We were particularly interested in investigating the effect of miR-31 on the cellular response to 5-FU. Our results showed that although the suppression of miR-31 alone had no effect on cell proliferation, it did increase the sensitivity to 5-FU in HCT-116 cells as early as 24 h after exposure. At the same time point, combined treatment by 5-FU plus negative control did not significantly reduce the viability of the cells. With prolongation of exposure to 5-FU, both the combined groups, either 5-FU plus negative control or anti-miR-31, significantly reduced the cells viability. The apoptosis rate in the anti-miR-31 plus 5-FU group was the highest among the four different treated groups of either cell line, indicating that the anti-miR-31 plus 5-FU inhibited the cells proliferation partly through the apoptotic mechanism. However, the increase in the sensitivity of HCT-116 cells to 5-FU is quite modest and further studies are required to validate this phenomenon on more colon cancer cell lines, and even on other kinds of cancer cell lines.
Accumulating evidence suggests that miRNAs could be key players in regulation of tumor cell invasion and metastasis . Our previous study showed that miR-31 expression was up-regulated in CRC compared to normal mucosa, and tumors which invaded adjacent tissues or organs had more miR-31 expression than those limited to the wall of the colon and rectum . It was reasonable to speculate that miR-31 might affect the migration or invasion in CRC.
In the present study, the suppression of miR-31 caused a reduction of the migratory cells by nearly 50% compared with the negative control in both HCT-116p53+/+ and HCT-116p53-/- cell lines. This was in line with a provious study that blockage of miR-31 expression significantly decreased viability and migration in a HNSCC cell line . Unexpectedly, our results showed that the invasive cells were increased by 8-fold in HCT-116p53+/+ cell line, and 2-fold in HCT-116p53-/- cell line. In HCT-116 cells, suppression of miR-31 decreased migration but increased invasion in vitro, this phenomenon might be partly explained by the fact that the migration assay detected the cell motility through an 8 μm pore polycarbonate membrane; however, the invasion assay not only evaluated cell motility but also the ability of tumor cells to invade through a basement membrane model (a reconstituted basement membrane matrix of proteins, including laminin and type IV collagen, derived from the Eangelbreth Holm-Swarm (EHS) mouse tumor ). So those cells that were capable of completing the entire process represented only a small subpopulation of the cells compared with the migration assay.
MiR-31 is down-regulated in serous ovarian cancer and miR-31 overexpression inhibits proliferation and induces apoptosis in a number of serous cancer cell lines with a dysfunctional p53 pathway. However, in other lines with functional p53, miR-31 has no effect . On the other hand, overexpression of miR-31 suppresses metastasis in breast cancer cell lines, and does not affect proliferation in vitro . In CRC, miR-31 was significantly up-regulated  and our present data showed that suppression of miR-31 increased the sensitivity of HCT-116 cells to 5-FU at an early stage, affected cell migration and invasion. Taken together, it seems that not only the expression but also the functions of miR-31 are cancer specific.
MiRNAs have been reported to be directly transactivated by p53; equally, p53 and components of its pathway have been shown to be targeted by miRNA thereby affecting p53 activities . We asked whether miR-31 had a relationship with p53 in CRC. The HCT-116p53+/+ and HCT-116p53-/- cell lines are suitable to investigate the relationship between miRNAs and p53. Our results showed that the suppression of miR-31 had almost the same effects on cell cycle and colony formation. However, as for the cell invasion, it had a stronger effect on HCT-116p53+/+ than HCT-116p53-/- cell line, the apoptotic rate in HCT-116p53+/+ was lower than that in HCT-116p53-/- cell line, suggesting miR-31 affected the colon cancer partly in a p53 dependent manner. In serous ovarian carcinomas, miR-31 is underexpressed, and, in a number of serous cancer cell lines with a dysfunctional p53 pathway, miR-31 overexpression inhibits proliferation and induces apoptosis; however, in other lines with functional p53, miR-31 has no effect . Taken together, it suggests that miR-31 may regulate different processes in different cancers, dependent on the cell of origin of the cancer.
The lack of knowledge about the targets for miR-31 in CRC hampers a full understanding on the biological functions of miR-31. Bioinformatic analysis reveals that miR-31 can control the expression of more than one hundred genes (miRBASE, http://microrna.sanger.ac.uk). It may be expected that the targets of miR-31 belong to the class of tumor suppressor genes or genes encoding proteins with potential tumor suppressor functions. BAP1, a tumor suppressor gene that functions in the BRCA1 growth pathway ; HIF1AN, inhibits HIF1A transcriptional activity ; MAPK transduction protein such as MAP4K5; RAS homologues RAB14, RAB6B, and RASA1 were found as potential targets of miR-31. MiR-31 is also up-regulated in HNSCC and lung cancer. More recently, factor-inhibiting hypoxia-inducible factor (FIH) was experimentally verified as the target of miR-31 in HNSCC cell and the tumor-suppressive genes, large tumor suppressor 2 (LATS2) and PP2A regulatory subunit B alpha isoform (PPP2R2A) were the targets of miR-31 in lung cancer [11, 14]. Whether these experimentally verified targets in other cancers are validated in CRC or there are other special targets in CRC need to be answered by further studies.