microRNA-10a抑制结肠癌肝转移肿瘤相关成纤维细胞活性的研究

目的探讨microRNA-10a(miR-10a)对肝脏微环境中肿瘤相关成纤维细胞(TAFs)的增殖、迁移以及促炎性因子白细胞介素6(IL-6)、白细胞介素8(IL-8)、白细胞介素1(IL-1)βmRNA表达水平的影响。方法收集同一结肠癌患者癌旁正常肝组织和转移至肝脏的病灶组织,采用组织块法建立原代人肝正常成纤维细胞(NFs)和原代TAFs。通过形态学观察和细胞免疫荧光染色对NFs和TAFs进行鉴定,并通过流式细胞术鉴定二者的纯度。用实时荧光定量聚合酶链反应(RT-qPCR)检测NFs和TAFs中miR-10a的表达水平,并在低表达miR-10a的细胞中过表达miR-10a。随后采用CCK-8实验、划痕实验和RT-qPCR分别检测miR-10a对该细胞增殖、迁移能力以及IL-6、IL-8、IL-1βmRNA表达水平的影响。结果细胞免疫荧光染色显示人细胞角蛋白18(CK-18)在NFs和TAFs中均不表达;成纤维细胞特异性蛋白-1(FSP-1)在两细胞中均表达;α-平滑肌肌动蛋白(α-SMA)在NFs中弱表达,在TAFs中强表达。流式细胞术显示NFs与TAFs中α-SMA阳性率为95.0%和95.3%。miR-10a在TAFs的表达水平为NFs的0.65倍(P<0.01)。过表达miR-10a后TAFs在第3、4和5天增殖能力显著低于同时期的阴性对照组细胞(P<0.05,P<0.05,P<0.01);TAFs的迁移能力在24 h和48 h分别较阴性对照组降低25%和15%(P<0.01,P<0.05),TAFs中IL-6、IL-8、IL-1β的表达水平分别较阴性对照组降低54%、27%和42%(P<0.01,P<0.01,P<0.05)。结论miR-10a在TAFs中呈低表达,miR-10a过表达抑制了TAFs的增殖和迁移,并降低炎性因子IL-6、IL-8、IL-1βmRNA的表达,这可能是TAFs抑制肝脏形成转移灶的重要因素。

Investigation of microRNA-10a suppressing the activity of tumor-associated fibroblasts from a patient with metastatic colon cancer to liver

Objective To investigate the effects of microRNA-10a(miR-10a)on the proliferation and migration of tumor-associated fibroblasts(TAFs)in the liver microenvironment,as well as on the mRNA expressions of interleukin(IL)-6,IL-8 and IL-1βin TAFs.Methods The normal liver tissues adjacent to cancer and focal tissues of metastatic colon cancer to the liver from the same patient were collected,and then primary normal fibroblasts(NFs)and the primary cell line of TAFs were established by tissue cultivation.The NFs and TAFs were identified by morphological observation and immunofluorescence staining,and their purity was determined by flow cytometry.The real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-10a in NFs and TAFs,and then miR-10a was over-expressed in the lower ones.Subsequently,the effects of miR-10a on cell proliferation,migration and the mRNA expression levels of IL-6,IL-8 and IL-1β were detected by the cholecystokinin(CCK-8)test,wound healing assay and RT-qPCR.Results Immunofluorescence staining showed that human cytokeratin 18(CK-18)was neither expressed in NFs nor in TAFs,while fibroblast-specific protein 1(FSP-1)was expressed in NFs and TAFs,and alpha-smooth muscle actin(α-SMA)was weakly expressed in NFs but strongly expressed in TAFs.The results of flow cytometry showed that the positive rates of α-SMA in NFs and TAFs were 95.6%and 95.3%,respectively.The mRNA expression of miR-10a in TAFs was 0.65 times of that in NFs(P<0.01).After overexpression of miR-10a,the proliferation abilities at the 3th,4th and 5th day were lower in TAFs than in NFs(P<0.05 and 0.01),the migration abilities at 24 h and 48 h were 25%and 15%lower in TAFs than in NF group(P<0.01 and 0.05),and the mRNA levels of IL-6,IL-8,IL-1βwere 54%,27%and 42%lower in TAFs than in NFs,respectively(P<0.01,0.01 and 0.05).Conclusions The overexpression of miR-10a in TAFs inhibits the cell proliferation and migration and reduces the mRNA expressions of inflammatory factors IL-6,IL-8 and IL-1β,which may be an important factor for TAFs’inhibiting liver metastasis.