著高于癌旁组织(1.168 ± 0.371) (P < 0.01)，其表达与患者TNM分期、分化程度、肿瘤大小及淋巴结转移呈正相关(P < 0.05)，与性别、年龄无显著相关性(P > 0.05)；③ CCK-8实验显示，过表达miR-325-3p可显著抑制CRC细胞增殖，抑制miR-325-3p表达可促进CRC细胞增殖；Transwell实验显示，过表达miR-325-3p可显著抑制CRC细胞侵袭、迁移能力，抑制miR-325-3p表达可促进CRC细胞侵袭、迁移；④ 生物信息学预测及双荧光素酶报告基因检测证实，miR-325-3p可直接靶向结合FOXP3的3’UTR特定位点；⑤ Spearman相关性分析显示，CRC组织中miR-325-3p与FOXP3表达水平呈显著负相关(P < 0.01)。结论：CRC组织中miR-325-3p低表达、FOXP3高表达，两者呈显著负相关，且均与CRC患者临床病理特征密切相关；miR-325-3p可能通过直接靶向负向调控FOXP3表达，参与CRC的发生、侵袭及迁移过程，可为CRC的靶向治疗及疾病监测提供新的分子靶点和研究思路。

Abstract: Objective: To investigate the expression of microRNA-325-3p (miR-325-3p) and forkhead box protein P3 (FOXP3) in colorectal cancer (CRC) tissues, analyze the relationship between their expression and clinicopathological features, and explore their clinical significance and potential internal relationship in the occurrence and development of CRC. Methods: The expression levels of miR-325-3p and FOXP3 in cancer tissues and paired adjacent tissues of 30 patients with CRC were detected by real-time fluorescence quantitative reverse transcription PCR (qRT-PCR). HCT116 colorectal cancer cells were transfected with miR-325-3p mimics (overexpression group) and miR-325-3p inhibitor (inhibition group), respectively. The effects of miR-325-3p on the proliferation, invasion and migration of CRC cells were detected by CCK-8 assay and Transwell assay. The targeting relationship between miR-325-3p and FOXP3 was predicted by bioinformatics software, and the targeting relationship between miR-325-3 p and FOXP3 was verified by dual luciferase reporter gene assay. SPSS20.0 software was used to analyze the correlation between the expression of miR-325-3p and FOXP3 and the clinicopathological features of CRC patients. Spearman correlation analysis was used to analyze the correlation between the expression levels of the two. Results: ① The relative expression of miR-325-3p in CRC tissues (0.630 ± 0.119) was significantly lower than that in adjacent tissues (1.109 ± 0.206) (P < 0.05). The expression of miR-325-3p was negatively correlated with lymph node metastasis, differentiation degree and TNM stage (P < 0.05), but not with gender, age and tumor size (P > 0.05). ② The relative expression of FOXP3 in CRC tissues (4.893 ± 1.753) was significantly higher than that in adjacent tissues (1.168 ± 0.371) (P < 0.01). The expression of FOXP3 was positively correlated with TNM stage, differentiation degree, tumor size and lymph node metastasis (P < 0.05), but not with gender and age (P > 0.05). ③ CCK-8 assay showed that overexpression of miR-325-3p could significantly inhibit the proliferation of CRC cells, and inhibition of miR-325-3p expression could promote the proliferation of CRC cells. Transwell experiments showed that overexpression of miR-325-3p could significantly inhibit the invasion and migration of CRC cells, and inhibition of miR-325-3p expression could promote the invasion and migration of CRC cells. ④ Bioinformatics prediction and dual luciferase reporter gene detection confirmed that miR-325-3 p could directly target the specific site of FOXP3 3’UTR. ⑤ Spearman correlation analysis showed that there was a significant negative correlation between miR-325-3p and FOXP3 expression in CRC tissues (P < 0.01). Conclusion: The low expression of miR-325-3p and the high expression of FOXP3 in CRC tissues are significantly negatively correlated, and both are closely related to the clinicopathological features of CRC patients; miR-325-3p may be involved in the occurrence, invasion and migration of CRC by directly targeting and negatively regulating FOXP3 expression, which can provide new molecular targets and research ideas for targeted therapy and disease monitoring of CRC.