Investigation of microRNA-10a suppressing the activity of tumor-associated fibroblasts from a patient with metastatic colon cancer to liver / 中华老年医学杂志

ABSTRACT

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-1β in 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-1 β were 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.