Overexpressing miR-873 suppressed proliferation and metastasis of CRC cells both and and methods

Overexpressing miR-873 suppressed proliferation and metastasis of CRC cells both and and methods. miR-873 expression was even lower than those without liver metastases (Figure 1B). And, the relationships of miR-873 expression with clinicopathological factors of CRC was shown in Table 1. The decrease of miR-873 expression was found to be significantly related to distant metastasis. However, no significant correlations were found between miR-873 Col11a1 expression and other factors including age, gender, clinical stage and lymph node metastasis. Interestingly, miR-873 levels in CRC cell lines with high metastatic potential (SW620, HCT116 and LoVo) were significantly lower than those cell lines with low metastatic potential (HCT8, SW480, LS174T, HT29 and RKO) and normal colon epithelial cell line NCM460 (Figure 1C). The AOM/DSS mouse model is a colitis-associated CRC model and the mouse model is a spontaneous CRC model. These two models can mimic most of the cases in human CRC progression [16C18]. We interrogated miR-873 expression in samples from these two kinds of mouse models. As shown in Figure 1D, miR-873 expression in tumor tissues from the AOM/DSS-administrated group was significantly lower than that in normal colon tissues from control group. Likewise, miR-873 expression was decreased in tumor tissues from mice compared with normal colon tissues from wild type mice (Figure 1E). These data indicated that miR-873 may be a tumor suppressor and is negatively correlated with the metastatic potential of CRC. Open in a separate window Figure 1 MiR-873 was downregulated in CRC clinical samples, mouse models and CRC cell lines. (A) qRT-PCR analysis of miR-873 levels in 55 paired CRC clinical specimens. (B) Corelation between miR-873 levels and the distant metastasis status of CRC samples. (C) qRT-PCR analysis of miR-873 levels in normal colon cell line and CRC cell lines with different metastatic potential. (D, E) qRT-PCR analysis of Clobetasol miR-873 expression in AOM/DSS mouse model (D) and mouse model (E). Data (mean SEM) are representative of three technique replicates. *< 0.05; **< 0.01; ***< 0.001. Table 1 Relationships between miR-873 expression levels with clinicopathological factors in CRC < 0.05 by Students significantly. Open in a separate window Figure 2 MiR-873 inhibits CRC cell proliferation, migration and invasion < 0.05; **< 0.01; ***< 0.001. Inhibition of miR-873 promotes CRC cell proliferation, migration and invasion < 0.05; **< 0.01; ***< 0.001. Overexpressing miR-873 suppresses CRC cell growth and liver metastasis gene, followed by infecting these two Luciferase-labeled cells Clobetasol with lentiviruses encoding the vector or pre-miR-873. Then, stable infected LoVo and HCT116 cells were subcutaneously injected into nude mice and bioluminescence imaging was performed after 4 weeks. As shown in Figure 4A, LoVo cells with miR-873 overexpression formed smaller tumors compared with control cells. We then isolated the xenograft tumors and found the weight of LoVo-miR-873 tumors was significantly decreased compared with LoVo-Control tumors (Figure 4A). Similarly, we observed ectopic expression of miR-873 in HCT16 cells also dramatically suppresses tumor growth (Figure 4B). And then, the expression of proliferation marker Ki67 in the isolated tumors was further detected. The proportion of Ki67-positive cells in tumors formed by miR-873 overexpressing cells were much lower than that in tumors formed by control cells (Figure 4C). Liver is the most vital target organ for metastatic CRC and liver metastasis is the direct cause of CRC death [21]. Thus, we further assessed the metastatic ability of miR-873-overexpressing cells by injecting them into nude mice intrasplenically to construct an experimentally metastatic model. Bioluminescence imaging results showed that LoVo (Figure 4D) and HCT116 (Figure 4E) cells with miR-873 overexpression formed less hepatic metastatic nodules which were validated by H&E staining of liver slices (Figure 4F). In summary, these above results indicated that miR-873 could inhibit CRC cell Clobetasol growth and metastasis < 0.05; **< 0.01. ELK1 and STRN4 are direct targets of miR-873 MiRNAs exert their biological roles by targeting the 3?UTR of mRNAs, resulting in mRNA degradation and/or translational inhibition. On the basis of above results which implied miR-873 may serve as a tumor-suppressive miRNA, we further aimed to identify its targets. Therefore, we applied the most common prediction algorithm TargetScan to mine the candidate targets of miR-873. And we selected seven oncogenes that have been proved to affect cell proliferation or/and mobility. The mRNA levels of CDK6, ELK1, MyoB1, STRN4, TRAF2 and WASF2 were revealed to be significantly decreased after ectopic expression of miR-873 in both LoVo and HCT116 cells (Figure 5A and ?and5B).5B). Moreover, we performed Dual Luciferase Reporter Assay to verify whether there.