Concomitant Up-Regulation of Hsa- Mir-374 and Down-Regulation of Its Targets, GSK-3β and APC, in Tissue Samples of Colorectal Cancer

Background: The WNT-pathway is involved in several cancers, including colorectal cancer (CRC). Many cell signaling components and pathways are controlled by microRNAs. The main purpose of the present study was to investigate the expression of hsa-miR-374, and its two target genes of the Wnt-pathway in CRC clinical samples. Methods: In this study, we predicted the miRNAs targeting key genes of WNT-pathway using bioinformatics algorithms. The expression levels of hsa-miR-374, APC and GSK-3β on 48 pairs of Formalin-Fixed ParaffinEmbedded (FFPE) CRC tumors and marginal-tumors were evaluated using real time-PCR. Additionally, the hsa-miR-374a-5p precursor sequence was amplified by whole-blood DNA as a template. This amplicon was cloned into pEGFP-c1 expression vector and transfected into SW742 cells. Aside from this, MTT assay was performed to evaluate the effect of miR-374 on cell viability. Results: The bioinformatics analysis indicated that hsa-miR-374 binds to the regulatory region the key components of WNT-pathway, including APC and GSK-3β considering the recognition elements and mirSVR scores. Our results revealed significant down-regulation of GSK-3β (0.94 times, p= 0.0098) and APC (0.96 times, p= 0.03) and up-regulation of miR-374 (1.22 times, p= 0.0071) on tumor samples compared with their normal pairs. Meanwhile, the results of the over-expression of miR-374 showed down-regulation of APC and GSK-3β. MTT-assay also indicated that the miR-374 increased cell survival. Conclusions: The results of our study indicated a concomitant change in the expression of miR-374 and its two related target genes, in clinical samples of CRC. Hsa-miR-374 might be as a helpful biomarker or therapeutic target in CRC.


Introduction
The genetic causes of colorectal cancer (CRC) are the occurrence of changes in several components in different signal transduction pathways, including P53, TGFB, JAK/STAT, PI3K/Akt, and Wnt pathways. These pathways are linked together in many cases. One of the important pathways associated with CRC cancer is the Wnt/β-catenin (1)(2)(3)(4)(5)(6). Wnt ligands can trigger two pathways, cononical Wnt-pathway and noncononical Wnt-pathway which are also referred to as β-catenin independent pathways (7). In cononical Wnt-pathway, following binding Wnt signal to the receptor, β-catenin (gene ID: 1499) is released, entered the nucleus and activated transcription factors (TFs) such as C-myc (gene ID: 4609) and cyclin D (gene ID: 595) etc. In the absence of the Wnt, β-catenin normally is destroyed by scaffolds of APC (gene ID: 324), GSK-3β (gene ID: 2932) and Axin2 (gene ID: 8313). The collapse of the genetic regulations caused by mutations in the cancer process leads to the survival of β-catenin and the expression of the proliferative genes (8), (9). Previous studies have investigated the deregulations of some components of the Wnt-signaling pathway in colon cancer samples (10)(11)(12). Recently, scientists have proposed the usage of molecular methods such as microarrays for analysis and identification of cancer molecular biomarkers. All components of signal transduction pathways are controlled by molecules called microRNAs (miRNAs) (13). The molecular network of carcinogenesis was determined by a systematic analysis between mRNAs and the expression of miRNAs. In some studies, based on their expression changes, classified tumors. miRNAs often interacted with their binding site in the 3'un-translated region (UTR) of target genes and affected their expression (14,15). The purposeful study on miRNAs in cancer requires a candidate gene. The aim of our study was to determine the expression change in miRNAs that are selected by bioinformatics studies and its association with two components of the Wnt pathway in CRC samples.

Bioinformatics analysis
Bioinformatics analysis using different software including microRNA.org, miRwalk, DIANA-microT, Targetscan and NCBI, were employed to predict miRNAs that target 3'-UTR region of 7 key component of WNT-signaling pathway including APC, GSK-3β, β-catenin, Axin1, Hnf4a, TCF, CyclinD and C-myc. We selected miRNAs that simultaneously target several upstream and downstream compounds of APC and GSK-3β in the WNT pathway. Furthermore, the main components of other KEGG signaling pathways including MAPK, PI3K/AKT and ErbB pathways, which are involved in CRC, were determined using miRwalk software.

Ethics statement
The clinical sample collection procedure was approved by the ethics committee of Arak University of medical sciences (Ethics committee ID: IR.ARAKMU.REC.1397.51(. In the present research, 48 pairs of formalinfixed embedded (FFPE) tissue samples with  ICD-O code 18 and 19 collected during the last 5 years, used from the archive of pathology departments of Khansarinejad hospital (Arak, Iran). Tumor and non-tumor samples obtained from the same CRC patients were confirmed using hematoxylin and eosin (H-E) staining.

The qPCR reaction
The synthesized cDNAs were used as a template for quantitative-PCR on a light Cycler 96 real-time PCR machine (Roche, Germany) with SYBR green master-mix kit (Yekta Tajhiz Azma, Iran). In this reaction addition to GSK-3β, APC, and miR-374 was evaluated the expressions of GAPDH and SNORD47 (U47) RNAs for normalization. The sequences of primers showed in Table 1.

Plasmid construction for functional analysis of miR-374
The genomic DNA was extracted from human blood cells using PZP DNA extraction kit (Iran). In order to cloning pri-miR-374 in pEGFP-c1 vector, primers were designed using specific software ( Table 2). The amplification reaction was carried out using these primers and PCR master-mix (Amplicon, Denmark) in a thermocycler (Eppendorf, Germany). The fragments were cloned in the vector using XhoI and SacII restriction enzymes (Fermentas, USA) and T4-ligase (Cinagene, Iran). Recombinant vectors were extracted by a plasmid extraction kit (Takara, Japan). In addition, a scramble fragment used as control. Verification of positive construct containing miR-374 precursor was performed by the sequencing method with an ABI Apply Biosystem 3730xl (Macrogen, South Korea).

Results
The results of bioinformatics analysis with specific software provided a list including 255 miRNAs related to 7 components of the WNT-signaling pathway that were chosen based on some criteria such as the miRNA recognition element (MRE sites) and mirSVR scores (Table 3). Here, we selected hsa-miR-374, which simultaneously target several key components of WNT-pathway and other components of the main pathways involved in CRC (Table 4). Binding site one of MRE of hsa-miR-374 were in nucleotide 359-366 at 3'-UTR of APC (mirSVR score: -1.1305 and PhastCons score: 0.6718) and nucleotide 4525-4532 at 3'-UTR of GSK-3Β (mirSVR score: -0.8140 and PhastCons score: 0.5930). Table 5 shows the clinic-pathological data of 48 collected samples.   (Fig. 1). Also, comparison of the expression level of aforementioned genes with clinic-pathological features revealed that down-regulation of GSK-3β and APC is related to high-grade tumors (T3) and age of patients (age< 51). Also, up-regulation of miR-374 is related to poor differentiation and lower age of the patient (age> 35). In H-E staining of tumor tissue were observed high nucleus/ cytoplasm (N/C) ratio and infiltration of mononuclear cells compared with margins of tumor (Fig. 1). The results of the cloning process indicated the proper designing of the primers and the correct implementation of the PCR reaction (Fig. 2).
The cloned fragment with the length of 290bp associated with miR precursor was transfected into colorectal cell line. The results of over-expression (transfection with a recombinant vector) showed changes in the expression of the studied genes (Fig.  3). SW742 transfected cells showed up-regulation of hsa-miR-374 (37.74 folds) and down-regulation of GSK-3β (0.68 folds) and APC (0.82 folds) than control cultures (Fig. 3).
The results of the MTT indicated that the cell survival by the transfection of the recombinant vector, containing the miR-374 sequence, was increased. These results indicate a direct relationship between the formation of violet crystals and the decrease of miR-374 levels (Fig. 4).

Discussion
More than 100,000 people annually are estimated to have died from CRC and more than 150,000 were affected by CRC worldwide. According to the Center for Disease Control and Prevention Statistics, CRC is the leading cause of death after lung cancer. The occurrence rate of this disease is increasing so that the incidence has doubled over the past 20 years (17,18).
The WNT-signaling pathway plays a role in key biological processes and controls the stages of development and tissue homeostasis. Changes in the components of this pathway lead to developmental disorders and various cancers. During metastasis, the expression changes of mesenchymal markers and tumor microenvironment (TME) in epithelial cancer cells, leading to the ability to attack adjacent tissues and create high-grade tumors (19). In general, the Wnt-signaling pathway has two cononical and non-cononical pathways. The cononical process leads to the accumulation of βcatenin. In the absence of Wnt, the cytoplasmic stability of β-catenin is usually disabled with a complex consisting of scaffolds including APC and GSK-3β (20). In the presence of Wnt, components of these scaffolds were dissociated, β-catenin was released, thereinafter entering the nucleus, were expressed the proliferative genes such as c-myc, c-jun and cyclineD1 (7). The defects in this pathway are associated with genetic mutations in cancer cells, including CRC, hepatocellular carcinoma and medulloblastoma. Previous studies revealed that APC containing mutation becomes unable to form a proper collection with GSK-3β and finally degradation of β-catenin (21). Aside from noncanonical Wnt pathways are classified into three other distinct pathways, Wnt/JNK, Wnt/Ca2+ and Wnt/planar cell polarity (PCP). The mechanisms of downstream signal transduction through these three pathways are poorly understood (19). Here, we could detect downregulation of two components of scaffold surrounding β-catenin, including APC in tumor tissues of CRC in comparison with non-tumor or marginal tumor tissues. The product of APC is a tumor suppressor protein that acts as an antagonist of the Wnt-signal transduction pathway. Also, GSK-3 has two isoforms named GSK-3α and GSK-3β which expressed a serine/threonine kinase. GSK-3 has multifaceted roles in several signal transduction pathways in cancer and autophagy-related diseases. Phosphorylation of GSK-3 by AKT and other kinases leads to its inactivation and consequently increased glycogen and protein synthesis. In addition, GSK-3 interaction with axin and APC regulated canonical WNT-signaling pathway (21). We revealed that GSK-3β was downregulated in CRC tumor tissues and probably after that, β-catenin was released and activated the proliferated genes in the nucleus.
In recent years, researches have uncovered that change in expression of many miRNAs and their target genes in CRC. For example, in CRC, hsa-miR-143 specifically inhibits the phosphorylation of KRAS kinase. Valeri et al. in 2014 reported that the inhibition of miR-135b in mouse models of CRC reduced tumor growth by controlling germ cell proliferation, invasion and apoptosis (5). In their study showed that miR-30b was able to suppress rectal tumors through KRAG, PIK3CD and BCL2 oncogenes. miR-135b affects apoptosis via the APC/ Wnt/ βcatenin signaling pathway. The silencing of miR-135b reduced the colony formation in mutated cells with PI3KCA.
In this study, hsa-miR-374 was selected using target prediction tools according to the scoring system of the software, MRE and conservation (22), which simultaneously targets several key components of the Wnt-pathway. Several studies revealed that miR-374 family members have the regulatory role in various cancers, cell growth, differentiation, reproductive disorders and epilepsy (23). The chromosomal locations of members of MiR-374 family members in humans are at Xq13.2 the X-inactivation center region (Xic). MiR-374b was significantly upregulated in hepatocellular carcinoma tissue (HCC) tissue and associated with grade and metastasis (24). In agreement with their findings, we revealed that miR-374 was up-regulated in tumor clinical samples compared with nontumor tissues. There are different conventional methods and new approaches to detect miRNAs Downloaded from rbmb.net at 12:13 +0330 on Thursday March 11th 2021 (25,26). We could optimize the cost-effective qRT-PCR as an efficient and precise quantitative method. Liao et al. conducted a study in 2014 to examine the relationship between miRNAs and tumor suppression (9). miR-374a activates Wntpathway to promote metastasis of breast cancer (27). it seems there is a logically contradiction between the expression of the miR-374 and two related targets. These two targets, APC and GSK-3β, are two key components of the Wnt pathway.
It is possible that the down-regulation of these two genes may be due to the up-regulation of miR-374 in clinical CRC tumor than non-tumor tissues. In addition, our data showed the upregulation of miR-374 and down-regulation of two its target genes have a correlation with a higher grade of the tumor. As shown in our data, the highest point (Fig. 1C) and lowest points of the scatter graph (Fig. 1A) are related to T3 grade of tumor blocks compared with marginal blocks of CRC FFPE tissues. Furthermore, down-regulation of APC and GSK-3β following precursor of miR-374 over-expression, indicated proper processing of the precursor molecules to mature miRNA in transfected cell lines and possibly targeting of APC and GSK-3β transcripts (Fig. 3). Although, several studies have been reported the expression alterations of different miRNAs related to CRC, but so far, the relationship between miR-374 and the key elements of the Wnt-pathway in CRC has not been studied. Aside from, with decreasing the recombinant vector containing pre-miR-374, decreases cell viability (Fig. 4). Taken together, possibly miR-374 conducted increasing in cell viability and proliferation. Therefore, the increase of the expression of this miRNA may be a reason to reduce the expression of these two genes in CRC tumor samples, and possibly the expression of this expression may be therapeutic application in CRC.
Bioinformatics analysis showed miR-374 is targeting the key components of the Wntpathway. Performing bioinformatics analysis before implementing laboratory processes is a simple and inexpensive method to reduce the number of targets of miRNAs. Given contradictory expression of miR-374 in tumor tissues relative to non-tumor samples, it might be considered as an onco-miR and a diagnostic biomarker in high-grade CRC.