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MicroRNAs miR-1 and miR-206 Regulate Monocarboxylate Transporter-4 and Vascular Endothelial Growth Factor Gene Expression in Colorectal Cancer

Background: Colorectal cancer (CRC) is currently the third most common cancer type in males and the second most occurring in females. The role of microRNA (miRNA) in the development of colorectal cancer is not fully elucidated. Therefore, understanding the mechanistic interaction between miRNA and their target oncogenes may hold great importance as a possible target for interventional anticancer therapy. Original Research Article Khaleel et al.; JPRI, 32(13): 141-152, 2020; Article no.JPRI.59474 142 Aims: To identify miRNAs that are part of the regulating pathway of Monocarboxylate Transporter4 (MCT4) and Vascular Endothelial Growth Factor (VEGF) oncogenes. Study Design: We used publicly available prediction tools (e.g. TargetScan, MicroCosm, PicTar, and DIANA-microT-CDS) to identify the possible miRNA that target the two oncogenes. Methodology: We used the GeneMania database to visualize the network and verify gene names and remove ambiguity and duplications. Furthermore, we used miRTarBase database to identify experimentally validated targets which we used to further confirm miRNA-oncogene relationships. Finally, we utilized miR-Mfold web-tool to further visualize the circular structures and the simulated miR-1 and miR-206 targeting arrangements. Results: We found two putative miRNA (miR-1 and miR-206) that may downregulate MCT4 coded by SLC16A3 gene and VEGF which is coded by VEGF gene. We found relationships between the validated target genes of miR-1 and miR-206 through GeneMania which we extracted from the literature. And we elucidated the proposed structure of these two miRNAs through miR-Mfold webtool. Conclusion: Our results elucidated a novel regulation pathway in CRC cells and may suggest a potential therapeutic approach for CRC therapy. MiR-1 and miR-206 may help cells go to apoptosis and inhibit the angiogenesis of colorectal cancer cells by down-regulation of MCT4 and VEGF proteins in tumor tissues.


INTRODUCTION
Monocarboxylate transporters (MCTs) are expressed in normal colonic epithelium and facilitate the transport of butyrate, the primary energy source for these cells [1]. However, in colorectal tumor cells, lactate is produced and transported via cell membranes during glycolysis and utilized for energy. The intracellular pH is regulated as the influx and efflux of lactate is controlled by MCTs. MCTs, hence have a vital role in the regulation of pH homeostasis [2]. If this balance is disrupted, the cells normally go through apoptosis. For carcinoma cells to survive by avoiding apoptosis, the control of lactate in glycolysis is considered necessary, and MCTs play an important role in this process [3]. In carcinogenesis, monocarboxylate transporter MCT-4 has a role in the efflux of lactate from tumor cells, which results in escaped apoptosis [4]. Moreover, MCT4 has been reported to be induced by the hypoxic conditions which are usually present in the tumor microenvironment [5].
VEGF is a well-known growth factor and numerous scientific evidences proved the indisputable role of Vascular Endothelial Growth Factor (VEGF) in angiogenesis as well as carcinogenesis [6]. Both MCT4 and VEGF were recently found to be overexpressed in colorectal cancer (CRC) [7].
To our knowledge MCT4 regulating miRNAs have not been described previously. In the current study, we aim to identify miRNAs that regulate MCT4 and VEGF using the miRNA target prediction web tools because these two genes are over expressed in CRC.

miRNA selection based on web tools prediction
We selected two miRNAs as candidates from the results of step 1 for our study. These miRNAs (miR-1 and miR-206) have never been investigated in CRC but have been reported to be involved in carcinomas.
PubMed search for experimentally validated targets of miR-1 and miR-206.
We did a PubMed database search of the literature looking for experimentally validated targets of miR-1 and miR-206. This search yielded 24 oncogenes that are targeted by miR-1 and miR-206.

Experimentally validated24 oncogenes targets uploaded to GeneMania
We uploaded the 24 oncogenes that we found in step 3 to the GeneMania databases [24] in order to visualize the network and verify gene names and remove ambiguity and duplications.

Pathway analysis and identifying overlapping target genes
To discover the 24 genes pathways and overlapping relationships/diseases. We utilized Gene set enrichment analysis (GSEA) [27] web tool. Furthermore, to identify overlapping target genes we used DIANA-microT-CDS web tool [23]. In the next step we uploaded those target genes to GeneMania which resulted in several genegene interactions including; co-expression (57.54%), physical interactions (19.62%), genetic pathway (18.92%), C-localization (3.63%), genetic interactions (0.15%), shared protein domains (0.13%). In addition, GeneMania yielded a genetic network presented in Fig. 1, where genes in black represent our 24 query genes targeted by miR-1 and miR-206.  The miRNA-target interactions (MTIs) that were extracted from miRTarBase database are shown in Table 2.

RESULTS AND DISCUSSION
To further visualize the circular structures and the simulated miR1 and miR2 targeting arrangements, we utilized miR-Mfold web the resulting structure is shown in Fig. 2.
MCT-4 is frequently deregulated in various cancer cells, it promotes their migration and proliferation and is associated with the level of malignancy and recurrence [56,57 other hand, the expression of reported more frequently in early compared to advanced-stage cancer types. Recent research suggests that VEGF is a negative prognostic factor for CRC [59,60]. Gotanda et al shown recently that increased expression is associated with tumor growth, infiltration, and angiogenesis in their CRC [7]. Previous research showed that miRNAs could serve as tumor suppressors or oncogenes Thus miR-1and miR-206 might help in targeting and regulating cancer cell proliferation, mi and angiogenesis by downregulating the expression of these two genes: MCT4 and VEGF Fig. 4 illustrates this theory.
In this study, we found two plausible miRNAs which were computationally validated to show that the mRNA of MCT4 and VEGF target of miR-1 and miR-206, by using publicly available miRNA target prediction web tools.
Pathway analysis by GSEA [27] most genes overlap were in PI3K/AKT cancer signaling pathway, and diseases of signal transduction by growth factor receptors and second messengers. As shown in Table 3. In this study, we found two plausible miRNAs which were computationally validated to show VEGF is a putative 206, by using publicly available miRNA target prediction webrevealed that most genes overlap were in PI3K/AKT cancer signaling pathway, and diseases of signal ion by growth factor receptors and second messengers. As shown in Table 3.

DIANA-microT-CDS
web tool overlapping target genes between the two miRNAs with miTG scores of 0.99 (Supplementary Table 1).
These two miRNAs, miR-1 and miR help in restoring apoptosis pathways by suppressing MCT4 along with inhibiting angiogenesis by targeting VEGF [61, Previous study has reported that miR 206 can regulate angiogenesis by targeting and reducing the levels of Vegf gene in zebrafish and the knocking down of miR-1and miR increased angiogenesis in the same Recent reports have also shown that both miR  reported that miR-1 and miR-206 can regulate angiogenesis by targeting and gene in zebrafish and 1and miR-206 increased angiogenesis in the same setting [63]. Recent reports have also shown that both miR-1 and miR-206 were down-regulated in many human cancer types including CRC Previous reports showed inhibitory roles of miRNAs of MCTs which could reduce tumor cell proliferation [66]. And some studies showed other potential roles of these small RNAs as negative regulators that may lead eventually to growth suppression in some malignancies [67].
In the future, our computational approach needs to be validated by in-vitro and in-vivo expr studies of both miRNAs and target genes in CRC cell-lines i.e. miR-1 and miR MCT4/VEGF. regulated in many human cancer types including CRC [64,34,30,65]. Previous reports showed inhibitory roles of miRNAs of MCTs which could reduce tumor cell And some studies showed other potential roles of these small RNAs as negative regulators that may lead eventually to growth suppression in some In the future, our computational approach needs vivo expression studies of both miRNAs and target genes in CRC 1 and miR-206 and

CONCLUSION
MiRNAs are pivotal regulators of gene expression, as they contribute to multiple critical biological processes, including cell proliferation, angiogenesis, and apoptosis. This study could help in deciphering the potential mechanism of acquired regulation of tumor growth and angiogenesis in CRC. In addition, this work sheds light on the involvement of miR-1 and miR-206 in the tumor inhibitory effect by targeting the two oncogenes VEGF/MCT4. Our results elucidated a novel regulatory pathway in CRC cells and could suggest a potential therapeutic approach for CRC. The possibility of metabolic modification of the tumor microenvironment via regulation or manipulation of MCT4 and VEGF may prove to be a promising target for future studies.

CONSENT
It is not applicable.