| Objectives This study aims to investigate the effect of exosomal-mi R-10 a on fibroblasts in the metastatic microenvironment of CRC and reveal the molecular mechanism,which will provide a new theoretical basis for the molecular mechanism of CRC metastasis and recurrence,as well as a new clue for the clinical diagnosis and treatment of CRC.Methods 1 We extracted venous blood from 20 healthy volunteers and 40 CRC patients(before surgery),and examined the expression of mi R-10 a in serums by real time fluorescene quantitative polymerase chain(RT-q PCR)assay,then analyzed the differential expression of mi R-10 a between CRC patients and healthy individuals.Subsequently,we randomly collected the cancer tissues and the paired normal intestinal wall tissues from 10 of 40 CRC patients,and analyzed the differential expression of mi R-10 a in tissues between the two groups.2 Establishment and identification of primary normal human lung fibroblasts(NHLF): We isolated and cultured NHLF from postoperative pathological tissues of patient with benign lung tumor.Flow cytometry and cellimmunofluorescence assays were used to detect the characteristic markers α-SMA and CK-18 of NHLF.3 The exosomes from SW480 cells,established from the primary CRC tissues,with mi R-10 a overexpression,transferred into NHLF.Then,CCK-8 assay assay was employed to detect the proliferation ability of CRC cells.Wound-healing assay and transwell assay were used to detect the migration ability of these cells.The expression levels of inflammatory cytokines(IL-6,IL-8,and IL-1β)of NHLF were detected by RT-q PCR assay.Results 1 mi R-10 a expression in venous blood from CRC patients was lower than that from healthy individuals(32/40)(P<0.001).Similarly,mi R-10 a expressed in the CRC tissues was significantly lower than in the paired normal intestinal wall tissues of the same patients(P<0.001).The difference of serous mi R-10 a from CRC patients was basically consistent with that of tissues(P<0.001).2 Cellular immunofluorescence assay showed that NHLF strongly expressed α-SMA in both cytoplasm and membrane,while weakly or not expressed in nucleus.CK-18 was weakly expressed in cell cytoplasm,membrane and nucleus in NHLF.The positive rates of NHLF expressing α-SMA and CK-18 were 98.0% and 69.3%,respectively.Flow cytometry assay indicated that the positive rates of NHLF expressing α-SMA and CK-18 in were 96.9% and 36.5%,respectively.3 CCK-8 assay,wound-healing assay and transwell assayshowed the proliferation and migration abilities of NHLF induced by exosomal-mi R-10 a for 48 h were significantly reduced(P<0.05).The expression levels of inflammatory cytokines IL-6,IL-8,and IL-1β of NHLF with the same treated were significantly down-regulated(IL-6: P<0.01,IL-8: P<0.05,IL-1β: P<0.01).Conclusions 1 mi R-10 a expression in venous blood from CRC patients was significantly lower than that from healthy individuals,and low expression of mi R-10 a in serums is associated with CRC.2 NHLF cell lines were established with high purity.3 mi R-10 a in exosomes derived from CRC cells inhibits cell proliferation and migration of NHLF,and down-regulates the expression levels of inflammatory cytokines IL-6,IL-8,and IL-1β of NHLF.In conclusion,The low expression of mi R-10 a in serum may be a potential biomarker for the prediction of colorectal cancer;mi R-10 a in exosomes derived from CRC cells in situ can inhibit cell proliferation,migration and secretion of IL-6,IL-8,and IL-1β of NHLF in the metastatic microenvironment,which may be an important factor contributes to preventing cancer metastasis.Figure4;Table2;Reference 101... |
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