In this paper, p62 was identified as aberrantly upregulated at both the mRNA and protein levels in human glioma tissues irrelevant to IDH status. We then focused on the functions of p62 and demonstrated that the accumulation of p62 could promote glioma by regulating autophagy, proliferation, migration, reactive oxygen species, TMZ resistance, glycolysis and NF-κB signalling pathway. In accordance with p62 overexpression, p62 knockdown exerted anti-tumour effects in U87 and U251 glioma cells. Subsequently, we determined that miR-124-3p directly targeted the mRNA 3′-UTR of p62, leading to the downregulation of p62 expression at both the mRNA and protein levels. Furthermore, p62 function could be partially reversed by miR-124-3p overexpression. Our findings suggest the potential value of p62 as a novel therapeutic target for glioma.
p62 is a multi-domain protein and thus exerts diverse functions through interacting with different molecules. First, p62 binds to mitogen-activated protein kinase kinase kinase 3 (MEKK3) through an N-terminal oligomerization domain (PB1), which leads to mTORC1 activation and c-Myc expression, thus promoting cancer cell proliferation in prostate cancer stromal fibroblasts and HCC [
5,
14]. In addition, p62 binds to LC3 on phagophore membranes through the LC3-interacting region (LIR) and delivers ubiquitylated cargos to the autophagosome for further degradation [
15]. GATA4, a tumour suppressor, is degraded by p62-dependent selective autophagy and is confirmed to be downregulated in GBM [
16]. Re-expression of GATA4 mediates sensitization of GBM cells to TMZ treatment through loss of APNG (alkylpurine-DNA-
N-glycosylase), a poorly characterized DNA repair enzyme, instead of O-6-methylguanine-DNA-methyltransferase (MGMT) [
17]. Moreover, p62 binds TNFR-associated factor 6 (TRAF6) with the TRAF6-binding domain (TB) to activate NF-κB signalling, resulting in the expression of inflammatory genes and cancer metastasis [
8,
18]. (4) Additionally, with the Keap1-interacting region (KIR), p62 binds to Kelch-like ECH-associated protein 1 (Keap1), leading to the activation of nuclear factor E2 related factor 2 (Nrf2), a transcription factor responsible for several antioxidant genes, which subsequently maintains a low level of ROS to protect cancer cells from oxidative damage [
5,
6,
19]. Constitutive p62 and Nrf2 overexpression is detected in many tumour types and can favour cancer cell survival, promote cell proliferation and protect tumour cells from chemotherapy, radiotherapy and oxidative stress [
6]. The overexpression of Nrf2 and p62 is observed in glioma samples and is identified to be closely related with the clinicopathological parameters and prognosis of patients with gliomas [
20]. Finally, p62 knockdown cells exhibited decreased lactate secretion and glucose uptake through the mTORC1/c-Myc pathway and F1F0-ATP synthase dysfunction [
14,
21]. On the other hand, high p62 expression induces high hexokinase 2 and hypoxia-inducible factor α expression via the upregulation of mTORC1 and NF-κB activity and the inhibition of von Hippel–Lindau E3 ubiquitin ligase activity, leading to enhancement of glycolysis in cancer [
22,
23]. In our research, we have found that p62 acts as a tumour promotor by regulating autophagy, proliferation, migration, reactive oxygen species, TMZ resistance-, glycolysis and NF-κB signalling pathway in glioma cells. However, it has to be noted that the control for p62-overexpressing plasmid is an empty vector in our experiment which may cause the cells grow faster than other control, since DNA length can also affect the cell growth. This may be the reason that some difference in our assays are not so profound. The expression of p62 can be regulated by various molecules with different mechanisms. Through the LC3-interacting region (LIR) domain, autophagy can bind p62 to LC3 on the membranes of autophagosomes and constantly degrade p62 via nonselective autophagy, thus playing a key role in the regulation of p62 levels [
24]. Autophagy has routinely been suggested to potentiate the response to conventional therapies and inhibit tumour progression in gliomas [
25]. 1L-6-hydroxymethylchiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate, an Akt inhibitor, reduces cell viability and radiosensitizes U87 glioma cells by inducing autophagy [
26]. Imipramine can induce autophagy-associated cell death and reduce the incidence of gliomas in gliomagenesis models [
27]. Gartanincan significantly induce autophagy and exhibit an anti-proliferation effect on T98G cells by inhibiting the PI3 K/Akt/mTOR signalling pathway [
28]. Furthermore, NF-κB, Nrf2and AP1 can induce SQSTM1 gene transcription, forming a positive feedback for p62 transcriptional regulation. As a result, inflammation and oxidative stress can induce p62 through NF-κB and Nfr2 to promote cell detoxification and selective autophagy, leading to the prevention of cell death and tumorigenesis [
29]. Guanylate-binding protein3 (GBP3) promotes cell growth through activating the p62-ERK1/2 signalling pathway in glioma [
30]. Furthermore, we found that there is no difference in the expression of p62 between IDH wild type group and IDH mutated group in our experiment, indicating that p62 function may be independent of IDH status. However, this still needs to be verified in future work. Additionally, p62 can be regulated by other molecules, such as microRNAs.
MiR-124-3p has been confirmed to be downregulated in glioma tissues, and loss of miR-124-3p is associated with high malignance and poor prognosis in patients with glioma [
31,
32]. Furthermore, the relative levels of miR-124-3p in serum exosomes could serve as a complementary diagnostic biomarker, providing a minimally invasive and innovative tool to diagnose gliomas at their onset and predict metastases and glioma grading before surgery [
33]. Because a single miRNA may target hundreds of mRNA targets, miR-124-3p exerts anti-tumour functions by suppressing a variety of target genes. MiR-124-3p inhibits glioma cell proliferation by blocking the expression of STAT3 [
34], iASPP [
35], Smad4 [
36], and Nur77 [
37]. MiR-124-3p restoration represses the migration and/or invasion of glioma cells by targeting Capn4 [
38], IQGAP1 [
39], iASPP [
35], and PIM1 [
13]. By directly targeting N-Ras and R-Ras, miR-124-3p impairs angiogenesis through inhibiting VEGF transcription activation [
40]. In this research, we demonstrate for the first time that miR-124-3p can directly regulate p62 in glioma. However, our experiment also has some flaws. The relatively low efficiency to inhibit miR-124-3p by anti-miR-124-3p may be the main reason why anti-miR-124-3p does not increase p62 levels considerably. In our future experiments, we will try to choose more efficient transfection tools to solve this problem.