Magic and mystery of microRNA‐32

Abstract MicroRNAs (miRNAs) are a group of endogenous, small (∼22 nts in length) noncoding RNA molecules that function specifically by base pairing with the mRNA of genes and regulate gene expression at the post‐transcriptional level. Alterations in miR‐32 expression have been found in numerous diseases and shown to play a vital role in cell proliferation, apoptosis, oncogenesis, invasion, metastasis and drug resistance. MiR‐32 has been documented as an oncomiR in the majority of related studies but has been also verified as a tumour suppressor miRNA in conflicting reports. Moreover, it has a crucial role in metabolic and cardiovascular disorders. This review provides an in‐depth look into the most recent finding regarding miR‐32, which is involved in the expression, regulation and functions in different diseases, especially tumours. Additionally, this review outlines novel findings suggesting that miR‐32 may be useful as a noninvasive biomarker and as a targeted therapeutic in several diseases.

the reference sequence) (Qing's master's thesis in Chinese). In the same year, we identified miR-32-5p as a potential key miRNA that is involved in vascular calcification (VC) in mice and humans. The mechanism of this involvement is as follows: miR-32 modulates VC progression by activating phosphoinositide 3-kinase (PI3K) signalling and increasing runt-related transcription factor-2 (RUNX2) expression and phosphorylation by targeting the 3′-UTR of phosphatase and tensin homolog (PTEN) mRNA in vascular smooth muscle cells (VSMCs). 6 In 2019, our group reported that miR-32-5p knockout eliminates lipopolysaccharide-induced depressive-like behaviour in mice through the inhibition of astrocyte overactivity. 7 Thus far, our group has found that miR-32 plays an important role in VC, atherosclerosis, diabetes, depression and inflammation (some data not shown).
An increasing number of studies on miR-32 have begun to focus on tumorigenesis and tumour progression in addition to vascular and metabolism-related diseases mainly because miR-32 regulates tumour cell apoptosis, proliferation and migration. MiR-32 has been documented as an oncomiR in most studies, although conflicting reports have verified miR-32 as a tumour suppressor miRNA. The contradictory role of miR-32 in cancers may impede its application as a diagnostic and therapeutic target. Thus, exploring the possible mechanisms behind these contradictory findings is of great importance. We wrote this comprehensive review article to discuss in detail the role of miR-32 in the pathogenesis and progression of vascular, metabolic and neoplastic diseases and the therapeutic potential of this molecule.

| OVERVIE W OF MIR-32
MiR-32 is an intronic miRNA that is located in intron 14 of c9orf5, the gene encoding transmembrane protein 245 (TMEM245), on chromosome 9. 8 To our knowledge, human miR-32 was first reported in 2001 by Dr. Thomas Tusachl's laboratory. 9 According to miRBase, the family of miR-32 includes 22 sequences. Among these sequences, hsa-miR-32-3p and hsa-miR-32-5p are located on chromosome 9 (chr9: 109 046 229-109 046 298, 9q31.3) and are highly conserved between species (according to miRcode, the miR-32 gene is 89% and 61% conserved among primates and mammals, respectively). 10 In further detail (Figure 2), miR-32 is expressed in a variety of tissues in humans and mice, including serum, 11 liver, 12 kidney, 13 breast 14 and brain tissues. 7 Wu et al. 15 revealed the structure and regulation of the hsa-miR-32 promoter F I G U R E 1 Schematic of miRNA biogenesis. The primary transcripts of miRNA genes (pri-miRNAs) are transcribed by RNA polymerase II (pol II/III). The initiation step is mediated by the Drosha-DGCR8 complex. The product of this nuclear processing step is an approximately 70-nt precursor miRNA (pre-miRNA). Then, pre-miRNAs are transported from the nucleus to the cytoplasm through nuclear export factor exportin-5. Once in the cytoplasm, pre-miRNAs are recognized and processing step to produce miRNA duplexes by another RNaseIII, Dicer.Then, the duplexes are separated. One strand is usually selected as the mature miRNA and is loaded onto the RISC. If perfect complementarity with the 3′-UTR of the target mRNAs, the target is cleaved and degraded, if not, the target is not cleaved, but translation is inhibited. In a few cases, miRNA can upregulate the transcription of the target mRNA affected by a miRNA on the basis of 3′ UTR luciferase reporter assay data to be considered as a target.

| Cardiovascular system
Cardiovascular diseases (CVDs), especially ischaemic heart disease, are the leading causes of deaths globally; approximately 17 million CVD-caused deaths occur annually worldwide, and acute myocardial infarction (AMI)-related mortality accounts for approximately 13% of these deaths. 21 AMI is a serious CVD caused by coronary artery occlusion. Serum miR-32-5p expression is elevated in patients with AMI and shows a positive correlation with the biomarker levels of myocardial damage, endothelial injury and proinflammatory cytokines of AMI via target KLF2. 22 Extracellular vehicles (EVs) including exosomes are nano-sized lipid-bound vesicles that are released from cells into the extracellular space. 23 The expression levels of miR-32-5p were significantly higher in circulating exosomes from patient with stable coronary artery disease (SCAD) than those from the control group. The diagnosis AUC value is 0.691, which suggest that serum exosomal miR-32-5p may serve as potential diagnostic biomarkers for SCAD. 24 Similar results were reported from another study, patient with CAD (All CAD patients were confirmed by angiographic evidence of >70% stenosis of at least 1 main coronary artery), plasma miR-32-3p was significantly higher than the control group, and the diagnosis AUC value is 0.745 (95%CI 0.649-0.84). 25  VC is a high-incidence and high-risk disease with increasing morbidity and high mortality. 30 Observations from a registry of 25,253 patients show that coronary calcification (CAC) is an independent predictor of mortality. CAC is associated with a 12-fold increased risk for hard coronary heart disease events. 31 We previously found that the expression of miR-32-5p increases during the occurrence of VC in mice and humans. Furthermore, miR-32-5p promotes VSMC calcification by inducing the expression of VC markers via activating the PI3K-Akt pathway by targeting PTEN and enhancing Runx2 expression and activity. Moreover, miR-32 is upregulated in plasma from patients exhibiting CAC, indicating that miR-32-5p can be used as a potential CAC biomarker. 6 Most recently, our group found that miR-32-5p promotes VSMCs calcification by upregulating TNFα in the microenvironment. 32

| Diabetes and its complications
The prevalence of type 2 diabetes mellitus (T2DM) increases in parallel with the ongoing global obesity epidemic. 33 Diabetic nephropathy (DN) is a serious microvascular complication of diabetes, which is the primary cause of end-stage renal disease (ESRD). 34 MiR-32-5p is highly expressed in kidney tissue, 35  and inflammation. Moreover, miR-32 overexpression magnificently reduction the expression of mothers against decapentaplegic homolog 7 (SMAD7), and the opposite effect was observed following F I G U R E 3 Role of miR-32 as an oncomiR or as a tumour suppressor miRNA. The yellow background indicates the miR-32 act as oncogenes, whereas the light blue background indicates the miR-32 act as tumour suppressor genes. MiR-32 is involved in the regulation of cell proliferation, migration, invasion, apoptosis and resistance to chemotherapeutic drugs by suppressing multiple targets knockdown of miR-32. These results showed that miR-32 may play roles in the progression of EMT and fibrosis in DN. 13 Dual-specificity protein phosphatase (DUSP) is known as a mitogen-activated protein kinase (MAPK) phosphatase and is expressed at low levels in the myocardium of diabetic rats. 37 High glucose levels result in miR-32-5p overexpression, which reduces the expression of DUSP1. The overexpression of miR-32-5p and the downregulation of DUSP1 promotes cell apoptosis and phenotypic changes in human cardiac fibroblasts, suggesting that miR-32 also may play a role in diabetic-related myocardial fibrosis. 38 Approximately 70% of patients with T2DM have fatty liver disease and exhibit a course of liver fibrosis with increased severity. 39 Growing evidence indicates that EMT serves a crucial role in the progression of liver fibrogenesis. MiR-32 expression is markedly increased in the liver tissue of STZ-induced diabetic rats and in AML12 cells under high glucose treatment and promotes liver fibrosis by directly targeting metastasis-associated proteins. 12

| MIR-32 IN C AN CER S
MiRNAs may act as oncogenes by targeting tumour suppressor genes or as tumour suppressors by either inhibiting cellular oncogene expression or regulating cell death ( Figure 3). Moreover, the same miRNA may have different roles in various tumours. 40 In the regulation of miRNA biogenesis, methylation and transcriptional changes in tumour tissues, the expression of some miRNAs changes accordingly partly due to the tumour microenvironment; these miR-NAs may affect tumour prognosis by regulating genes related to tumorigenesis, regulating apoptosis, autophagy or affecting the tumour microenvironment and can thus be used as potential biomarkers. 41,42 MiR-32 expression is changed in numerous cancer types.
MiR-32 has been identified as an oncomiR in the majority of research, but it has also been identified as a tumour suppressor miRNA in other findings. The molecular processes behind the alteration of miR-32 in cancer have piqued the curiosity of researchers. Given that the same individual miRNAs can operate as tumour suppressors in certain cancer types and as oncomiRs in others, it is important to investigate their phenotypic effects and target genes independently in various malignancies. Table 3 shows direct target genes that have been validated in various cancers.

| Breast cancer
Breast cancer is the most common malignant tumour in women worldwide and is curable in ~70%-80% of patients with nonmetastasis breast cancer. However, advanced breast cancer with distant organ metastases is considered incurable with current strategies and agents. 43 Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor prognosis. 44 LncRNAs are an extraordinary group of nonprotein-coding RNAs that are longer than 200 nts in length but absence of protein-coding potential. 45 Certain lncRNAs can act as competing endogenous RNAs (ceRNAs), by competitively occupying the shared binding sequences of miRNAs, thus sequestering the miRNAs and altering the expression of their downstream target genes. 46 Evidence showing that miR-32 can interact with lncRNAs exists. LncRNA WEE2-AS1 is often considered to be an oncogene. 47 Interestingly, WEE2-AS1 is also considered to be an atherosclerosis-related gene. Antisense WEE2-AS1 can regulate human vascular endothelial cell viability via the cell cycle G2/M transition in arteriosclerosis obliterans. 48 Wang et al. 14

| Osteosarcoma
Osteosarcoma (OS) is a primary malignant tumour in children and adolescents. 49 MiR-32-5p is present at decreased levels in OS tissues and cells. LncRNA HNF1A antisense RNA 1 (HNF1A-AS1) is connected to the development of a range of cancers, such as hepatocellular cancer 50 and OS. 51 Moreover, HNF1A-AS1 binds to miR-32-5p to regulate the expression of HMGB1-induced cell apoptosis and impedes proliferation, migration and invasion in OS cells, indicating that HNF1A-AS1 and miR-32-5p may be a potential biomarker and therapeutic target for the diagnosis and treatment of OS. 52

| Retinoblastoma
Retinoblastoma is a highly malignant tumour that appears in retinal development and is the most common primary intraocular tumour in childhood and infancy. 53 Recent studies have shown that lncRNA-ROR may contribute to the tumorigenesis and metastasis. [54][55][56] LncRNA-ROR is significantly upregulated in retinoblastoma tissues, and its overexpression is significantly correlated with optic nerve invasion, nodal or distant metastasis, and recurrence. LncRNA-ROR modulates the EMT programme by competitively binding to endogenous miR-32-5p and regulating Notch signalling pathway activity in retinoblastoma cells; these behaviours may provide new insights into novel molecular therapeutic targets for retinoblastoma. 57

| Ovarian cancer
The incidence of ovarian cancer ranks sixth among female tumours.
Meanwhile, its mortality ranks first among gynaecological tumours, with at least 120,000 deaths worldwide annually. 58   30%. 59 Zhang et al. 60 reported that miR-32 is significantly downregulated in ovarian cancer tissues and cells. The overexpression of miR-32 significantly inhibits the proliferation, migration and invasion of ovarian cancer cells by regulating its target genes, namely, B and T lymphocytes attenuator (BTLA).

| Haematological oncology
Acute myeloid leukaemia (AML) is an aggressive haematopoietic malignancy and the most common form of acute leukaemia in adults.
Resistance to chemotherapy contributes to the poor outcome of in T-ALL. 64 The expression levels of miR-32 are significantly higher in patients with T-ALL than in healthy individuals, and miR-32 inhibits FBXW7 expression by targeting the 3′-UTR of FBXW7; thus, miR-32 and FBXW7 may become potential targets for the diagnosis and treatment of acute leukaemia. 65 Myeloma remains an incurable plasma-cell cancer. 66 Sun et al. 67 showed that in patients with myeloma, miR-32, miR-126, miR-123 and miR-183 are significantly highly expressed, whereas miR-5, miR-76 and miR-50 are expressed at remarkably low levels.
In myeloma cells, the overexpression of miR-32 can significantly enhance proliferation capability and inhibit apoptosis by targeting PTEN, indicating the positive association between miR-32 and myeloma.

| Colorectal cancer
Colorectal cancer (CRC) is one of the most malignant cancers worldwide, which had caused several millions of deaths annually due to its late-stage diagnosis, metastasis trend and high recurrence. 68 The level of miR-32-5p is significantly increased in CRC tissues

| Glioma
Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2), which participates in cell cycle regulation and carcinogenesis through methylating H3K27. 87 Zhang et al. 88 reported that miR-32 is downregulated in glioma tissues and cells and has an important role in inhibiting glioma cell proliferation and metastasis by suppressing the expression of EZH2 by directly targeting its 3′-UTR.

| Cervical cancer
Cervical cancer (CCa) is one of the most common female cancers globally. 92 The World Health Organization states that approximately 530,000 patients with CCa are diagnosed every year around the world; this number progressively increases annually at a rate of 5% of the total female population and skews young. 93 Liu et al. 94 posted data showing that in CCa tissue and cell lines, miR-32-5p is expressed at significantly reduced levels and can inhibit cellular malignant behaviour by regulating the expression of HOXB8.

| Prostate cancer
Prostate cancer (PC) is the second most common urological malignancy and the sixth leading cause of cancer-associated mortality in males worldwide. 95 Early diagnosis and treatment are crucial because the localized PC can be cured by the radical prostatectomy or definitive radiation therapy. However, castration-resistant PC, an advanced form of the disease, lacks curative treatment. 96 Prostatespecific antigen (PSA) screening for prostate cancer in men of average risk remains controversial. Moreover, the use of PSA also is associated with overdiagnosis. 97 Therefore, more specific biomarkers are required for PC diagnosis and prognosis, as well as for thera-

| Melanoma
Cutaneous malignant melanoma is among the deadliest human cancers that is broadly resistant to most clinical therapies. 104 MiR-32 is downregulated in primary and metastatic melanoma, and the overexpression of miR-32 induced apoptosis and reducing anchorage-independent growth in melanoma cells via downregulates pMEK levels by targeting MCL-1 3′UTR. Furthermore, the efficacy of miR-32 expression in inhibiting tumour growth in vivo has been validated. This tumour growth inhibitory effect of miR-32 is more effective than that of vemurafenib, a BRAFV600E inhibitor.
Moreover, the combination of miR-32 and vemurafenib is more effective than either that of vemurafenib or miR-32 treatment alone, therefore suggesting that miR-32 acts as a tumour suppressor in melanoma cells. that KLF4 mRNA is expressed at significantly lower levels in GC than in adjacent tissues. Moreover, the authors identified a significant inverse correlation between miR-32 and KLF4 in GC, 106,107 suggesting that the miR-32-KLF4 axis may be useful targets for GC diagnosis and therapeutics.

| Clear cell renal cell carcinoma
Although clear-cell renal cell carcinoma (ccRCC) is the most common histologic subtype of renal cell carcinoma and accounts for 70% of the cases of this malignancy, its detailed metastasis mechanisms remain unclear. 108 NR2C2 (nuclear receptor subfamily 2, group C, member 2), also known as testicular orphan nuclear receptor 4 (TR4), is a transcription factor and a member of the nuclear receptor family. 109

| CON CLUS I ON AND PER S PEC TIVE S
In the past decade, we and other groups have found through deep and extensive studies that miR-32 has a broad regulatory role in biological events, especially in tumorigenesis and cardiovascular system. The upstream of miR-32 is mainly regulated by a series of lncRNAs that act as competing endogenous RNAs (Figure 4). The expression of miR-32 may be upregulated or downregulated in different cancers, which may act as a tumour suppressor miR or on-comiR. The opposite expression trends and effects of miR-32 have been reported even in the same cancer. The roles of miR-32 in different cancers, as suggested by the literature, are shown in Table 3.
Moreover, as shown in Table 4

CO N FLI C T O F I NTE R E S T
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Abbreviations: ALL, acute lymphoblastic leukemia; AMI, acute myocardial infarction; AML, acute myeloid leukemia; CAC, coronary artery calcification; ESCC, esophageal squamous cell carcinoma; OS, osteosarcoma; PBMCs, peripheral blood mononuclear cells.