The Role of miRNA-424 in Various Cancers: Focusing on Drug Resistance and Sensitivity

However, advanced technologies have been developed in the treatment of various cancers the mortality rate of this disease is still very high. Drug resistance is a major problem for cancer patients which causes the treatment process to fail. In addition to inhibiting drug resistance, targeted therapy is also very important in its treatment. Nowadays, miRNAs have gained increasing interest in recent years which play a major role in both drug resistance and target therapy. MicroRNA (miRNA) is an important part of non-coding RNA that regulates gene expression at a posttranscriptional level. One of these microRNAs is miR-424 that by targeting genes involved in various cellular processes can participate in proliferation, differentiation, apoptosis, invasion, angiogenesis, and drug resistance and sensitivity. In this study, we collected the role of miR-424 in a variety of cancers and by identifying the role of miR-424 in drug resistance we can reduce the effect of drug resistance in many cancers.


Introduction
Cancer is the second leading cause of death in the world. There were an estimated 18 million cancer cases around the world in 2018, of these 9.5 million cases were in men and 8.5 million in women [1]. The highest prevalence of cancers is related to lung cancers (12.5%), breast cancers (12.3%), colorectal cancers (10.6%), and prostate cancers (7.5%). About 50% of cancers can be treated before the onset of clinical symptoms due to rapid diagnosis. There have been many successes in the diagnosis and treatment of cancer in recent years [1]. Damage to healthy cells during chemotherapy and resistance to chemotherapy drugs are major problems in the treatment of this disease. So targeted therapy and reduction of drug resistance are the most important reasons for successful cancer treatment. [2]. One of the factors involved in both targeted therapy and reduction of drug resistance is microRNA. MicroRNAs are small, single-stranded, untranslatable RNAs and have between 18-24 nucleotides whose function is to bind the 3 ' UTR region of their target gene and regulate its expression by impairing the translation. miRNAs are important regulators in diverse biological processes of cancer, such as cell proliferation, apoptosis, angiogenesis, cell differentiation, adhesion and metastasis [3,4].

Properties and functions of miR-424
miR-424 is a member of the family of miR15/107. Members of this family have AGCAGC sequences in the Seed area and are involved in the cell division, apoptosis, stress responses, and cancer. miR-424 or its autologous miR-322 and miR-503 are encoded as a cluster by H19X on the Xq2603 chromosome. miR-424 is involved in cell cycle regulation, EMT, differentiation, hypoxia, proliferation, apoptosis, invasion, angiogenesis, and drug resistance and sensitivity. Transcription factor PU1 is one of the inducers of miR-424 [5]. In this study, we looked at the role of miR-424 in a variety of cancers and described the target genes of miR-424 and we have speci cally focused on its role in drug resistance and sensitivity.

Mirna Biogenesis
The construction of miRNAs begins with the transcription of RNA polymerase II. The resulting miRNA is the primary miRNA (pri-miRNA). Then there is Drosha enzyme which is a type III RNase. It is connected to the pri-miRNA with the help of cofactor protein DGCR8. The two RNase sites present in Drosha mediate the cleavage of 3/ and 5/ strand of pri-miRNA to generate pre-miRNA. Then, exportin 5 causes pre-miRNA to exit the nucleus into the cytosol. In the cytosol, the Dicer and TAR RNA binding protein (TRBP) bind to the pre-miRNA and cut the terminal loop and resulting in a miRNA duplex. In the next step, miRNA Duplex enters the RNA-induced silencing complex (RISC). Finally, the miRNA Duplex processor is performed in the RISK complex by argonaute (AGO) family of proteins and a mature miRNA is obtained [5].

Drug Resistance Or Sensitivity And Micrornas
Drug resistance is a major problem in patients with advanced cancer. It is the cause of 90% of deaths in patients who are resistant to chemotherapy drugs. MicroRNAs can play a role in drug resistance or sensitivity by targeting genes that are effective in responding to chemotherapy drugs. Another way that microRNAs can affect drug resistance and sensitivity is survival and apoptosis signaling pathways and drug transport routes [6]. The function of miR-424 in tumor inhibition or tumor induction as well as in drug resistance and sensitivity in a variety of cancers is listed.

Target Genes Of Mir-424 And Its Role In Drug Resistance And Sensitivity In Various Cancers
According to multiple studies and investigations, miR-424 plays a vital role in various cancers. (Fig. 1 The role of miR-424 through inhibition or induction of various genes in inhibition of proliferation, cell cycle, EMT and induction of apoptosis and differentiation), (Table 1)  In vitro [74] 4.1 Role of miR-424 in drug resistance and sensitivity of breast cancer The most common cancer diagnosed among women around the world is breast cancer. After lung cancer, breast cancer is the second leading cause of death among women. Epigenetic events as well as miRNA expression are the master regulators of tumorigenesis and add a further layer to the complexity of breast cancer pathogenesis. Studies show that miR-424 is highly expressed in breast cancer patients compared to healthy people and can discriminate early-stage breast cancer patients from healthy controls [7,8]. Even the presence of miR-424 in urine helps to differentiate between patients and healthy individuals [9,10]. Regarding the role of miR-424 in cell proliferation, it has been shown that miR-424 has an inhibitory role in cell proliferation by inhibiting CDK1 and YAP from the Hippo pathway and inhibiting p-ERK1/2 from the ERK pathway [11]. MiR-424 levels in breast cancer cells that express TRb ( thyroid receptor ) are increased by the T3 hormone and targets of that like CCND2, CDK6, Cdc25, E2F3, c-Myb and CHK1 that all of them involved in proliferation are declining [12].
However, in breast cancer patients with a negative lymph node that constitutes 60% of all breast cancer cases, it has been shown that there is no association between proliferation and miR-424 [13]. Hyperglycemic conditions reduce miR-424 and the inhibitory effect of miR-424 is removed from cdc42 in this way, STAT5 is activated and causes the expression of Prdm14 gene. Increased Prdm14 expression is associated with invasion and poor prognosis [14]. MiR-424 increases metastasis by targeting Smad7 and Smurf2 because these are two negative regulators of the TGFß pathway. Binding of TGFß to its receptors is one of the signaling pathways involved in metastasis [14]. Another role of miR-424 is inhibition of CDC25A, BCL2, IGF1R genes. Increased expression of these three genes is associated with a poor prognosis [15]. Some chemotherapy drugs, such as paclitaxel (PTX),

Role of miR-424 in drug resistance and sensitivity of gastric cancer
Gastric Cancer is one of the most common and lethal malignancies worldwide [18]. The survival time in these patients is 5 years. Because of the high rates of postsurgical recurrence and metastasis, the prognosis of GC patients diagnosed as advanced-stage is pessimistically bad [5,19] There are many signaling pathways that play a role in the onset and progression of cancers. The Hippo pathway plays an important role in cell growth and metastasis and LATS1 (Large tumor suppressor kinase 1) is one of the main members of this pathway.
CircRNAs are a type of ncRNAs that controls gene expression [20]. CircLARP4 is a type of circRNAs that in normal cells LARP4 is high and decreases miR-424. Finally, LATS1 increases and YAP pathway decreases. But in gastric cancer, the opposite happens. CircLARP4 may function as a tumor suppressive factor in GC via regulation of miR-424/LATS1/YAP signaling pathway [5]. LncRNANNT-AS1 is a type of lncRNA (Long-noncoding RNA) that has a high expression in GC and this high expression is accompanied by a bad prognosis. LncRNAs are involved in regulating gene expression and can activate or suppress gene expression through a variety of mechanisms [19]. NNT-AS1 has been found to act as oncogene in human cancer and is a powerful cell cycle regulator through the miR-424/E2F1. LncRNANNT-AS1 inhibition inhibits the cell cycle in the G0/G1 phase and also inhibits tumor proliferation and invasion. NNT-AS1 connects to the miR-424 and NNT-AS1/miR-424 targeted E2F1 in the cycle progression regulation of GC cells. E2F1 is an important transcription factor in regulating cell cycle and apoptosis [19]. MiR-424 is involved in the drug resistance of GC patients who are being treated based on platinum chemotherapy drugs. One of the target genes of miR-424 is SMURF1, which belongs to the NEDD4 family and is involved in ubiquitinase activity. In patients who resistance to cisplatin, the reduction of miR-424 increased the SMURF1, and it also stimulated the RhoA. RhoA belongs to the Rho GTPase family and many studies have shown that it plays a role in drug resistance [21]. Another study showed that a decrease

Role of miR-424 in drug resistance and sensitivity of nonsmall cell lung cancer (NSCLC)
The rst common cancer in the world is lung cancer. Non-small Cell Lung Cancer (NSCLC) is a type of lung cancer whose survival time is only 15%. NSCLC accounts for approximately 85% of lung cancer cases [25,27].  [30,31]. and expression of that was inversely correlated with Ki-67. Ki-67 protein is strictly associated with cell proliferation and is frequently used as a proliferation biomarker. MiR-424 represses cell cycle/E2F signaling by direct targeting Akt3 and E2F3 to suppress HCC growth. As well as it has been proved that cyclin D and GSK3 were regulated by miR-424 and Akt3, while cyclin E, c-Myc and Cdc-2 were regulated by miR-424 and E2F3 [31]. recurrent tumors in patients following liver transplantation (LT) have been shown to reduce the expression of miR-424. Collected information demonstrates that miR-424 expression is an important indicator for predicting tumor recurrence in patients with HCC following LT [32]. MiR-424 levels in HCC cells that express TRb (thyroid receptor) are increased by the T3 hormone and targets of miR-424 like CCND2, CDK6, Cdc25, E2F3, c-Myb and CHK1 that all of them involved in proliferation are declining [12]. The rst line of treatment of HCC patients is chemotherapy with Sorafenib. Resistance to sorafenib is one of the problems facing these patients. The protein associated with drug resistance in this patient is CBX4 (choromobox homolog 4). CBX4 is a Polycomb protein and has high expression and poor prognosis in HCC patients but miR424 reduces CBX4 expression and causes drug sensitivity [33].

Role of miR-424 in drug resistance and sensitivity of glioma
Glioma is one of the most important tumors of the nervous system. About 15% of patients die 1 year after diagnosis [34,35]. One of the genes involved in the molecular mechanism of glioma is PVT1 (Plasma-cytoma and dCCA (40%) represent the majority of cholangiocarcinoma cases, while iCCA is less than 10% of total. In iCCA, ARK5 expression is high, which causes metastasis in these patients. But miR-424-5p prevents EMT and metastasis by targeting ARK5 [41]. Chemotherapy with cisplatin and gemcitabine is the rst line of treatment for CCA patients. But it has been found that an lncRNA called LIN00665 makes these cells resistant to gemcitabine.

Blood Malignancy
5.1 Role of miR-424 in drug resistance and sensitivity of acute myeloid leukemia (AML) Acute myeloid leukemia (AML) is a clonal and heterogeneous malignancy characterized by deregulated proliferation and inhibited differentiation of hematopoietic progenitors [43]. NPM1 (Nucleophosmin 1) mutation is seen in 60% of CN-AML (Cytogenetics normal) cases and miR-424 levels are reduced in CN-AMLs that have the NPM1 mutation. This reduction indicates the role of miR-424 in leukemogenesis [44]. Chronic myeloid leukemia (CML) is a biphasic hematopoietic stem cell (HSC) myeloproliferative disorder. Its main feature is ABL/BCR oncogene. The ABL-BCR gene fusion produces a protein that is an active tyrosine kinase that causes proliferation and reduction of apoptosis. Therefore, by inhibiting the activity of this tyrosine kinase, proliferation can be prevented and apoptosis can be induced. miR-424 targets ABL and induces apoptosis and inhibits proliferation. And also in this way it sensitizes cells to imatinib [47]. Another factor related to drug resistance CML patients is Cobll1, which increases in blast crisis. MiR-424 destroys drug resistance by targeting this agent [48].

Future Perspective
As stated above miR-424 has been studied more in solid tumors. One of our hypotheses is the use of this microRNA in the treatment of leukemia patients. The most important mechanisms that can be used are to induce apoptosis and inhibit cancer cell proliferation. Forrest et al. Showed that increased expression of miR-424 induces cycle arrest in the G1 phase in THP1 cell line. Also, Oshrat Hershkovitz-Rokah and colleagues showed that miR-424 inhibits proliferation in the K562 cell line. MiR-424 induces these effects by acting on cell cycle regulators. Other effects of miR-424 in the treatment of leukemia include inhibition of expressed oncogenes. For example, LYPLAs increase expression in CLL and inhibit apoptosis by binding to CD95 so miR424 induces apoptosis by inhibiting LYPLAs. WEE1 is one of the genes that increase in AML and it is one of the miR-424 targets Therefore, by increasing the expression of miR-424, WEE can be inhibited and its effects can be prevented. We know that miR-424 has 4 methylated CpG sites and methylation reduces gene expression.
Therefore, one of the ways to increase the expression of miR-424 is the use of hypomethylation drugs such as 5-Azacitidine. Has been shown using of azacitidine increase the expression of miR-127 and 149 in breast cancer it also increases the expression of miR-130 in ovarian cancer. AZA is monophosphated by the uridine cytidine kinase and then diphosphate and trisphosphates by pyrimidine monophosphate kinase and pyrimidine diphosphate kinase, respectively. 3-phosphate AZA (5-aza-CTP) enters RNA. The introduction of 5-aza-CTP into RNA disrupts protein synthesis, which promotes apoptosis. A minority of this drug is converted to 5-aza-dCTP, the 3-phosphate form of decitabine, by the enzyme ribonucleotide reductase, and enters DNA during replication, binds to DNMT1, and inhibits this enzyme. Chun-Te Wu et al. Showed that expression of miR-424 increased in cancer cells that had DNMT inactivated. Therefore, the use of 5-AZA strongly increases the expression of miR-resistance or sensitivity in various cancers drug resistance can be eliminated by increasing or decreasing its expression.

Conclusion
microRNAs play a major role in tumorigenesis, proliferation, cell differentiation, apoptosis and metastasis. miR-424 plays a major role in important processes such as cell cycle, EMT, hypoxia, tissue differentiation, tumor onset and progression, and tumor inhibition. In this study, we showed that miR-424 exhibits different functions in different cancers by targeting different genes. Due to the fact that miR-424 has 4 CpG sites and is a hypermethylated microRNA it can be used to increase its expression and increase its function by using hypomethylating drugs such as 5-AZA or decitabine …And by targeting genes that play an oncogenic role in leukemia, it can play an important role in inhibiting cancer.
According to the above, miR-424 can inhibit proliferation in cancer cells by targeting cell cycle regulators such as cyclin E1, cyclin D2, cyclin D, cyclin A2, CDK1, CDK6, and CDC25A. Each of these plays an important role in cell cycle. E2F1 gene is also a regulator of cell cycle, which can be one of the important goals of miR-424 in reducing cell growth. on the other hand, could play an important role in increasing cancer cell death by targeting genes involved in apoptosis these genes include Bcl2 and Akt3. In relation to the role of miR424 in the treatment of cancers, we can mention the suppression of oncogenes. WEE1 and PLAG1 are genes that are overexpressed in blood cancers miR-424 by targeting these can play a role in cancer control. One of the important recent ndings is the targeting of ABL-BCR in CML patients, which makes patients sensitive to imatinib. miR424 disrupts the junction between PD1-PDL1 and CTLA4-CD80 Eventually, the cells of the immune system will be able to ght the cancer cells more e ciently. MiR-424 is also involved in drug resistance, which can be eliminated by increasing or decreasing its expression according to the type of cancer. (Fig. 2   The role of miR-424 through inhibition or induction of various genes in inhibition of proliferation, cell cycle, EMT and induction of apoptosis and differentiation