miR-29a靶向调控COL6A6对垂体腺瘤细胞增殖和侵袭的影响及其与患者预后的相关性

Effect of miR-29a targeting COL6A6 on the proliferation and invasion of pituitary adenoma cells and its correlation with prognosis of patients

摘要:

目的 探讨miR-29a靶向调控胶原蛋白VIA6(COL6A6)在垂体腺瘤(PA)发生、发展过程中的作用机制及其与患者预后的相关性。方法 选取2022年3月—2023年3月昆明医科大学第一附属医院鼻颅底外科收治的非侵袭性垂体腺瘤(NIPA)患者25例和侵袭性垂体腺瘤(IPA)患者25例。采用逆转录实时荧光定量PCR(RT-qPCR)、Western blot检测两组患者PA组织及正常垂体细胞、PA细胞系(AtT-20、HP75)中miR-29a、COL6A6的表达水平，将miR-29a mimic、miR-29a inhibitor和对照miRNA mimic、miRNA inhibitor分别转染至PA细胞系中，通过RT-qPCR检测转染后miR-29a、COL6A6的表达水平；双荧光素酶报告基因实验验证miR-29a与COL6A6之间存在结合位点关系；CCK-8检测细胞增殖能力；使用SPSS 27.0统计软件分析miR-29a表达差异与IPA的相关性以及其临床诊断预后价值。结果 miR-29a在IPA组织中的表达显著高于NIPA组织，差异具有统计学意义(P<0.001)；与正常垂体细胞(MPC)相比，miR-29a在PA细胞系(AtT-20、HP75)中均呈高表达，差异具有统计学意义(P<0.01)；miR-29a与COL6A6呈负相关的靶向结合关系。miR-29a过表达可促进PA细胞的增殖与侵袭；miR-29a的表达水平与肿瘤大小(χ²=4.056，P=0.021)、Knosp分级(χ²=25.873，P<0.001)、肿瘤全切率(χ²=4.903，P=0.027)密切相关。ROC曲线分析显示，miR-29a表达水平评估PA的不良预后具有一定价值(AUC=0.779，95%CI：0.638~0.920)；Kaplan-Meier生存曲线显示，miR-29a低表达组与高表达组患者复发率分别为13.0%、51.9%，差异具有统计学意义(HR=3.882，95%CI：1.45~10.39，P=0.0069)；多因素非条件Logistic回归分析显示，肿瘤全切率(OR=0.009，95%CI：0.001~0.184，P=0.002)、miR-29a表达水平(OR=15.325，95%CI：1.147~204.716，P=0.039)是影响PA患者预后的独立危险因素。结论 miR-29a通过靶向调控COL6A6促进PA细胞增殖与侵袭，这一靶向调控通路可能是PA发生、发展及侵袭性表型形成的重要分子机制；同时，miR-29a是影响PA患者预后的独立危险因素，对评估患者不良预后具有可靠价值，可能为该疾病的诊断和治疗提供新的分子标志物和治疗靶点。

Abstract:

Objective To explore the mechanism by which miR-29a targets and regulates collagen type VI alpha 6 chain (COL6A6) in the occurrence and development of pituitary adenoma (PA) and its correlation with patient prognosis. Methods Twenty-five patients with non-invasive pituitary adenoma (NIPA) and 25 patients with invasive pituitary adenoma (IPA) who were treated in the Department of Rhinocranial Base Surgery, the First Affiliated Hospital of Kunming Medical University from March 2022 to March 2023 were selected. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the expression levels of miR-29a and COL6A6 in PA tissues of the two groups, as well as in normal pituitary cells (MPC) and PA cell lines (AtT-20, HP75). miR-29a mimic, miR-29a inhibitor, control miRNA mimic and control miRNA inhibitor were transfected into PA cell lines, and RT-qPCR was used to detect the expressions of miR-29a and COL6A6. Dual-luciferase reporter gene assay was performed to verify the binding site relationship between miR-29a and COL6A6. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation ability. SPSS 27.0 was used for statistical analysis of the correlation between the differential expression of miR-29a and IPA, as well as the prognostic value of clinical diagnosis. Results The expression of miR-29a in IPA tissues was significantly higher than that in NIPA tissues, and the difference was statistically significant (P<0.001). Compared with MPC, miR-29a was highly expressed in PA cells (AtT-20, HP75), and the difference was statistically significant (P<0.01). There was a negatively correlated targeted binding relationship between miR-29a and COL6A6. Overexpression of miR-29a could promote the proliferation and invasion of PA cells. The expression of miR-29a was closely associated with tumor size (χ²=4.056, P=0.021), Knosp grade (χ²=25.873, P<0.001) and total tumor resection rate (χ²=4.903, P=0.027). Receiver operating characteristic (ROC) curve analysis showed that the expression level of miR-29a had a certain value in evaluating the poor prognosis of PA (AUC=0.779, 95%CI: 0.638-0.920). Kaplan-Meier survival curve showed that the recurrence rate of patients in the low-expression group of miR-29a and the high-expression group was 13.0% and 51.9%, respectively, and the difference was statistically significant (HR=3.882, 95%CI: 1.45-10.39, P=0.0069). Multivariate unconditional logistic regression analysis showed that total tumor resection rate (OR=0.009, 95%CI: 0.001-0.184, P=0.002) and miR-29a expression level (OR=15.325, 95%CI: 1.147-204.716, P=0.039) were independent risk factors affecting the prognosis of PA patients. Conclusions miR-29a promotes the proliferation and invasion of PA cells by targeting and regulating COL6A6, and this targeted regulatory pathway may serve as an important molecular mechanism for the occurrence, development, and formation of the invasive phenotype of PA. Meanwhile, miR-29a serves as an independent risk factor affecting patient prognosis in PA, providing reliable value for predicting poor outcomes and potentially offering a novel molecular marker and therapeutic target for the diagnosis and treatment of this disease.