Pathology & Oncology Research (2019) 25:559–566 https://doi.org/10.1007/s12253-018-0502-9 ORIGINAL ARTICLE Non-target Genes Regulate miRNAs-Mediated Migration Steering of Colorectal Carcinoma Sohair M. Salem1 & Ahmed R. Hamed2,3 & Alaaeldin G. Fayez1 & Ghada Nour Eldeen1,4 Received: 20 January 2018 /Accepted: 15 October 2018 /Published online: 25 October 2018 # Arányi Lajos Foundation 2018 Abstract MicroRNAs (miRNAs) trigger a two-layer regulatory network directly or through transcription factors and their co-regulators. Unlike miR-375, the role of miR-145 and miR-224 in inhibiting or driving cancer cell migration is controversial. This study is a step towards addressing the potential of miR-375, miR-145 and miR-224 expression modulation to inhibit colorectal carcinoma (CRC) cells migration in vitro through regulation of non-target genes VEGFA, TGFβ1, IGF1, CD105 and CD44. Transwell migration assay results revealed a significant subdue of migration ability of cells transfected with miR-375 and miR-145 mimics and miR-224 inhibitor. Real time PCR data showed that expression of VEGFA, TGFβ1, IGF1, CD105 and CD44 was downreg- ulated as a consequence of exogenous re-expression of miR-375 and inhibition of miR-224. On the other hand, ectopic expression of miR-145 did not affect VEGFA, TGFβ1 and CD44 expression, while it elevated CD105 and suppressed IGF1 expression. MAP4K4, a predicted target of miR-145, was validated as a target that could play a role in miR-145-mediated regulation of migration. At mRNA level, no change was observed in expression of MAP4K4 in cells with restored expression of miR-145, while western blotting analysis revealed a 25% reduction of protein level. By applying luciferase reporter assay, a significant decrease in luciferase activity was observed, supporting that miR-145 directly target 3’ UTR of MAP4K4. The study highlighted the involvement of non-target genes VEGFA, TGFβ1, IGF1, CD105 and CD44 in mediating anti- and pro-migratory effect of dmiR-375 an miR-224, respectively, and validated MAP4K4 as a direct target of anti-migratory miR-145. Keywords MiRNAs, colorectal carcinoma . Non-target genes . MAP4K4 . Migration Introduction (miRNAs) are small non-coding RNAs that usually bind to recognition sites on target mRNAs leading to either degrada- Colorectal cancer (CRC) is the third leading cause of cancer tion of the transcript, inhibition of translation or rapid death among adults. Targeting dysregulated pathways repre- deadenylation [20]. Many studies have linked a well-known sents the best way to improve cancer treatment and increase sub-cellular structure, the cytoplasmic processing bodies the overall survival in cancer patients [43]. MicroRNAs (PBs), to miRNA pathway [30]. MiRNAs have been shown to act as tumor suppressors by negatively regulating gene expression of oncogenic factors. Alternatively, oncomirs, * Sohair M. Salem miRNAs which down-regulate expression of tumor suppres- [email protected] sor genes, have been identified in a variety of cancer types [14]. MiRNAs provide new therapeutic targets for many dis- 1 Molecular Genetics and Enzymology Department, National eases, including cancer. The progress in selecting specific Research Centre, 33 El-Buhouth St., Dokki, Giza 12622, Egypt anti-miRNA inhibitor chemistries and delivery system sug- 2 Phytochemistry Department, National Research Centre, 33 gests that miRNA-based therapeutics may soon be applied in El-Buhouth St., Dokki, Giza 12622, Egypt the clinic [5, 32]. In the context of CRC, several miRNAs have 3 Biology Unit - Central Laboratory of Pharmaceutical and Drug been reported to regulate different cellular processes that con- Industries Research Division, National Research Centre, 33 trol migration and metastasis by inhibition of multiple target El-Buhouth St., Dokki, Giza 12622, Egypt genes [9, 37, 52, 53]. 4 Stem Cell Research Unit, National Research Centre, 33 El-Buhouth MiR-375 is down-regulated and evinces significant tumor St., Dokki, Giza 12622, Egypt suppressive properties in CRC tissue [12] and could predict 560 S. M. Salem et al. the metastatic potential of CRC patients [45]. It inhibited the MAP4K4 was demonstrated to play a central role in cell mi- proliferation, invasion and migration by directly targeting Sp1 gration and invasion through the JNK pathway [7, 26], and is transcription factor and regulating matrix metalloproteinase 2 regulated by miR-141 [16]andmiR-194 [43]. (MMP2) and epithelial-mesenchymal transition (EMT)-asso- The complexity of miRNAs is attributed to the ability of ciated genes [8]. Tumor suppressor miR-145 inhibited invasion single miRNA to fine-tune the expression of hundreds and metastasis in many cancers by targeting several genes [10] mRNAs, and the potential of each mRNA to be regulated by and was expressed at reduced levels in colon cancer epithelial hundreds of miRNAs [1] through the two-layer regulatory cells [27]. However, a study by Yuan et al. [47]demonstrateda network. in this network, miRNAs directly regulate the genes significant relation between miR-145 expression and lymph node by mRNA degradation or translational inhibition at the prima- metastasis of CRC. The potential function of miR-224 as onco- ry level of the regulatory cascade; whereas the secondary tar- gene or tumor suppressor remains contradictory. Previous data gets of miRNAs are the genes that regulated by miRNA- have founded that miR-224 was significantly up-regulated in targeted TFs [38]. This study is a step towards highlighting CRC tissue samples and associated with disease relapse and a the involvement of non-target genes VEGFA, TGFβ1, IGF1, relative poorer disease-free survival rate, suggesting the oncogen- CD105 and CD44 in mediating the anti - or pro-migratory ic role of miR-224 in the tumorigensis of CRC, and its prognostic effect of miR375, miR-145 and miR-224, and validating function as a novel biomarker for patients relapse [50]. In con- MAP4K4 as target of miR-145. trast, another study indicated a significantly lower expression level of miR-224 in CRC tissues than those in non-tumor tissues. The differences in miR-224 expression levels in the pathogenesis Methods of cancer were suggested to be related to mutation status of the adenomatous polyposis coli (APC) gene and racial and environ- Cell Line Propagation and Transient Transfection mental diversity [21]. Molecularly-targeted inhibitors that blocked migration me- Human CRC cell line HCT116 (ATCC®) was maintained as diators, therefore subdue cancer cell migration, would be ex- monolayer culture in RPMI-1640 medium supplemented with pected to be clinically beneficial to cancer patients [54]. One 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml strep- of the most prominent angiogenic factors is vascular endothe- tomycin sulfate and 2 mM L-glutamine at 37 °C under a hu- lial growth factor (VEGF) that widely studied for its signifi- midified atmosphere of 5% CO2. HiPerFect transfection re- cant role in tumor growth, migration and metastasis [41]. agent, miScript miRNA mimic of miR-375 and miR-145 and Transforming growth factor β (TGFβ) pathway also plays a miScript miRNA inhibitor of miR-224 were purchased from pro-tumoral role [28] and promote migration and metastasis of Qiagen (Germany). Transfection was carried out according to colon cancer cell into the liver and lung [29]. TGFβ was found the manufacturer’s instructions. Briefly, cells were seeded, to cooperate in an autocrine/paracrine fashion with other sig- transfected with transfection complex of HiPerFect transfec- naling cascades, including VEGF, basic fibroblast growth fac- tion reagent and 5 nM miRNA mimic or 50 nM miRNA tor (bFGF), platelet-derived growth factor (PDGF), inhibitor diluted in serum free medium, and incubated until angiopoietin and Notch, to regulate migration [29, 33]. monitoring gene or protein expression. For co-transfection of Insulin-like growth factor (IGF) family molecules act through mimic and plasmid, HiPerFect transfection reagent was com- binding to IGF receptors (IGFRs) which are receptor tyrosine plexed with 400 ng plasmid DNA and 5 nM miR-145 mimic kinases (RTKs) and most frequently signal through phos- diluted in medium without serum or antibiotics. phatidylinositol 3-kinase (PI3K) and mitogen-activated pro- tein kinase (MAPK) dependent mechanisms [19, 40]. It was MiRNAs Target Prediction demonstrated that IGF1 signaling is important in mediating protease activation and cell migration and invasion, and it Bioinformatics algorithms MIRDB (http://mirdb.org)and combined with TGFβ signaling to bring about an EMT [40]. Target Scan (http://targetscan.org) were used to predict Endoglin/CD105 functions as a co-receptor for TGFβ1, it downstream targets of miRNAs. Mature sequences of specifically enhanced TGFβ1-induced phosphorylation of in- miRNAs were downloaded from miRBase and the 3’ UTR tracellular effectors Smad 1/5/8 and increased a Smad 1/5/8 sequence of human MAP4K4 was downloaded from Ensembl responsive promoter [22]. Expression of both TGFβ1 and genome browser. CD105 correlated with increased expression of tumor vascular marker CD44 [3]. Reverse Transcription and Real-Time PCR MAPK kinase kinase kinase isoform 4 (MAP4K4) is a serine/threonine (S/T) kinase belongs to the mammalian Total RNA, containing miRNA, was extracted from STE20/MAP4K family, which is associated with cell motility, transfected cells using Direct-zol™ RNA MiniPrep (Zymo rearrangement of the cytoskeleton, and cell growth [18]. Research) according to the manufacturer’s instructions. The