The functions and targets of miR‐212 as a potential biomarker of cancer diagnosis and therapy

Abstract Cancer is a major health problem worldwide. An increasing number of researchers are studying the diagnosis, therapy and mechanisms underlying the development and progression of cancer. The study of noncoding RNA has attracted a lot of attention in recent years. It was found that frequent alterations of miRNA expression not only have various functions in cancer but also that miRNAs can act as clinical markers of diagnosis, stage and progression of cancer. MiR‐212 is an important example of miRNAs involved in cancer. According to recent studies, miR‐212 may serve as an oncogene or tumour suppressor by influencing different targets or pathways during the oncogenesis and the development and metastasis of cancer. Its deregulation may serve as a marker for the diagnosis or prognosis of cancer. In addition, it was recently reported that miR‐212 was related to the sensitivity or resistance of cancer cells to chemotherapy or radiotherapy. Here, we summarize the current understanding of miR‐212 functions in cancer by describing the relevant signalling pathways and targets. The role of miR‐212 as a biomarker and its therapeutic potential in cancer is also described. The aim of this review was to identify new methods for the diagnosis and treatment of human cancers.


| INTRODUC TI ON
For many years, cancer has been the main cause of death worldwide. 1 Recently, a large number of researchers have devoted themselves to the identification of targets or pathways involved in cancer and the development of targeted therapies that may efficiently kill cancer cells. Abnormal expression of miRNAs can be seen in different types of human cancers and correlates with proliferation, differentiation, invasion and other biological aspects of cancer. 2 miRNAs, about 18-25 nucleotides in length, are non-coding RNA molecules that can regulate the expression of genes involved in various biological processes, including proliferation, apoptosis, differentiation and survival of cellular processes, at the post-transcriptionally level. 3,4 miRNAs can participate in both physiological and pathological processes, such as tumours development, via interaction with the 3′ untranslated regions (UTRs) of their target mRNAs. 5,6 According to the role they play in cancers, miRNAs are classified as tumour promoters or tumour suppressors. 7 For instance, miR-802, as an oncogenic miRNA, promotes the proliferation of osteosarcoma (OS) cells via the suppression of p-27, which is a kind of cell-cycle inhibitor, 8 while miRNA-223 acts as a tumour suppressor in non-small-cell lung cancer (NSCLC) by interacting with the insulin-like growth factor-1 receptor. 9 It was also recently demonstrated that miRNAs may play a role in drug resistance. 10 miR-212, found on chromosome 17q13.3, shares highly conserved sequences with miR-132 among vertebrates. miR-132 and miR-212 play an essential role in different aspects of neural development, maturation, morphogenesis and function. Therefore, the abnormal expression of miR-132 and miR-212 may cause a series of neurodegenerative diseases, such as Alzheimer's disease, epilepsy, tauopathies and schizophrenia. Thus, miR-132/212 is sometimes called 'neurimmiR'. 11 Although the initial research on miR-212 emerged from studies performed in the neuronal context as well as studies on inflammation and other biological (dys)functions, studies on tumorigenesis and cell transformation have become the most popular in recent miR-212 research. 11 More and more studies are reporting the abnormal expression of miR-212 in various cancers and that miR-212 can act as a marker to diagnose or predict the outcome of cancer. In previous studies, miR-212-3p/5p was reported to be suppressed in hepatocellular carcinoma (HCC), 12 ovarian cancer 13 and gastric cancer (GC) 14 and showed antitumour effect. Two other studies indicated that miR-212 functions as an oncogene, with a higher expression in pancreatic cancer. 15,16 The different functions performed by miR-212 in cancer depend on the tumour types, targets or pathways involved. 17 In addition, evidence has emerged that the decreased expression of miR-212-3p may be due to DNA hypermethylation, which weakens the effect of miR-212-3p and regulates the biological characteristics of tumour cells. 18 However, another study found that it was histone modifications, not DNA hypermethylation that led to the decreased expression of miR-212-3p in lung cancer. 19 Recently, growing evidence has indicated that miR-212 affects the response to radiotherapy or chemotherapy. This review discusses the pathways or targets of miR-212 involved in cancer, summarizes its function as a marker for the diagnosis or prediction of the outcome of cancer and, finally, analyses the therapeutic potential of miR-212 for malignant tumours.

| miR-212 and cell apoptosis
Apoptosis, a process characterized by programmed cell death, occurs in both the physiological state, such as for maintaining cell populations in tissues, as well as pathological states, such as during injured cells. 20,21 Both intrinsic and extrinsic pathways may lead to apoptosis. When stimulated by specific growth factors or cytokines, apoptosis usually occurs through the extrinsic pathway. To our knowledge, not only precancerous cells, but also tumour cells can be induced to undergo apoptosis by p53. 22 Recent research showed that miR-212 is associated with cancer cells apoptosis through various targets.  25 It was also reported that CCND3, encoding protein cyclin D3, played an important role in controlling the advancement through the G0/G1 phase of the cell cycle. 26 XIAP, a key molecule of the cell apoptosis pathway, could prohibit cell apoptosis by inhibiting caspase-3, caspase-7 and caspase-9. [27][28][29] In renal cell carcinoma (RCC), it was found that miR-212-5p inhibited the progression of RCC cells via interaction with XIAP. 30 The remarkable mechanism of competing endogenous RNA (ceRNA) has recently attracted a lot of attention. One study found that miR-212-5p may induce degradation or inhibit the expression of NDUFA4. Moreover, LncMIF-AS1 was found to promote the expression of NDUFA4 in GC cells by binding miR-212-5p, and the recovered NDUFA4 strengthened the proliferative ability and prevented apoptosis of GC cells in vitro. 31

| miR-212 and the cell cycle
A number of molecular pathways and checkpoints regulate the cell cycle in most adult mammalian cells. Many miRNAs take part in these pathways. For example, some miRNAs are involved in the anti-proliferation of cells by inhibiting mitogenic pathways responsive to the activation of cyclin-CDK complexes. In contrast, other miRNAs may facilitate tumour cell proliferation by interacting with CDK inhibitors, such as p53 and ATM/ATR. 32 SIRT1, which was shown to modulate both physiological and pathological processes in cells via acting on cell cycle proteins, including Rb, is now verified to be targeted by miR-212 in some cancers. 33,34 miR-212-3p was shown by Ramalinga et al 35 to prevent autophagy and angiogenesis while inducing cellular senescence by targeting SIRT1 in PCa. Likewise, it was demonstrated that miR-212-3p can suppress thyroid cancer cell growth through SIRT1. 36 One study conducted by Bo Hu demonstrated that miR-212-3p has an antitumour role in PCa by means of targeting MAPK1 37 . MAPK1, which belongs to the MAPK family, is closely associated with PCa, especially in the aspects of cell proliferation, cell cycle and apoptosis. 38 Ji-ping Zeng demonstrated that miR-212-3p suppressed GC growth by modulating P21 CIP1 and P27 kip1 . 39 It was verified that P21 CIP1 and P27 kip1 are associated with cell cycle arrest, and they showed a tendency for lower expression in GC. 40 Jong-Kook Park found that upregulated miR-132-3p/-212-3p in pancreatic ductal adenocarcinoma (PDAC) increased cell proliferation via targeting the tumour suppressor Rb1. 15 pRb, a protein produced by the gene Rb1, participates in the regulation of the cell cycle, specifically the transition of G1/S and G2/M. 41 In HCC, it is suggested that downregulated hsa-miR-212-3p expression can lead to overexpression of RBP2 and promote cell proliferation. The authors of the study that revealed this concluded that there was a link between the hsa-miR-212-3p/RBP2/CDKI pathways and progression of HCC. 42 Likewise, for lung cancer cells, miR-212/132 can inhibit the development of tumour cells and affect the cell cycle by targeting p21 and cyclin D1. 43 Thus, functional studies of miR-212 can help us better understand the role of miR-212 in cell cycle regulation.

| miR-212 and immune responses
miRNAs participate in both innate and adaptive immunity by interacting with various immune cells, including monocytes, macrophages, NK cells and T helper cells, in differentiation, activation and other functions. The relationship between miRNAs and immunity has important effects on cancer progression. 44 An earlier study on the biological functions of miRNA-212 found it to interact with the neurons as well as inflammation. 11 Studies performed recently demonstrated that miRNA-212-3p can interact with immune cells and inflammatory cytokines in the occurrence or development of cancer. It was shown that downregulated miRNA-212-3p regulates the development of PCa through the secretion of inflammatory cytokines via the NF-κB pathway. 45 Another study showed that the regulatory effect of miR-212-3p in CD80 expression can be disrupted by the SNP rs1599795 in the 3′-UTR of CD80, which may induce GC tumorigenesis. 46 Ding et al 47 suggested that IFN-γ could be used as an immunological method to treat pancreatic cancer as it inhibits miR-212-3p expression and the subsequent upregulation of RFXAP and MHC class II. It was also found that miR-212-3p may induce immunologic tolerance of pancreatic tumour cells by affecting dendritic cell functions. 48 The findings of these studies indicate that targeting miR-212 will become a promising cancer immunotherapy approach.

| miR-212 as a diagnostic or prognostic biomarker
Multiple data indicate that miRNAs participate in human carcinogenesis. It was shown that some miRNAs were significantly upregulated or downregulated in comparison with paired normal tissues in various cancers, emphasizing the tremendous potential of miR-NAs as diagnostic and therapeutic markers in cancer. Additionally, some miRNAs were found to be linked with the outcome of certain cancers. Importantly, these miRNAs can exist either in cells or in cell-free body fluids such as urine and saliva. This stability of miR-NAs in body fluids allows them to be easily tested as biomarkers of disease. 49 Emerging researches have reported that the aberrant expression of miR-212 can be seen in different cancers and can be used for the diagnosis or prediction of outcome of cancer. One study on 386 patients revealed that a miRNA signature, including hsa-miR-212, may help identify early-stage breast cancer. 50 After examining 45 PDAC and 20 adjacent normal pancreatic tissue specimens, miR-212-3p was found to correlate with tumour growth and disease stage in PDAC, and high levels of miR-212-3p were associated with poor outcome. 51 In oesophageal cancer (EC), one study involving 46 patients revealed that, with other factors, such as treatment or staging of comparable disease, high miR-212 expression was accompanied with poor outcome. 52 Contrarily, in acute myeloid leukaemia (AML), according to a study involving 576 patients, the high expression of miR-212 was associated with a better outcome of patients and its role in predicting cancer was found to not be affected by other factors. 53 One study, including 15 cases associated with lymph node (LN) metastasis and another 15 cases without LN metastasis, found that there was a link between downregulated hsa-miR-212 expression and LN metastasis in GC and suggested that the level of hsa-miR-212 may serve as a clinic marker for GC. 54 The effect of miR-212 on lung cancer is controversial. One study involving 418 lung adenocarcinoma (LUAD) patients found that high miR-212 levels predicted a poor recurrence-free survival (RFS), 55 Table 1.

| The Wnt signalling pathway
The dysregulation of the Wnt/β catenin pathway is believed to be central to the pathogenesis of cancer, chronic inflammation and F I G U R E 2 miR212 and Hedgehog pathway degenerative diseases. The phosphorylation, degradation and regulation of β catenin by Wnt are considered to form the core of this pathway. Without Wnt, Wnt target genes are significantly inhibited because of the persistent degradation of the β catenin protein by the Axin complex. [64][65][66] It was reported that the dysregulation of miR-212 participated in different cancers through the Wnt/β catenin pathway via specific targets (Figure 1).
Zhi-dong Lv and colleagues showed that miR-212-5p may target Prrx2, which plays an important role in the proliferation, invasion and migration of breast cancer cells via the Wnt/β-catenin signalling pathway. 56 Recently, miR-212-3p was found to regulate Wnt signalling pathways by acting on LEF-1, c-Myc and β-catenin in HCC, which is why miR-212-3p can inhibit the tumorigenesis and growth of HCC cells. 67 The inhibition of the Wnt/β-catenin signalling pathway by miR-212-5p was also found in AML, and FZD5 was reported as the target gene of miR-212-5p. 68

| The Hedgehog signalling pathway
The Hedgehog (Hh) pathway has as a role in embryonic development, tissue patterning and wound healing. 72 Aberrant functioning of this pathway is connected with the development of cancer in several organs. 73,74 The Hh pathway is often triggered by ligands. SHH, one of the identified ligands of the Hh pathway, is the most studied. 75 Without SHH, PTCH1 can hinder the expression of smoothened (SMO), a target protein of the Hh pathway. As a result of the interaction between SHH and PTCH1, SMO and the subsequent activation of GLi transcription factors can be reduced, and the functions of Gli1, Gli2 and Gli3 are affected in the end. 75 Thus, PTCH1 is called the "gatekeeper" of the Hh pathway.
In some kinds of cancer, miR-212 may play a role as an oncogene targeting PTCH1 (Figure 2). miR-212-3p was shown by Chen-chao Ma and colleagues to promote PDAC progression and metastasis via modulation of the Hh signalling pathway receptor PTCH1. 16 In the case of NSCLC, Yuan Li found miR-212-3p may act as an oncogene to promote cell proliferation and other aggressive behaviour of tumour cells by acting on PTCH1. 76

| The Hippo/YAP signalling pathway
The Hippo/YAP signalling pathway was found to be associated with liver size. 77 Table 2.
According to the

| CONCLUSION AND PERSPECTIVES
In general, a plenty of evidences show that miR-212 can interact with different targets and participate in multiple pathways, as can also be detected in serum and plasma, which are much more readily obtainable than tissues, it attracts increased clinical attention as a biomarker. The controversial findings regarding miR-212 in some cancers require further study. miR-212 participates in chemoresistance and radioresistance, significantly affecting cancer treatment. Identifying vital miR-212 targets and developing safe, effective methods to address miR-212 involvement in the resistance to chemotherapy or radiotherapy will become an important focus in the field of cancer therapy.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflicts of interest.