宫颈脱落细胞microRNA-508-3p、ROCK1的表达及其对宫颈癌的诊断价值研究

Expression of microRNA-508-3p and ROCK1 in exfoliated cervical cells and their diagnostic values in cervical cancer

摘要:

目的 探究宫颈脱落细胞microRNA-508-3p（miR-508-3p）、ROCK1的表达及其对宫颈癌的诊断价值。方法 选取2016年2月—2019年9月诸暨市人民医院收集的宫颈脱落细胞标本172例，其中正常组70例、宫颈上皮内瘤变组54例、宫颈癌组48例。通过qRT-PCR、免疫组织化学、Western blotting检测各组细胞miR-508-3p、ROCK1的表达，荧光素酶报告实验验证宫颈病变脱落细胞miR-508-3p与ROCK1的相关性，观察宫颈癌脱落细胞miR-508-3p、ROCK1表达与宫颈癌患者临床资料的关系，通过受试者工作特征（ROC）曲线评估宫颈病变脱落细胞miR-508-3p、ROCK1表达对宫颈病变的诊断价值。结果 与正常组比较，宫颈癌组和宫颈上皮内瘤变组miR-508-3p降低（P <0.05），ROCK1 mRNA、ROCK1 mRNA阳性率、ROCK1蛋白相对表达量升高（P <0.05）；与宫颈上皮内瘤变组比较，宫颈癌组miR-508-3p降低，ROCK1 mRNA、ROCK1 mRNA阳性率、ROCK1蛋白相对表达量升高（P <0.05）。宫颈癌组、宫颈上皮内瘤变组miR-508-3p与ROCK1 mRNA呈负相关（r =-0.6678和-0.5234，均P =0.000），ROCK1是miR-508-3p的直接靶基因。miR-508-3p、ROCK1水平与宫颈癌患者FIGO分期、淋巴结转移有关（P <0.05），与年龄、绝经情况、肿瘤直径及病理类型无关（P >0.05）。在诊断宫颈癌和宫颈上皮内瘤变时，miR-508-3p的ROC曲线下面积（AUC）分别为0.946（95% CI：0.899，0.993）和0.851（95% CI：0.782，0.921），敏感性为87.1%（95% CI：0.776，1.654）和84.3%（95% CI：0.746，1.589），特异性为100.0%（95% CI：1.000，2.000）和79.6%（95% CI：0.702，1.497）；ROCK1的AUC分别为0.949（95% CI：0.892，1.000）和0.905（95% CI：0.852，0.958），敏感性为89.6%（95% CI：0.810，1.706）和77.8（95% CI：0.667，1.445），特异性为100.0%（95% CI：1.000，2.000）和88.6%（95% CI：0.812，1.698）。随访1年后，48例宫颈癌患者中生存42例，死亡6例。生存组miR-508-3p高于死亡组（P <0.05），ROCK1 mRNA相对表达量低于死亡组（P <0.05）。结论 宫颈脱落细胞miR-508-3p、ROCK1在宫颈癌患者中表达异常，其中miR-508-3p下调，ROCK1上调，两者呈靶向负调控关系，并与宫颈癌患者FIGO分期及淋巴结转移密切相关，具有一定的宫颈癌诊断价值，可作为早期宫颈癌筛查的潜在生物学指标。

关键词:宫颈脱落细胞;miR-508-3p;ROCK1;宫颈癌;诊断价值

Abstract:

Objective To investigate the expression of microRNA-508-3p (miR-508-3p) and Rho-associated protein kinase 1 (ROCK1) in exfoliated cervical cells and their diagnostic values in cervical cancer.Methods From February 2016 to September 2019, 172 cases of exfoliated cervical cells were collected in our hospital, including 70 cases in the normal group, 54 cases in the cervical intraepithelial neoplasia group, and 48 cases in the cervical cancer group. The expression of miR-508-3p and ROCK1 in each group was detected by qRT-PCR, immunohistochemistry, and Western blotting. Luciferase assay was used to verify the interaction between miR-508-3p and ROCK1 in the exfoliated cervical cells. The relationship between the expression of miR-508-3p and ROCK1 in the exfoliated cervical cells and the clinical data of cervical cancer patients were analyzed, and the diagnostic value of miR-508-3p and ROCK1 expression in the exfoliated cervical cells was evaluated via receiver operating characteristic (ROC) curve.Results Compared with the normal group, the content of miR-508-3p in cervical cancer group (P < 0.05) and cervical intraepithelial neoplasia group (P < 0.05) was decreased, while ROCK1 mRNA level, the positive rate of ROCK1 mRNA and ROCK1 protein content were increased (P < 0.05). Compared with the cervical intraepithelial neoplasia group, the content of miR-508-3p in the cervical cancer group was decreased (P < 0.05), while ROCK1 mRNA level, the positive rate of ROCK1 mRNA and ROCK1 protein content were increased (P < 0.05). MiR-508-3p was negatively correlated with ROCK1 in the cervical cancer group (r = -0.6678, P = 0.000) and the cervical intraepithelial neoplasia group (r =-0.5234, P = 0.000), and ROCK1 was the direct target gene of miR-508-3p. The relative expression levels of miR-508-3p and ROCK1 were correlated with FIGO stage and lymph node metastasis of cervical cancer (P < 0.05), but not with age and menopausal status of patients, or diameter and pathological type of tumor (P > 0.05). In the diagnosis of cervical cancer and cervical intraepithelial neoplasia, the area under the ROC curve (AUC) of miR-508-3p was 0.946 (95% CI: 0.899, 0.993) and 0.851 (95% CI: 0.782, 0.921), the sensitivity was 87.1% (95% CI: 0.776, 1.654) and 84.3% (0.746, 1.589), and the specificity was 100.0% (1.000, 2.000) and 79.6% (95% CI: 0.702, 1.497), respectively. The AUC of ROCK1 was 0.949 (95% CI: 0.892, 1.000) and 0.905 (95% CI: 0.852, 0.958), the sensitivity was 89.6% (95% CI: 0.810, 1.706) and 77.8% (95% CI: 0.667, 1.445), and specificity was 100.0% (95% CI: 1.000, 2.000) and 88.6% (95% CI: 0.812, 1.698), respectively. After 1-year follow-up, 42 cases survived and 6 cases died in 48 cases of cervical cancer. The expression level of miR-508-3p in the survival group was higher than that in the death group, and the expression level of ROCK1 mRNA in the survival group was lower than that in the death group (P < 0.05).Conclusions The expression of miR-508-3p and ROCK1 in exfoliated cervical cells of patients with cervical cancer is severely abnormal, in which miR-508-3p is downregulated while ROCK1 is upregulated. The miR-508-3p targets ROCK1 and negatively regulates ROCK1, and both of them are closely associated with the pathological staging and lymph node metastasis in cervical cancer patients. Besides, they are of diagnostic value and may be potential biomarkers for cervical cancer screening.

Key words:exfoliated cervical cells;miR-508-3p;ROCK1;cervical cancer;diagnostic value