Long non-coding RNA MIR4435-2HG: a key molecule in progression of cancer and non-cancerous disorders

MIR4435-2HG (LINC00978) is a long non-coding RNA (lncRNA) that acts as an oncogene in almost all cancers. This lncRNA participates in the molecular cascades involved in other disorders such as coronary artery diseases, osteonecrosis, osteoarthritis, osteoporosis, and periodontitis. MIR4435-2HG exerts its functions via the spectrum of different mechanisms, including inhibition of apoptosis, sponging microRNAs (miRNAs), promoting cell proliferation, increasing cell invasion and migration, and enhancing epithelial to mesenchymal transition (EMT). MIR4435-2HG can regulate several signaling pathways, including Wnt, TGF-β/SMAD, Nrf2/HO-1, PI3K/AKT, MAPK/ERK, and FAK/AKT/β‑catenin signaling pathways; therefore, it can lead to tumor progression. In the present review, we aimed to discuss the potential roles of lncRNA MIR4435-2HG in developing cancerous and non-cancerous conditions. Due to its pivotal role in different disorders, this lncRNA can serve as a potential biomarker in future investigations. Moreover, it may serve as a potential therapeutic target for the treatment of various diseases.


Introduction
Genetic alterations are one of the primary causes of cancer, leading to the deregulation of gene networks [1][2][3]. In recent years following developments in RNA sequencing technologies, this insight came into being that a large part of the genome transcribes into non-protein-coding RNAs [4]. Long non-coding RNAs (lncRNAs) are a subclass of functional RNAs which are longer than 200 nucleotides in sequence length without a protein-coding capacity [5][6][7]. In the beginning, lncRNA transcripts were regarded as 'transcriptional noise' or 'junk' . Subsequent investigations revealed that lncRNAs are key players in human disorders, particularly in malignant conditions [8,9]. Although lncRNAs do not translate into proteins, they play a meaningful function in regulating gene expression through different mechanisms such as remodeling of chromatin, modulating the activity of transcription factors, epigenetic regulation, post-transcriptional, and cell cycle regulation [10,11]. MIR4435-2 Host Gene (MIR4435-2HG), also named LINC00978, AK001796, AWPPH, MIR4435-1HG, MORRBID, and AGD2, is an lncRNA that resides on chromosome 2q13 region and includes ten exons. MIR4435-2HG has 108 transcripts produced through alternative splicing (https:// www. ensem bl. org/ Homo_ sapie ns/ Gene/ Summa ry? db= core;g= ENSG0 00001 72965;r= 2: 11100 6015-11152 3376). Previous studies have reported that the MIR4435-2HG has an oncogenic role in the progression of different cancer types. In addition to the role of MIR4435-2HG in tumorigenesis, some studies suggest that it is involved in the pathogenesis of non-cancerous conditions such as coronary artery diseases [12], osteonecrosis [13], osteoarthritis [14], osteoporosis [15], and periodontitis [16]. Due to the important regulatory roles of MIR4435-2HG, in the present review, we provide comprehensive information about its function in cancer and other diseases.

MIR4435-2HG and cancer
Previous studies have shown that the expression level of MIR4435-2HG was upregulated in almost all cancers. MIR4435-2HG upregulation can promote tumor progression by increasing cell proliferation, invasion, migration, epithelial-mesenchymal transition (EMT), chemoresistance and suppression of apoptosis.

Colorectal cancer (CRC)
Overexpression of MIR4435-2HG has been reported in CRC tissues and cell lines in several studies [17][18][19][20][21][22]. Wen et al. have demonstrated that upregulation of MIR4435-2HG in CRC tissues was significantly correlated with the TNM stage [17]. Cancer-developing conditions such as chemoresistance, invasion, metastasis, migration, cancer stemness, and EMT can be regulated by Yes-related protein 1 (YAP1) transcription factor [23]. Dong et al. showed that MIR4435-2HG could regulate the expression of miR-206. On the other hand, they also indicated that YAP1 was a potential target for miR-206 ( Fig. 1 and Table 1). MIR4435-2HG knockdown could block invasion, migration, and cell proliferation through the miR-206/YAP1 axis in the HCT116 and SW620 cell lines [18]. Previous studies reported that expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its regulator, heme oxygenase-1 (HO-1), increased after treating cancer cells with chemotherapeutic agents. These factors regulate the detoxification process and antioxidant enzymes, which results in the reduction of drug effects and an increase in drug resistance [24,25]. In HCT116R cells (a cisplatin-resistant cell line), knockdown of MIR4435-2HG significantly induced cisplatin sensitivity, enhanced apoptosis, and inhibited cell proliferation via modulating Nrf2/HO-1 cascade (Fig. 1). Hence, it seems that MIR4435-2HG is involved in oxidative stress [19]. Another experiment has indicated that in patients with colon cancer, serum levels of glucose transporter 1 (GLUT-1) and MIR4435-2HG were significantly higher than healthy controls. Moreover, silencing MIR4435-2HG inhibits cell proliferation through downregulation of GLUT-1 in the HT-29 cancerous cell line ( Fig. 1) [20]. In our previous study, we showed a positive correlation between β-catenin and MIR4435-2HG expression that indicated mentioned lncRNA might regulate the Wnt signaling pathway via stabilization of β-catenin, which can lead to the progression of CRC [21]. Shen et al. showed that high expression of MIR4435-2HG was remarkably related to clinicopathological features, including stage, tumor size, tumor node and lymph node metastasis. Their results showed that the patients Fig. 1 Schematic representation of MIR4435-2HG functions in CRC. Increased level of MIR4435-2HG blocks miR-206 which leads to elevation of YAP1 and enhanced cell invasion, migration, proliferation and chemoresistance. In HCT116R cells, the activation of Nrf2 pathway leads to drug resistance; however, knockdown of MIR4435-2HG sensetives cancer cell to chemotherapy through inhibition of Nrf2. Furthermore, MIR4435-2HG can increase CRC progression via promoted GLUT-1 with higher levels of MIR4435-2HG had a worse prognosis than the patients with lower expression levels. In addition, MIR4435-2HG silencing remarkably reduced cell proliferation and enhanced cell apoptosis [22]. To conclude, MIR4435-2HG can promote CRC via different mechanisms.

Gastric cancer (GC)
Several studies reported that the expression level of MIR4435-2HG was significantly increased in GC tissues, plasma samples, and different cell lines compared to the normal controls [26][27][28][29]. TGF-β/SMAD is one of the pathways involved in the progression of metastasis in gastric cancer [30]. Min et al. showed that MIR4435-2HG expression was significantly correlated with TNM stage, tumor size, and lymphatic metastasis. Knockdown of MIR4435-2HG elevated the expression of E-cadherin protein while the expression levels of vimentin, slug, N-cadherin, and twist proteins were inhibited. On the other hand, MIR4435-2HG knockdown leads to the inhibition of transforming growth factor beta (TGF-β) and phosphorylated SMAD2 (p-SMAD2) in gastric cancer cell lines. This observation suggests that knockdown of MIR4435-2HG can elevate EMT, and apoptosis and inhibit cell cycle progression, invasion, and migration via the regulation of TGF-β/SMAD signaling pathway ( Fig. 2) [27,31,32]. Yuan et al. reported that MIR4435-2HG could target miR-497. Interestingly, tropomyosin receptor kinase C (NTRK3) plays a critical role in cancer progression and is a direct target of miR-497. MIR4435-2HG acts as a molecular sponge of miR-497, which leads to an increase in NTRK3. It can be concluded that the elevation of MIR4435-2HG could enhance tumorigenesis via miR-497/NTRK3 axis ( Fig. 2 and Table 1) [28]. Gao et al. demonstrated that high expression of MIR4435-2HG was associated with poor survival rate in GC patients. They also reported the enhancement of apoptosis and suppression of cell proliferation, migration, invasion and EMT after MIR4435-2HG knockout in gastric carcinoma cells. It was suggested that overexpression of MIR4435-2HG affects the expression of SRY-box transcription factor 4(SOX4) via sponging miR-138-5p. Therefore, MIR4435-2HG plays an oncogenic role in GC by targeting the miR-138-5p/SOX4 axis ( Fig. 2 and Table 1) [29].
In vitro and in vivo studies performed by Zhao et al. showed that upregulation of MIR4435-2HG increased migration, cell proliferation, metastasis, and tumor growth in hepatocellular carcinoma cells via regulating the interaction of Y-box binding protein 1 (YBX1) with snail family transcriptional repressor 1 (SNAIL1) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha)PIK3CA(. Previous studies reported that YBX1 could induce EMT, SNAIL1 mRNA translation, and promote metastasis. YBX1 can stimulate PIK3CA transcription and enhance the PI3K/AKT signaling pathway by binding its promoter in cancer cells (Fig. 3) [34,39,40]. Another study showed that miRNA-487a and MIR4435-2HG were elevated in HCC tumor samples compared to adjacent tissues, and a positive correlation was detected between the genes expression. The overexpression of MIR4435-2HG in the HCC SNU-398 and SNU-182 cell lines promoted cell proliferation through upregulation of miRNA-487a ( Fig. 3 and Table 1 Table 1) [38].

Lung cancer (LC)
Qiaoyuan et al. showed that the MIR4435-2HG expression was downregulated after treating LC cells with resveratrol. They showed that cell cycle arrest occurred in G0/G1 phase following MIR4435-2HG knockdown. They also indicated that inhibition of MIR4435-2HG in lung cancerous cell lines enhanced the anticancer effects of resveratrol [43]. Another experiment revealed EMT suppression following MIR4435-2HG knockdown. Notably, MIR4435-2HG prevents the destruction of β-catenin by the proteasome system, however, MIR4435-2HG knockdown resulted in the decreased β-catenin transactivation and subsequent inhibition of the Wnt/β-catenin signaling pathway [44]. Silencing of MIR4435-2HG promoted the expression of miR-204 and therefore decreased the expression of cyclin dependent kinase 6 (CDK6), resulting in the enhancement of cell proliferation, invasion and migration in the A549 cell line [46].

Breast cancer (BC)
One of the pioneer investigations for the assessment of MIR4435-2HG has been conducted in the BC tissues and cell lines by Lin et al. They indicated that MIR4435-2HG was over-expressed in breast cancer tissues and cell lines compared with corresponding controls and therefore may act as an oncogene. They reported that hormone receptor status and MIR4435-2HG expression were negatively correlated [47]. Consistent with the above study, Jing et al. showed that MIR4435-2HG was upregulated in breast cancer tissues and cell lines. They indicated that MIR4435-2HG could enhance many cellular parameters such as proliferation, EMT, migration, and invasion via regulating the miR-22-3p/TMEM9B axis ( Fig. 4 and Table 1) [48]. Liu et al. demonstrated that the plasma level of MIR4435-2HG was remarkably higher in patients with Triple-negative breast cancer (TNBC) than healthy  [49].

Prostate cancer (PC)
The expression level of MIR4435-2HG is reported to be enhanced in prostate cancer. It is suggested that this lncRNA causes cancer progression through various mechanisms such as FAK/AKT/β-catenin and TGF-β1 pathways [52,53]  and TGF-β1 expression level was positively correlated to MIR4435-2HG. They reported that the effects of MIR4435-2HG on cell migration and invasion decreased following the inhibition of TGF-β1 (Fig. 5) [53].

Glioma cancer
One of the members of the TGF-β/Smad signaling pathway is transforming growth factor-beta receptor type II (TGFBR2) that acts as a cancer suppressor [57]. Xu et al. reported that the expression level of MIR4435-2HG was upregulated in patients with glioblastoma (GBM). In contrast, the expression level of miR-1224-5p was suppressed in GBM cancer cell lines. They used bioinformatics predictions and in vitro methods to show that this lncRNA acts as a sponge for miR-1224-5p. On the other hand, one of the direct targets of miR-1224-5p is TGFBR2 gene, and the mRNA level of its gene was enhanced in GBM cancer cell lines. Taken together, it can be argued that MIR4435-2HG can promote cancer progression by targeting the miR-1224-5p/TGFBR2 axis (Table 1) [58]. TGF-β signaling pathway is an essential factor for EMT. In patients with glioma cancer, a positive correlation was detected between the plasma levels of TGF-β and MIR4435-2HG. Therefore, it may be concluded that MIR4435-2HG is involved in the progression of glioma occur through TGF-β signaling pathway [59]. As a transcription coactivator, tafazzin, phospholipid-lysophospholipid transacylase (TAZ) is one of the most important downstream effectors of the Hippo signaling pathway that regulates cell proliferation, migration, and apoptosis [60]. The expression level of TAZ was upregulated in the brain tissue of glioma patients. Shen et al. indicated that the expression of MIR4435-2HG was positively correlated with TAZ expression, while miR-125a-5p expression was negatively correlated with TAZ expression in the brain tissue (Table 1) [61].

Leukemia cancers
Rho-associated protein kinase 2 (ROCK2) is an important therapeutic target, and its upregulation was confirmed in many cancers, including T-cell acute lymphoblastic leukemia (T-ALL) [62,63]. The expression of MIR4435-2HG and ROCK2 was positively correlated in patients with T-ALL. The overexpression of MIR4435-2HG remarkably increased ROCK2 expression at both protein and mRNA levels; also, the overexpression of ROCK2 significantly upregulated MIR4435-2HG expression in T-ALL cells. It seems that MIR4435-2HG inhibits apoptosis and increases cell proliferation in T-ALL cells through interactions with ROCK2 [64]. Zhigang et al. reported that MIR4435-2HG was overexpressed in human acute myeloid leukemia (AML), which was correlated with a poor survival rate. They showed that MIR4435-2HG acts as a transcriptional repressor of BIM pro-apoptotic gene and, via this mechanism, regulates the lifespan of myeloid cells. In vivo study demonstrated that the loss of MIR4435-2HG in genetic mice models promoted the expression levels of BIM that increased cell death in mature and immature myeloid cells [65].
Knockdown of lncRNA MIR4435-2HG regulates cell cycle, cell proliferation, and cells growth via modulating MDM2/p53 signaling pathway in patients with ESCC [69]. Wang et al. demonstrated that the expression level of MIR4435-2HG was upregulated in BCa tissues and cell lines compared to the control samples. They indicated that MIR4435-2HG served as a competing endogenous RNA (ceRNA) and sponged miR-4288. Using in vitro methods, they showed that knockdown of MIR4435-2HG significantly inhibited tumor growth by sponging miR-4288 [71]. Shu et al. reported that the high expression level of MIR4435-2HG was significantly associated with advanced tumor metastasis node in patients with HNSCC. In vivo and in vitro investigations indicated that knockdown of MIR4435-2HG decreased invasion, cell proliferation, and EMT in HNSCC cancer cell lines. As shown in Table 1, MIR4435-2HG executes these functions via modulating miR-383-5p. On the other hand, one target of miR-383-5p is RNA binding motif protein 3 (RBM3). It can be concluded that HNSCC progression can be regulated by MIR4435-2HG/miR-383-5p/RBM3 axis [70]. It has been established that flotillin-2 (FLOT2) has a critical role in the progression of human cancers through different mechanisms [74,75]. According to bioinformatics analysis, miR-802 targets FLOT2 gene. Han et al. showed that MIR4435-2HG sponged miR-802 which leads to increased expression of FLOT2 and tumor progression (Table 1) [72]. The experimental studies showed that MIR4435-2HG inhibited apoptosis while facilitating migration, and cell proliferation in NPC cells. The mentioned lncRNA exerts this function by inhibiting of phosphatase and tensin homolog (PTEN) as a tumor suppressor gene [73].

MIR4435-2HG and non-cancerous diseases
Accumulating evidence reveals that MIR4435-2HG not only is involved in cancer progression but also plays a critical role in the development of other diseases. In this section, we investigated the role of MIR4435-2HG in non-cancerous disorders.

Coronary artery diseases (CAD)
The serum level of MIR4435-2HG remarkably increased in CAD patients compared to healthy controls. Clinical studies revealed that treatment with statins drugs, atorvastatin, and rosuvastatin, reduced MIR4435-2HG level significantly in CAD patients. This reduction was mainly observed in patients treated with rosuvastatin [12].

Osteoarthritis
The MIR4435-2HG transcript level was lower in plasma samples of patients with osteoarthritis than in healthy controls. Knockdown of MIR4435-2HG decreased proliferation and promoted cell apoptosis in chondrocytes, while overexpression of MIR4435-2HG enhanced proliferation of chondrocytes and suppressed apoptosis. After treatment with anti-inflammatory drugs (such as naproxen), reducing the joint burden and exercise, the expression level of MIR4435-2HG was increased [14].

Osteoporosis
Guang et al. reported that MIR4435-2HG was downregulated in plasma of patients suffering osteoporosis compared to healthy controls. They also showed a positive correlation between MIR4435-2HG and bone turnover markers, procollagen-1 N-terminal peptide (P1NP) and tartrate-resistant acid phosphatase 5b (TRACP-5b). The phenotype of osteoblasts can be regulated by type I collagen α1/α2 ratio. Knockdown of MIR4435-2HG suppressed α1 expression but upregulated α2. In contrast, upregulation of MIR4435-2HG elevated α1 but decreased α2. It can be concluded that MIR4435-2HG can affect the phenotype of osteoblasts via alteration in type I collagen α1/α2 ratio [15].

Osteonecrosis of the femoral head (ONFH)
Runt-related transcription factor 2 (RUNX2) has been identified as a marker of osteoblastic differentiation [76,77]. Decreased expression of RUNX2 led to the development of non-traumatic ONFH [78]. The investigation showed that the expression level of MIR4435-2HG in both serum and mesenchymal stem cells (MSCs) samples was significantly downregulated. Silencing and overexpression of MIR4435-2HG in hMSC-BM cells could lead to inhibition and promotion of RUNX2 expression, respectively. To conclude, MIR4435-2HG participates in the progression of non-traumatic ONFH through elevated RUNX2 [13].

Periodontitis
Xiaofang et al. demonstrated that the expression level of MIR4435-2HG was elevated in plasma samples of patients with periodontitis compared to healthy controls. They showed that the expression level of MIR4435-2HG was remarkably downregulated after treatment (administration of both oral and topical antibiotics, root planning and scaling). However, after two years of follow-up, the expression of MIR4435-2HG was significantly elevated in patients with recurrence of periodontitis [16].

Human immunodeficiency virus (HIV) infection
Expression of MIR4435-2HG is also involved in immune responses against HIV-1 infection. Hartana et al. investigated the expression level of this lncRNA in myeloid dendritic cells (mDCs) obtained from HIV-1 elite controllers (ECs), in whom the virus replication is under control in the absence of antiretroviral treatment, compared to HIV-1-negative healthy controls and those who were treated using antiretroviral therapy. They found that regulatory associated protein of MTOR complex 1 (RPTOR) gene, a major component of the mammalian target of rapamycin (mTOR) signaling pathway, via induction of an epigenetic alteration. Taken together, upregulation of MIR4435-2HG in mDCs from ECs influences immunometabolic activities through different mechanisms, including altered glycolysis, oxidative phosphorylation, epigenetic modifications [79].

Diagnostic value of MIR4435-2HG
Several studies have shown that evaluating lncRNAs expression in serum, plasma, and other body fluids may serve as diagnostic or prognostic biomarkers in different disorders that are non-invasive and convenient compared to biopsy and imaging methods. For example, Fu et al. showed the elevated levels of MIR4435-2HG both in tumor tissues and serum samples of gastric cancer patients. They suggested that this lncRNA may be a potential biomarker in gastric cancer [27]. Receiver Operating Characteristic (ROC) Curve Analysis plays a central role in evaluating the diagnostic ability of tests to discriminate the true state of subjects. The diagnostic value of MIR4435-2HG has been evaluated in some tumors and other diseases. MIR4435-2HG can differentiate tumor samples from corresponding controls and distinguish disease status in other non-cancerous conditions. As shown in Table 2, MIR4435-2HG has the best diagnostic power in osteoarthritis subjects.
The overexpression of MIR4435-2HG in all cancer types that have been studied so far indicates the key role of this lncRNA in cancer progression as an oncogene. Cell proliferation, EMT, invasion, migration, and suppressed apoptosis are key hallmarks of cancer that can be affected by MIR4435-2HG expression. Besides, several studies confirmed the effectiveness of MIR4435-2HG silencing in inhibiting tumor growth in colorectal cancer, esophageal squamous cell carcinoma, gastric cancer, hepatocellular carcinoma, lung cancer, neuroglioma, and prostate cancer.
In contrast, in non-cancerous conditions such as periodontitis, osteoporosis, osteoarthritis, and osteonecrosis of the femoral head, the expression level of MIR4435-2HG has been downregulated. However, in coronary artery diseases, the expression level of MIR4435-2HG was elevated.
The expression level of MIR4435-2HG alters in response to many drugs, including statins (atorvastatin and rosuvastatin), oral and topical antibiotics, anti-inflammatory drugs (such as naproxen) and chemopreventive agent resveratrol. This subject indicates that MIR4435-2HG has a pivotal function in molecular mechanisms involved in disease development. Therefore, it can be concluded that MIR4435-2HG may serve as a potential therapeutic target for the treatment of various diseases.
Despite fundamental improvement in cancer diagnosis methods, recurrence and metastasis occur in many patients suffering from cancer, therefore, the discovery of new diagnostic biomarkers could be helpful in this regard [80,81]. Moreover, according to the literature, the diagnostic value of MIR4435-2HG was acceptable in both cancerous and non-cancerous conditions. Detection and measurement of MIR4435-2HG in body fluids such as serum, plasma, and joint fluid suggest that this lncRNA could be used as a non-invasive marker.
Although previous studies have emphasized the role of MIR4435-2HG in the progression of different diseases, few studies has been conducted to describe the possible mechanisms involved in its regulation. Therefore, understanding the mechanisms involved in MIR4435-2HG regulation may shed light on the diagnosis and treatment of several related diseases.
In conclusion, MIR4435-2HG has a pivotal role in cancer progression and critical function in non-neoplastic conditions. Future studies may explain the role of this lncRNA as a potential biomarker and therapeutic target in human disorders, especially in tumors.