Downregulated miR-495-3p in colorectal cancer targets TGFβR1, TGFβR2, SMAD4 and BUB1 genes and induces cell cycle arrest CURRENT STATUS: POSTED

• Background: Hsa-miR-495 (miR-495) has been extensively investigated in cancer initiation and progression. On the other hand, our bioinformatics analysis suggested that miR-495 exert its effects through targeting of TGFβ signaling components. Methods In order to investigate such effect, miR-495 precursor was overexpressed in HEK293T and HCT116 cells, that it was followed by downregulation of TGFβR1, TGFβR2, SMAD4 and BUB1 putative target genes, detected by RT-qPCR. Also, luciferase assay supported direct interaction of miR-495 with 3’UTR sequences of TGFβR1, TGFβR2, SMAD4 and BUB1 genes. Furthermore, a negative correlation of expression between miR-495-3p and these target genes was deduced in a set of colorectal and breast cancer cell lines. Then, flow cytometry analysis showed that the overexpression of miR-495 in HCT116 and HEK293T resulted in an arrest at the G1 phase. Consistently, western blotting analysis showed a significant reduction of the Cyclin D1 protein in the cells overexpressing miR-495, pointing to downregulation of TGFβ signaling pathway and cell cycle arrest. Finally, microarray data analysis showed that miR-495-3p is significantly downregulated in colorectal tumors, compared to the normal pairs. as a cell cycle progression suppressor, regulates TGFβR2, SMAD4 and BUB1 genes. This finding suggests miR-495-3p as a tumor suppressor for further


Introduction
MicroRNAs are small non-coding RNAs that regulate gene expression post-transcriptionally [1]. After being transcribed and processed in the nucleus and cytoplasm, miRNAs are loaded into a complex called the miRISC (miRNA-induced silencing complex), and guide it to the 3'UTRs their target transcripts leading to their degradation or translational inhibition [2]. One miRNA can target even hundreds of genes, and a single gene transcript could be targeted by various miRNAs [3]. This characteristic makes the mechanism of regulating genetic expression by miRNAs an extremely complex process and requires the discovery of all the target genes of each miRNA to determine its functions in the various physiological and pathological contexts. miR-495 is a member of the human 14q32.31 miRNA cluster that encodes a set of important miRNAs [4]. Various studies have been done to investigate the role of miR-495-3p in cancer progression [4]. However, the role of this miRNA in this field is still controversial. Wang et al. reported a downregulation of miR-495-3p expression in breast tumors and showed that this miRNA inhibits G1-S phase transition of breast cancer cells by targeting the BMI-1 oncogene [5]. Li et al. showed that miR-495-3p is downregulated in prostate cancer and regulates migration and invasion in prostate cancer cells by targeting AKT and mTOR transcripts [6]. In contrast, Lin et al. analysis revealed that miR-495-3p expression level is elevated in colon tumors and confirmed an oncogenic role for this miRNA exerted by targeting the WIF1 gene, a Wnt/β-catenin signaling pathway inhibitor [7]. The various roles of miR-495 in cancer progression have been reviewed recently by Chen et al. [4].
TGFβ (transforming growth factor β) signaling pathway plays critical roles in cell biology including cell growth, differentiation, apoptosis, homeostasis and other cellular functions [8]. Canonical TGFβ signaling involves the activation of R-SMADs and coSMADs that form heterodimeric complexes and translocate into the nucleus where they regulate the expression of target genes [9]. The non-canonical branch of this pathway includes the 4 activation of Mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) signaling pathways [9]. Canonical and non-canonical modes of TGFβ signaling exert opposite effects on the cellular fates, with the first inhibits cellular proliferation and survival while the latest activates these processes [10]. The BUB1 protein has been recently introduced as an important TGFβ signaling component that is responsible for stabilizing the interaction between TGFβR1 and TGFβR2 receptors and R-SMADs in the cytoplasm [11]. In addition, BUB1 is known to plays an essential role in promoting proper chromosome alignment during cell preparation for division [12].
In the current study, we intended to perform additional investigations concerning the role of miR-495-3p by focusing on its potential uncovered target genes. To this aim, several bioinformatics software was used to predict the potential targets of this miRNA. Additional gene-ontology enrichment analysis of the obtained target lists revealed that a set of the predicted genes represents key components of the TGFβ signaling pathway, including the BUB1 gene. Considering the dual role played by TGF-β signaling during cancer initiation and progression, and its implication in various developmental processes, we selected these genes for further experimental validations as important potential targets for miR-495-3p.

Bioinformatics analysis
Prediction of miR-495-3p target genes was performed using TargetScan [13] and DIANAmicroT [14,15] web servers. RNAhybrid web server [16] was used to predict the interaction strength between the miR-495-3p and the selected target genes.

Vector construction
The genomic region containing pre-miR-495, in addition to the target genes' 3'UTRs regions containing the predicted MREs of miR-495-3p were PCR-amplified using pairs of specific primers (primers listed in table 1). PCR products were cloned into pTG19-T vector, and later sub-cloned into the mammalian expression vector pEGFPC1 (pre-miRNA) or

RNA and protein extraction
Total cellular RNA was extracted using RiboX reagent according to the manufacturer's instructions. Protein extraction was performed using the RiboX-precipitated cell extracts according to the protocol recently described by Kopec et al. [17]. Quantitative assignment of the extracted proteins was done using Bradford assay [18].

cDNA synthesis and qPCR analysis
Poly-A tailing was performed by incubating 1 µg of total RNA with 2.5 U Poly-A polymerase (NEB, UK) and 2 µl of 10 mM ATP at 37 o C for 30 minutes. Polyadenylated or nonpolyadenylated RNA were subjected to DNase I treatment (Takara, Japan) at 37 o C for 30 min, followed by heat inactivation of the enzyme at 72 o C for 10 minutes. After that, cDNA synthesis was performed by utilizing PrimeScript II reverse transcriptase (Takara, Japan) and anchored or universal oligo-dT universal primers (primers shown in Table 1). Real-time PCR was done using the StepOne™ system (Applied Biosystems, USA) and SYBR Premix Kit (Takara Biotechnology, Japan). Primers used in the PCRs are presented in Table 1. software.

Statistical analysis
The results of RT-qPCR were normalized against the expression of U48 or GAPDH genes.
Gene expression results were analyzed according to the Delta CT and Delta-Delta CT algorithms by Microsoft Excel. GSE18392 (colon cancer) and GSE10259 (colorectal cancer) microarray data were downloaded from NCBI-GEO database and normalized using R program. GraphPad Prism 7 (San Diego, CA) was used to perform statistical analysis and graph construction. Differences with p-values<0.05 were considered statistically significant.

7
Important TGFβ signaling components are potential targets of miR-495-3p Target prediction of miR-495-3p was done using DIANA micro-T and TargetScan and RNAhybrid web servers. Four of the predicted targets grabbed our attention because they represent important components of the TGFβ signaling pathway (Fig. 1A). The four genes are TGFβR1, TGFβR2, SMAD4 and BUB1. Multiple conserved miRNA responsive elements (MREs) for miR-495-3p were predicted within each of the 3'UTR sequences of the potential target genes (Fig. 1A). The used software showed important interactions between miR-495-3p seed sequence and the predicted MREs (Fig. 1B).
Overexpression of miR-495 was followed by the downregulation of the predicted target genes To investigate the predicted results at the experimental level, pre-miR-495was cloned in the pEGFPC1 mammalian expression vector and transfected into HEK293T and HCT116 human cell lines. These cells were selected because they are known as TGFβ signalingactive cell lines. RT-qPCR showed that mature miR-495-3p has been successfully overexpressed in both HEK293T and HCT116 cells, in comparison to the cells transfected with mock control vector (Fig. 2A&C).
RT-qPCR against TGFβR1, TGFβR2, SMAD4 and BUB1 putative target genes showed significant reduction in their expression levels in both HEK293T and HCT116 cells overexpressing miR-495-3p, in comparison to the related controls ( Fig. 2B & D).
Overexpression of this miRNA in HCT116 cells also resulted in reduced cyclin D1 (CCND1) protein level, detected through western blotting analysis (Fig. 2E). Furthermore, dual luciferase assay was performed after co-transfection of HEK393T cells, using the vector ensuring pre-miR-495 overexpression and the vector which carried luciferase ORF fused with one of the candidate genes 3'UTR sequences. The 3'UTR sequence of K-RAS gene was used as an off-target to check the accuracy of the test. Luciferase assay results supported direct interaction of miR-495 with 3'UTR sequences of TGFβR1, TGFβR2, SMAD4 and BUB1 putative target genes (Fig 3). Overall, RT-qPCR along with dual luciferase assay results 8 introduced miR-495-3p as a common regulator for the genes that are involved in TGFβ signaling pathway.
Negative correlation between miR-495-3p and its target genes in various human cell lines To gain more evidence about the targeting of TGFβR1, TGFβR2, SMAD4 and BUB1 genes by miR-495-3p, we determined the expression levels of this miRNA and the four target genes in SW480 and HCT116 colorectal cancer cells and MDA-MB-231and SKBR3 breast cancer cell lines (Fig. 4A). A significant negative correlation was obtained between the expression levels of miR-495-3p and each of the target genes in the four tested cell lines (Fig. 4B).
These results provide additional evidence about the targeting of TGFβR1, TGFβR2, SMAD4 and BUB1 genes by miR-495-3p.

Overexpression of miR-495-3p arrests cell cycle in HEK293T and HCT116 cells
The impact of miR-495 overexpression on cell cycle status was investigated in HEK293T and HCT116 cells using Propidium Iodide flow cytometry. Results indicated a significant increase of the sub-G1 cell population in both HEK293T and HCT116 cells (~10 % and ~5 %, respectively) overexpressing miR-495, in comparison to untransfected cells or those transfected with mock control (Fig 5A & B). These results suggested a tumor suppressive role for miR-495-3p. Thus, we intended to investigate the expression of this miRNA in some tumors. Analysis of available microarray expression data of colon (GSE18392, 116 cancerous and 29 normal samples) and colorectal (GSE10259 58 cancerous and 8 normal samples) tumors showed that miR-495-3phasbeensignificantly downregulated in the cancerous samples of both groups in comparison to the adjacent normal tissues (Fig. 6).
These results suggest miR-495-3p as an important tumors suppressor miRNA in cancer research.

Discussion
MicroRNAs are small non-coding RNAs that are implicated in most biological processes through regulating genetic expression [19]. Although of the various studies that aimed to determine the role of miR-495-3p in cancer initiation and progression, the essential role of miR-495-3p in this field is still not clear [4]. Several studies showed that miR-495-3p inhibits the progression of prostate cancer, colorectal cancer and melanoma [6,20,21]. In contrast, recent study provided important evidence about an oncogenic role of miR-495-3p in colon cancer [7].
TGFβ signaling pathway plays critical roles in many developmental and physiological processes [8]. Recent studies revealed that this pathway exerts its effects contextually depending on cell types [22]. For instance, few genes are regulated by TGFβ signaling in pluripotent embryonic cells versus hundreds in differentiated cells [22]. Additional complexity of this pathway is that it can activate or suppress the expression of the same gene depending on the cellular context [22]. Thus, elaborating the molecular regulators of this pathway helps in understanding the mechanisms that leads to diseases. TGFβ signaling pathway has been reported to be regulated by several known miRNAs [23][24][25][26].
In the current study, we provided important evidences about the targeting of TGFβ showed that BUB1 knockdown reduces TGFβ-mediated cancerous cells proliferation [11].
In addition to its role in promoting proper chromosome alignment during cell preparation for division [27], BUB1 is proved to interact with TGFβR1 and TGFβR2 receptors and R-SMADs in the cytoplasm [11]. This interaction stabilizes the heteromeric interaction between TGFβR1 and TGFβR2 receptors and R-SMADs and promotes the activation of TGFβ signaling [11]. An important characteristic of BUB1 gene is its short 3'UTR (~266 nucleotides) that results in reducing the chance of its posttranscriptional regulation by miRNAs. However, in the current study, we predicted the presence of an MRE for miR-495-3p in the BUB1 3'UTR sequence, and supported this prediction by experimental evidences.
These results revealed additional important aspects of miR-495-3p function and made it important candidate for further research in the field of cancer molecular studies.
TGFβ signaling pathway is known to play important role in the control of cell cycle [28].
This notion promoted us to investigate the effect of miR-495-3p on cell cycle status. Flow cytometry analysis showed that the overexpression of miR-495 in HCT116 and HEK293T resulted in an arrest at the G1 phase (Fig. 5). Additional western blotting against Cyclin D1 protein confirmed the flow cytometry analysis (Fig. 2E & F). Moreover, analysis of GSE18392 and GSE10259 colon and colorectal cancer microarray data showed that miR-495-3p is downregulated in tumor samples in comparison to normal adjacent tissues (Fig.   6). Considering the big number of samples that have been analyzed in the microarray data, analysis results represent important evidence about the role of miR-495-3p in cancer progression. These results are consistent with other studies that showed that miR-495-3p is an inhibitor of cell cycle progression [20,21,29,30]. Finally, miR-495-3p represents an important candidate for further studies in the field of cancer and development due to its targeting of essential components of TGFβ signaling that plays crucial roles in these processes.

Conflicts of interest
The authors declare that the present research was conducted with no commercial or financial relationships that could be construed as a potential conflict of interest.

Repositories
All datasets on which the conclusions of the paper rely on, are available to readers. Whole datasets are presented in the main manuscript.