| Objectives This study aims to investigate the effect and molecular mechanism of miR-10 a on the liver fibroblasts in the microenvironment of colon cancer liver metastasis,and reveal the effect of function change of liver fibroblasts induced by miR-10 a on colon cancer cells.This research will provide a new theoretical basis for the molecular mechanism of colon cancer liver metastasis.Methods 1 The liver stellate cell line LX-2(named TGF-LX-2)was treated with TGF-?1 for 48 hours.The expression of ?-SMA and miR-10 a in LX-2 cells and TGF-LX-2 cells were detected by qRT-PCR assay;miR-10 a were overexpressed in TGF-LX-2 cells.Then,the proliferation,migration abilites and secretion levels of pro-inflammatory factors IL-6 and IL-8 of each group cells were dectected by CCK-8 assay,transwell migration assay and ELISA assay,respectively.2 The conditioned medium of TGF-LX-2 cells overexpressed miR-10 a and its control group were collected to act on the colon cancer cell line SW480.Then,CCK-8 assay,wound assay transwell invasion assay,cell-matrix adhesion assay and sphere formation assay were used to evaluate the effects of the conditioned medium of the two groups on the proliferation,migration,invasion,adhesion abilities and cell stemness of SW480 cells.3 We cultured primary cell from normal liver tissue at a distance of ?5 cm from the periphery of the tumor and liver metastasis of colon cancer tissue in the same patient;Normal human liver fibroblasts(NHLF)and tumor associated human liver fibroblasts(THLF)were identified by morphological observation and immunofluorescence staining,and the purity of them was identified by flow cytometry;Then,qRT-PCR assay and ELISA assay were used to evaluate the expression of miR-10 a and secretion of pro-inflammatory factors in the two groups cells.4 miR-10 a were overexpressed in THLF cells.Then,the proliferation,migration abilites and expression levels of IL-6 and IL-8 of each group cells were dectected by CCK-8 assay,transwell migration assay and qRT-PCR assay,respectively.5 The conditioned medium of NHLF and THLF were collected to act on the colon cancer cell line SW480.Then,CCK-8 assay,wound assay transwell invasion assay,cell-matrix adhesion assay and sphere formation assay were used to evaluate the effects of the conditioned medium of the two groups on the proliferation,migration,invasion,adhesion abilities and cell stemness of SW480 cells.Results 1 Compared with the untreated group,the expression level of ?-SMA mRNA in LX-2 cells treated with TGF-?1 was significantly increased(t=5.168,P=0.007),while the expression level of miR-10 a was significantly decreased(t=6.469,P=0.003);Compared with the control group,the proliferation ability of the TGF-LX-2 cells in the overexpression miR-10 a group was significantly decreased(t=3.323,P=0.029;t=3.711,P=0.021;t=6.498,P=0.003),the number of cells passing through the transwell chamber was notably decreased(without matrixgel)(t=7.861,P=0.0014),and the secretion levels of IL-6 and IL-8 were decreased significantly(IL-6: t=7.415,P=0.002;IL-8: t=3.471,P=0.026).The results showed that miR-10 a can negatively regulate the proliferation,migration and pro-inflammatory factor secretion of TGF-LX-2 cells.2 Compared with the control group,the migration rate of SW480 cells treated with the conditioned medium of TGF-LX-2 cells overexpressing miR-10 a was significantly decreased(t=5.645,P=0.005;t=5.866,P=0.004),the number of cells passing through the transwell chamber were notably decreased(with matrixgel)(t=15.43,P=0.0001),and the spherical volume was decreased,however there was no significant difference in proliferation and adhesion(P >0.05 and P >0.05).The results showed that the inhibition of TGF-LX-2 cell activity mediated by miR-10 a could inhibit the metastasis of colon cancer cells.3 The primary cell lines NHLF exhibited a long fusiform shape in bundles or braids with consistent size,while THLF showed a flat fusiform shape with different?sizes and disorder growth;Immunofluorescence staining showed that FSP-1 was expressed in both cells,and ?-SMA was weakly expressed in NHLF but strongly expressed in THLF;The results of flow cytometry showed that the positive rates of ?-SMA in NHLF and THLF were higher than 95%;Compared with NHLF,the expression level of miR-10 a in THLF was significantly lower(t=4.638,P=0.009),and the secretion levels of IL-6 and IL-8 were increased significantly(IL-6: t=4.326,P=0.012;IL-8: t=6.486,P=0.003).The results showed that the primary cell lines were NHLF and THLF,and the expression level of miR-10 a in THLF was low,but more IL-6 and IL-8 were secreted,which confirmed that the low expression of miR-10 a was related to the activation of fibroblasts.4 Compared with the control group,the proliferation ability of the THLF cells in the overexpression miR-10 a group was significantly decreased(t=5.835,P=0.004;t=4.410,P=0.012;t=4.703,P=0.009),the migration rate was notably decreased(t=4.766,P=0.009;t=3.478,P=0.026),and the expression levels of IL-6 and IL-8 were decreased significantly(IL-6: t=8.151,P=0.001;IL-8: t=5.081,P=0.007).The results showed that miR-10 a can negatively regulate the proliferation,migration and pro-inflammatory factor expression of THLF cells.5 Compared with the NHLF group,the migration rate of SW480 cells treated with the conditioned medium of THLF was significantly increased(t=4.988,P=0.008;t=3.404,P=0.027),the number of cells passing through the transwell chamber were notably increased(with matrixgel)(t=7.477,P=0.002),and the spherical volume was increased,however there was no significant difference in proliferation and adhesion(P>0.05 and P>0.05).The results showed that tumor associated fibroblasts could promote the metastasis of colon cancer cells.Conclusions 1 Overexpression of miR-10 a inhibited the proliferation and migration of TGF-LX-2 cells and the release of pro-inflammatory factors IL-6 and IL-8.2 The conditional medium of TGF-LX-2 cells overexpression miR-10 a could inhibit the migration,invasion and stemness of SW480 cells,but had no effect on cell proliferation and adhesion.3 The expression level of miR-10 a in THLF was lower than that in NHLF,but the secretion of IL-6 and IL-8 were higher than that in NHLF.4 Overexpression of miR-10 a inhibited the proliferation and migration of THLF and the expression of pro-inflammatory factors.5 The conditional medium of THLF could promote the migration,invasion and stemness of SW480 cells,but had no effect on the proliferation and adhesion.In conclusion,miR-10 a could inhibit the activation of fibroblasts.It is an important factor in preventing liver metastasis of colon cancer cells.Figure 26;Table 0;Reference 74... |
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