Here in our study, we find miR-320a is commonly downregulated in CRC tissues compared with normal colonic epithelial. Restoration of miR-320a suppresses cell growth, clone formation ability, invasiveness of CRC cells in vitro, and tumor growth in xenografted mice model. miR-320a is predicted in silico and confirmed by luciferase reporter assay and western blotting, to target SP1 by binding to its mRNA 3’UTR. Rescue of SP1 expression in CRC cells could partly abrogate miR-320a-induced inhibition of cell behaviors, indicating that miR-320a exert tumor-suppressive role through at least partly, inhibiting SP1 activity. On the other hand, we show that SP1 induces downregulation of miR-320a by functioning as a transcription repressor binding to miR-320a promoter. Targeting SP1 using shRNA leads to restoration of miR-320a and concomitantly repression of cell growth, clone formation ability, invasiveness of CRC cells, which is partly antagonized upon miR-320a inhibitor treatment. Moreover, in mechanism analysis, we show that MACC1, previously described as a master regulator of oncogenic HGF/MET signaling [34], is a transcription target of SP1. By western blotting, we observe MACC1 expression is expectedly repressed along with SP1 inhibition induced by miR-320a. As downstream effectors of MACC1, MET expression and ERK1/2, AKT phosphor-activity are concomitantly observed being attenuated. Our data hence illustrate a double-negative feedback loop between SP1 and miR-320a in CRC cells, which retains miR-320a expressing at low level but elevating level of SP1, thus leading to malignant cell phenotypes through inducing enforced oncogenic MACC1/MET signaling.
Since most of coding mRNAs harbor different complementary seed sequences for miRNAs recognition, a cluster of mRNAs may be regulated by a single miRNA, and vice versa. Moreover, different miRNAs could co-target cooperatively a cluster of protein coding mRNAs with relevant function or belonging to a cellular pathway [35]. On the other hand, from the perspective of gene expression regulated by TFs, miRNA as a sort of non-coding genes, is presumably transcriptionally regulated by TFs. Therefore, a substantial number of regulatory interactions between TFs and miRNAs are presumably conserved across cancer entities. Recent studies have experimentally disclosed a number of TF-miRNA reciprocities involved in tumorigenesis of multiple cancers. For example, miR-23 ~ 24 ~ 27 cluster, upregulated as an oncogene in breast cancer, can target directly HIC, while HIC can in turn repress miR-23 ~ 24 ~ 27 cluster transcription, thus indicating a double negative feedback loop that promote tumor growth [36]. In liver cancer, EZH2 is engaged a reciprocal negative circuit with miR-101-1, thus attenuating tumor-suppressive role by inducing downregulation of miR-101-1 [37]. In addition, interplay that implicates more than a single TF or miRNA, is often described in diverse cancerous processes, especially in EMT-regulating mechanism. It has been reported that EMT-inducing TFs such as ZEB1/2, Snail, Slug, etc., often form reciprocal regulatory network with some certain miRNAs consist of miR-34, miR-200 and miR-17-92 cluster [38]. Take miR-200 family for example: p53-inducible miR-200 family is the first identified EMT-inhibiting miRNAs, mainly through targeting EMT-inducing TFs ZEB1/2 by posttranscriptional modification, whereas ZEB1/2 can bind to promoter of miR-200 family thus directly repressing expression of them, which means frequent loss of p53 and/or miR-200 family in tumor shift the equilibrium of the reciprocal regulation among p53, miR-200 and ZEB1/2, towards mesenchymal state thereby confer tumor cell with disseminated feature [39].
It is well characterized that SP1 and its modification (phosphorylation, acetylation, glycosylation) are crucial for maintaining cell homeostasis and early development of embryo by retaining basal transcriptional machinery [40]. We therefore speculate SP1 and miR-320a reciprocity represent a manner of sustaining conventional adjustive activity of SP1 and/or miR-320a in normal cellular processes. However, in tumor context, the equilibrium may be shifted by certain tumor-related stimuli, thus giving rise to elevating level and activity of SP1, and miR-320a expressing in low level, eventually promoting malignant feature of tumor cells. This role change in question implies SP1 a non-oncogene addiction (NOA) gene [40], and underscores one of causes for miR-320a downregulation in CRC.
MACC1 has been established as a key player and biomarker in tumor progression first in CRC [34], then across multiple tumor entities [41]. Recent mechanism studies have unveiled possible upstream transcriptional regulation of MACC1 by β-catenin, YB1 [42, 43], while TWIST1/VEGFA, Nanog/Oct4 axis, and SPON2 [44–46] as its downstream effectors, hence illustrating a MACC1-centered regulatory network in tumor.
In our study, we newly describe a regulatory approach upstream MACC1. It is illustrated that, deregulation of SP1 in CRC due to disequilibrium of SP1/miR-320a negative reciprocity, promotes MACC1 transcription, thus leading to activation of MACC1-mediated c-MET and downstream signaling pathway, eventually influencing malignant features of CRC (Fig. 7). Targeting either SP1 or miR-320a may presumably represent potential strategy to prevent MACC1-driven tumor progression.