Role of miR-424 in the carcinogenesis

Recent studies have revealed the impact of microRNAs (miRNAs) in the carcinogenic process. miR-424 is a miRNA whose role in this process is being to be identified. Experiments in the ovarian cancer, cervical cancer, hepatocellular carcinoma, neuroblastoma, breast cancer, osteosarcoma, intrahepatic cholangiocarcinoma, prostate cancer, endometrial cancer, non-small cell lung cancer, hemangioma and gastric cancer have reported down-regulation of miR-424. On the other hand, this miRNA has been found to be up-regulated in melanoma, laryngeal and esophageal squamous cell carcinomas, glioma, multiple myeloma and thyroid cancer. Expression of this miRNA is regulated by methylation status of its promoter. Besides, LINC00641, CCAT2, PVT1, LIN00657, LINC00511 and NNT-AS1 are among lncRNAs that act as molecular sponges for miR-424, thus regulating its expression. Moreover, several members of SNHG family of lncRNAs have been found to regulate expression of miR-424. This miRNA is also involved in the regulation of E2F transcription factors. The current review aims at summarization of the role of miR-424 in the process of cancer evolution and its impact on clinical outcome of patients in order to find appropriate markers for malignancies.


Introduction
MicroRNAs (miRNAs) are transcripts with sizes around 23 nt that can influence expression of genes at post-transcriptional level.These small, highly conserved transcripts are transcribed by RNA polymerases II and III.The miRNA precursors generated by these polymerases go through a group of cleavage actions to make mature miRNAs [1].miRNA precursors are lengthy polycistronic RNAs that share some features with mRNAs since they possess distinctive 5′ and 3′ borders, 7-methyl guanylate caps and poly(A) tails.
The function of miRNAs in the regulation of genes expression is accomplished via the RNA-induced silencing complex (RISC) [2].After assemblage into RISC, miRNAs activate this complex to target mRNAs in a specific manner [1].miRNAs which control oncogenes and tumor suppressor genes, are differentially expressed in various human malignancies and play a central role in all cancer hallmarks, especially in their real targets [3][4][5][6].Moreover, miRNAs differ in their transcriptional units and the mechanisms of their regulation within genomic loci.Those being located within an intron of a host gene are transcribed in the identical orientation with primary transcript by the promoter of the host gene [7].On the other hand, miRNAs located in the intergenic loci have their own promoters [7,8].
miR-424 is encoded by a gene located on chr Xq26.3.This miRNA has been demonstrated to be dysregulated in different cancers.Notably, dysregulation of miR-424 in cancer samples have been associated with invasive behavior of malignant cells.However, different studies have reported various results regarding its expression in different cancers.Mechanistically, several lncRNAs act as molecular sponge for miR-424 to regulate its expression.The current review aims at summarization of the role of miR-424 in the process of cancer evolution and its impact on clinical outcome of patients in order to find appropriate markers for malignancies.

Cancer cell lines
An experiment in neuroblastoma cell lines has revealed down-regulation of miR-424 and up-regulation of its target gene DCLK1 in these cells compared with normal spongiocyte cells.Mechanistical studies has confirmed that the role of miR-424 in suppression of cell viability, invasive properties, and epithelial-mesenchymal transition (EMT) is mediated through targeting DCLK1.In fact, DCLK1 could partially reverse function of miR-424 in neuroblastoma cells [9].
In MG-63 and SaOS2 osteosarcoma cells, expression of miR-424-5p has been increased upon treatment with melatonin leading to inhibition of VEGFA.Moreover, melatonin could suppress neoangiogenesis, affecting proliferation and migration of neighboring endothelial cells as well as release of angiogenic growth factors.It has also induced morphological changes in blood vessels, and.Taken together, melatonin has a tumor suppressive role through influencing miR-424-5p/VEGFA axis [10].
In silico studies in glioblastoma have predicted a tumor suppressor role for miR-424.This miRNA has also been predicted to target several genes from the ERBB signaling pathway that are activated in the majority of glioblastoma patients.Cell line studies have also confirmed the impact of miR-424 overexpression in suppression of proliferation and migratory potential of glioblastoma cells.Moreover, miR-424 has been shown to promote apoptosis and induce cell-cycle arrest in glioblastoma cells.As predicted by in silico assays, miR-424 could decrease expressions of KRAS, RAF1, MAP2K1, EGFR, PDGFRA, AKT1, and mTOR.Direct interactions between miR-424-5p and RAF1 and AKT1 oncogenes has been verified by dual-luciferase reporter assay [11].
Contrary to glioblastoma cells, expression of miR-424-5p has been reported to be increased in colorectal cancer cell lines.miR-424-5p can promote proliferation and metastaticrelated phenotypes through directly binding with SCN4B [12].In laryngeal squamous cell carcinoma cells, up-regulation of miR-424-5p has enhanced proliferation, migratory aptitude, invasiveness, and adhesion of cancer cells with an important effect on cell cycle progression.In addition, CADM1 has been identified a direct target of miR-424-5p in these cells [13].
An experiment in lung cancer cells has shown that both miR-424-3p and miR-424-5p can prevent proliferation, migration, and invasiveness of these cells.In addition, miR-424-3p but not miR-424-5p could enhance chemosensitivity of lung cancer cells via influencing expression of YAP1 [14].
Different types of Small nucleolar RNA host gene (SNHG) family members have been shown to regulate expression levels of miR-424 in multiple cancers.SNHG family belongs to lncRNAs and have oncogenic roles in the malignancies [15].For evaluation of their relation to miR-424, following studies have been conducted: In osteosarcoma cell lines Saos-2, MG63, HOS and U2OS, SNHG1 acts as a molecular sponge for miR-424-5p.After knocking down SNHG1, expression levels of miR-424-5p rises and this miRNA can target 3′-UTR of FGF2, resulting in reduced proliferation, migration and invasion [16].The same molecular mechanism also applies for cervical cancer cells, but the difference is that sponging molecule is SNHG12 in this case and there is no FGF2 targeting [17].Finally, in T98G and LN229 glioma cells, treatment with Ropivacaine causes SNHG16 levels to drop, and subsequent up-regulation of miR-424-5p happens, which not only suppresses proliferation and migration, but also induces apoptosis in glioma cells [18].
E2F family of TFs (transcription factors) are TFs that were firstly studied in 1987 [19].This family can either be activators or suppressors of transcription [20].Interestingly, E2Fs are dysregulated in a variety of cancers and it has been demonstrated that they can be regulated by miR-424.For instance, in hepatocellular carcinoma, forced expression of miR-424-5p and miR-424 is followed by downregulation of E2F7 and E2F3, respectively [21,22].Downregulation of these two TFs is favorable and contributes to normallike cell properties.In cases of endometrial carcinoma, upregulation of miR-424 diminishes E2F6 and E2F7 levels in HEC-1A, Ishikawa and HEC-1B cells, which in turn inhibits migration, invasion, and colony formation of cells [23,24].Lastly, in non-small cell lung cancer cell lines A549 and H460, elevation of miR-424 directly targets expression of E2F6, and consequently reduced proliferation occurs [25].
X-inactive specific transcript (XIST) belong to lncR-NAs, and is famously known as X chromosome inactivator in females [26].It is of great importance to know that this lncRNA can act as a molecular sponge for miR-424-5p in two types of cancer: firstly, in pituitary adenoma cell lines, depletion of XIST is followed by up-regulation of miR-424-5p, and as expected, reduced proliferation, migration and invasion occurs because of bFGF targeting by miR-424-5p [27].A more detailed mechanism of XIST is demonstrated in hepatocellular carcinoma, in which inhibiting XIST expression contributes to overexpression of miR-424-5p.This miRNA degrades OGT and suppresses RAF1 glycosylation, resulting in favorable normal features in HCC cell lines [28].
Table 1 summarizes the role of miR-424 in different cancer cell lines.

Animal models
Animal models of different types of cancers, including mammary tumors, aggressive osteosarcoma, gastric cancer, esophageal squamous cell carcinoma, thyroid cancer and glioma have been established to assess the impact miR-424 dysregulation on the tumor burden (Table 2).In gastric cancer xenograft models, the results of two studies are contradictory.While up-regulation of miR-424-3p has led to reduction of tumor growth and metastasis in one study [52], another study has reported that over-expression of mir-424-5p has the opposite effect [53].In esophageal squamous cell carcinoma, both conducted studies have confirmed an oncogenic role for this miR-424 [56,93].The results of other studies in xenograft models are shown in Table 2.

Assays in clinical samples
Expression of miR-424 has been evaluated in a variety of malignant tissues.Experiments in ovarian cancer, cervical cancer, hepatocellular carcinoma, neuroblastoma, breast cancer, osteosarcoma, intrahepatic cholangiocarcinoma, prostate cancer, endometrioid endometrial cancer, nonsmall cell lung cancer, hemangioma and gastric cancer have reported down-regulation of miR-424.On the other hand, this miRNA has been found to be up-regulated in melanoma, laryngeal and esophageal squamous cell carcinomas, glioma, multiple myeloma and thyroid cancer.In colorectal cancer, both patterns have been reported (Table 3).In neuroblastoma tissues, down-regulation of miR-424 has been found to be accompanied by up-regulation of its target gene DCLK1 [9].On the other hand, miR-424-5p has been found to be upregulated in laryngeal squamous cell carcinoma samples versus adjacent normal margin tissues.Over-expression of miR-424-5p has been associated with poor differentiation, advanced tumor stages and cervical lymph node involvement.In silico analyses have shown that target genes of this miRNA are principally enriched in cell cycle, cell division, and negative regulation of cell migration [13].In patients with cervical cancer, down-regulation of miR-424 has been reported to be associated with low level of differentiation of cancer cells, advanced clinical stages and metastasis to lymph nodes [95].Furthermore, in patients with non-small cell lung cancer, low levels of miR-424-3p have been associated with disease progression and overall prognosis [14].
Out of 4 different studies conducted on ovarian cancer/ epithelial ovarian cancer patients, two have reported downregulation of miR-424 and other two verified downregulation of miR-424-5p in tumor tissues compared with adjacent normal tissues [81][82][83]96].
In Oral squamous cell carcinoma (OSCC), three different studies confirmed up-regulation of miR-424-5p both in OSCC tissues and blood samples [29,30,97].Discordant to mentioned studies, miR-424-3p has shown to be underexpressed in saliva of OSCC patients, and its levels can be used as a diagnostic marker to differentiate OSCC patients and healthy controls, with an AUC value of 0.732, sensitivity of 0.605 and 0.818 specificity.
It is worth mentioning that a great number of research on clinical specimens have been conducted in hepatocellular carcinoma (HCC) patients.In total, after evaluating expression of miR-424 and miR-424-5p in 886 pairs of HCC tissues in 11 independent studies, it is concluded that both of these miRNAs are downregulated in HCC tissues.According to Kaplan-Meier analysis, downregulated levels of miR-424-5p is associated with shorter over-all survival in HCC patients [65].In addition, diminished levels of this miRNA is associated with alpha-fetoprotein content and HBV infection in HCC [65].
In gastric cancer (GC) patients, miR-424 has been verified to be downregulated [98] but miR-424-5p is upregulated in tumor expression profile [54,55].In case of miR-424 downregulation, NNT-AS1 acts as a molecular sponge and inhibits miR-424 and this phenomenon is accompanied by activation of E2F1, a transforming transcription factor [98].
Associations between dysregulation of miR-424 and clinical outcome have been demonstrated in ovarian, cervical, breast, prostate, lung, melanoma, colorectal and other cancers (Table 3).
In patients with colorectal cancer, cancer cells have been shown to release miR-424-5p into peripheral blood in the form of exosomes.Notably circulating exosomal levels of miR-424-5p can separate patients with early stage of colorectal cancer from healthy individuals with area under the ROC curve (AUC) value of 0.82 [12].In patients with multiple myeloma, serum levels of this miRNA could be used as a diagnostic marker with AUC value of 0.95 [111].Diagnostic role of miR-424 has also been evaluated in hepatocellular carcinoma, prostate cancer and renal cell carcinoma revealing different AUC values (Table 4).

Discussion
miR-424 is an example of miRNAs with crucial roles in the carcinogenesis.However, its role in this process may depend on the type of tissue, since in some tissues it makes cancer cells grow and in others it prevents cancer cells from         growing.Moreover, it is possible that miR-424-3p and miR-424-5p exert different roles in the process of carcinogenesis in some cases (115) (Fig. 1).This is mostly related with the specific targets of miR-424 in each tissue.The interactions between miR-424 and tissue specific transcription factors might also been involved in the specificity of miR-424 functions in each tissue.Expression of this miRNA is regulated by methylation status of its promoter and lncRNAs that sponge this miRNA [64].LINC00641 [115], CCAT2 [116], PVT1 [117], LINC00511 [102] and NNT-AS1 [98] are among lncRNAs that act as molecular sponges for miR-424.The interactions between lncRNAs and miRNAs forms a huge and complex regulation network for regulation of gene expression at transcriptional, post-transcriptional, and post-translational levels [118].This multilevel regulatory network is involved in the carcinogenesis.miR-424/lncRNA axes might represent potential targets for design of novel therapeutics for cancers.However, the function of these axes should be individually assessed in each type of cancer.miR-424 has a regulatory role on activity of VEGFA, ERBB, mTOR, TGF-β and PTEN/PI3K/AKT pathways.Since miR-424 can influence expression of VEGFA, it has a pivotal role in the regulation of tumor angiogenesis [119].Moreover, it has been shown to influence EMT process through inhibiting mTOR phosphorylation [73].miR-424 can affect cell-cycle transition and cell apoptosis.KRAS, RAF1, MAP2K1, EGFR, PDGFRA, AKT1, and mTOR expressions can also been affected by miR-424 [11].Therefore, a wide array cancer-related genes, pathways and cellular functions are controlled by this miRNA.
Serum levels of miR-424 can distinguish cancer patients from healthy controls with variable diagnostic power values.Diagnostic role of miR-424 has been evaluated in hepatocellular carcinoma [112], multiple myeloma [120], prostate cancer [121], oral squamous cell carcinoma [107], renal cell carcinoma [50] with the best values being obtained in multiple myeloma.Confirmation of these results in larger sample sizes from different types of cancer can broaden application of this miRNA in non-invasive methods for cancer detection.SPF BALB/c nude mice ↓ XIST: ↑ miR-424-5p: ↓ tumor growth [28] BALB/c nude mice ↑ miR-424-5p: ↓ tumor growth [69] Nude mice ↑ miR-424-5p: ↓ tumor growth [66] Nude mice ↑ miR-424-5p: ↓ tumor growth [22] Colorectal cancer BALB/c nude mice ↓ miR-424-5p ↓ tumor growth [33] Glioma BALB/c nude mice ↑ miR-424: ↓ tumor growth   Since early diagnosis of cancer is the most efficient way to increase survival of patients, these findings have significance in this regard.Moreover, serum levels of miR-424 can be used for follow-up of patients with different types of cancers after conduction of anti-cancer therapies to detect cancer recurrence.
The association between expression levels of miR-424 and clinical outcome of patients further highlights the potential of this miRNA as a predictive biomarker in cancer patients.However, since miR-424 has opposite roles in different cancers [13], the patterns and direction of these associations depends on the role of miR-424 in each type of cancer.This note should also be considered when designing miR-424-targeting strategies in the treatment of cancer.Although there are ongoing clinical trials in phase 1 and 2 regarding their therapeutic application, there is no FDA approved miRNA-based drug in the market [122].However, patisiran, and givosiran are two FDA approved siRNA-based drugs [123] and because of the similarity in the mechanism of action, we can anticipate miRNA-based drugs in the near future.In the cases of miR-424, the wide range of molecules being affected by this miRNA enhances the efficacy of targeted therapies in the field of cancer.However, this feature also increases the possibility of unwanted side effects.
Since the majority of studies, especially cell line studies are conducted on colorectal cancer (7 studies), osteosarcoma (5 studies), breast cancer (7 studies), gastric cancer (4 studies), hepatocellular carcinoma (11 studies), endometrial carcinoma (5 studies), ovarian cancer (6 studies), glioma (5 studies) and non-small cell lung cancer (4 studies), it might be wise to be more focused on these conditions when considering therapeutic approaches of miR-424.
In brief, miR-424 is an example of miRNAs with tissue-specific impacts in the carcinogenesis.Experiments in cancer cell lines and animal models of cancer have shown feasibility and efficacy of miR-424-targeting strategies in decreasing invasiveness of cancer cells and tumor burden, respectively.Applicability of these strategies in clinical setting has not been evaluated yet.Future studies are needed to elaborate this aspect.

Fig. 1
Fig. 1 miRNA-424 can either inhibit or stimulate cancer cell growth depending on the context.For instance, overexpression of miR-424 can inhibit glioblastoma cell growth and migration.In addition, miR-424 has been demonstrated to induce cell-cycle arrest and enhance