Colorectal cancer is one of the most common and deadly cancers that is caused by many genetic and environmental factors(Aran, Victorino et al. 2016, Rawla, Sunkara et al. 2019). Therefore, identifying genetic factors can help us understand the gene network that is involved in cancer formation. MiRNA as one of the regulatory factors for gene expression can play important roles in regulating this gene network(Plaisier, Pan et al. 2012).
Others have shown that miR-3120 regulates Wnt signaling pathway through direct targeting of Axin2 gene (Hongdan, Feng et al. 2018). It also upregulates the expression of Sox2, Nanog and Oct4 genes stemness markers and increases the invasion of tumor cells and is considered as an oncomiR(Hongdan, Feng et al. 2018). Interestingly, this microRNA is a mirror microRNA for hsa-miR-214 (Scott, Howarth et al. 2012). No such mirror genes were reported before, and the presence of these two microRNAs with nearly identical 3p and 5p sequences due to the palindrome sequence of this region of genome suggests that the expression of these two microRNAs and their target genes may be influenced by each other's effects. In the present study, using bioinformatics and experimental approaches, we tried to investigate the molecular and cellular effects of miR-3120 and its mirror miRNA (miR-214) on colorectal cancer.
Microarray data analysis showed that miR-3120 is up-regulated in colorectal cancer tumors compared with normal groups. However, in contrary to miR-3120 expression, miR-214 expression is down-regulated in colorectal tumors. Consistently, expression analysis of these two miRNAs in three CRC cell lines (HT29, HCT116 and SW480), showed opposite expression pattern for miR-3120 and miR-214. Also, RT-qPCR result indicated that miR-3120 and miR-214 affects each other’s expression. This data is in consistent with previous data, which shows that the antisense gene can affect the expression of the sense gene(Su, Xiong et al. 2010, Balbin, Malik et al. 2015).
Different bioinformatics databases were used to find the molecular pathways regulated by miR-3120 and miR-214. Some of the predicted target genes were specific to the miR-3120, some were specific to miR-214, and some were common between the two miRNAs. To evaluate the effect of miR-3120/miR-214 on the predicted target genes expression, overexpression strategy was applied. The results of RT-qPCR of the genes expression showed that in most cases, these two miRNAs have distinctive effects on the target genes expression. Overexpression of miR-3120 in HCT-116 cell line followed by qRT-PCR indicated increased expression of SMAD3, SMAD4 and AKT2 genes, whereas overexpression of miR-214 inversed this effect. Previous studies have shown that miR-3120(Scott, Howarth et al. 2012, Hongdan, Feng et al. 2018) and miR-214(Penna, Orso et al. 2015) displays specific and even contrasting functions in different tumor types by differential mRNA targeting. Thus, these mirror miRNAs may play different roles according to co-expression with its own direct or indirect target genes such as SMAD3, SMAD4 and AKT2. To investigate cellular effect of miR-3120/miR-214 colorectal cancer, cell cycle analysis was performed and the result showed that forced expression of miR-3120 and miR-214, decreased the number of cells in phase G1 and increased the number of cells in phase S. In other word, these miRNAs promote cell cycle progression in HCT116 cells.
According to the evidence obtained in this study, with respect to miR-3120 target genes and its increased expression in colon cancer compared to normal, this miRNA seems to have an oncomeric role. These data suggest that miR-3120 and its mirror miRNA, miR-214, may be involved in regulating the molecular pathways of colorectal cancer, and part of this regulation could be related to the interaction of these miRNAs with each other. Further studies are needed to investigate the exact molecular mechanism of these two mirror miRNAs in cancer.