HELLPAR/RRM2 axis related to HMMR as novel prognostic biomarker in gliomas

Gliomas are the most frequent type of central nervous system(CNS) tumor, accounting for more than 70% of all malignant CNS tumors, with an annual incidence rate of 6.6 per 100,000 population [1‐3]. Unfortunately, half of newly diagnosed gliomas are the most malignant glioblastoma, with median patient survival duration of approximately 14 to 17 months [4‐7]. Low-grade gliomas have the potential to evolve into more aggressive and glioblastoma (GBM) in spite of its encouraging prognosis [8, 9]. Over the last decade isocitrate dehydrogenase (IDH) mutation, chromosome 1p/19q deletion, MGMT promoter methylation, TERT promoter mutation and histone mutation have been identified as biomarkers and play a central role for classification of gliomas and treatment decisions [10‐12]. However, the molecular understanding of gliomas is still limited, the treatment of gliomas is full of challenges and prognosis is not optimistic. Therefore, it is important to find new biomarkers to provide a highly reliable prediction of a patient,s survival and more aggressive treatment.

The cell membrane receptor hyaluronan-mediated motility receptor (HMMR), which is linked to the Glycosaminoglycan hyaluronic acid (HA), is significantly expressed in a variety of malignant tumors, including breast cancer, stomach carcinoma, bladder cancer, prostate carcinoma, colorectal cancer, and others [13‐17]. According to several research, increased HMMR expression is linked to a bad prognosis because it speeds up tumor growth and metastasis. However, for malignant peripheral nerve sheath tumor and other tumors, poor patient survival associates with low HMMR expression [18, 19]. Although HMMR was shown to be overexpressed in GBM stem cells and might be used as a biomarker for gliomas, its predictive usefulness and putative function in gliomas were unknown and unproven [20].

Noncoding RNAs, including long noncoding RNAs (lncRNAs), short microRNAs (miRNAs), and circular RNAs (circRNAs), account for 95% of total eukaryotic transcripts [21]. LncRNAs are ncRNA subtypes with a length more than 200 nt and no or limited ability to code for proteins [22]. LncRNAs have been shown to play both direct and indirect regulatory roles in cancer biology in a number of studies [23‐25]. Specific lncRNAs have been linked to cancer recurrence, metastasis, and poor prognosis in several cancer types, including gliomas [26‐28]. During the disease process of gliomas, lncRNA LINC01057 can promote mesenchymal differentiation by activating NF-kB signaling in glioblastoma, lncRNA BCYRN1 can prevent gliomas carcinogenesis by competitively interacting with miR-619-5p to modulate CUEDC2 expression, lncRNA PVT1 can enhance gliomas tumorigenesis and progression by modulating the MiR-128-3p/GREM1 axis and the BMP signaling pathway and the like [29‐31].

MicroRNAs (miRNAs) are short single-stranded noncoding RNAs (ncRNAs) with 19–25 nucleotides that can attach to target mRNAs and prevent gene destruction or translation [32‐34]. MiRNAs also control roughly 30% of the genes in the human genome [35, 36]. At present, it is mainly believed that in the cytoplasm, lncRNAs regulate mRNA by adsorbing miRNAs through a competitive endogenous RNA (ceRNA) regulatory mechanism [37, 38]. The lncRNA KTN1-AS1 negatively regulates miR-505-3p through ceRNA action to promote glioma cell proliferation and invasion [39]. LncRNA NEAT1 and lncRNA NFIA-AS2 can promote gliomas progression by regulating miR-98-5p/BZW1 and miR-655-3p/ZFX axis, respectively [40, 41]. These findings suggest that the ceRNA network plays an important role in the disease progression of gliomas.

In this study, we comprehensively evaluated the predictive value of HMMR and constructed a HMMR related ceRNA network in patients with gliomas using data from the Cancer Genome Atlas (TCGA) database, Chinese Gliomas Genome Atlas (CGGA) databases and Gene Expression Omnibus (GEO) database (Fig. 1). First, we used expression analysis, survival analysis, cox regression analysis to demonstrate that the level of HMMR expression was found to be substantially linked to the poor prognosis of glioma patients. Based on the high and low HMMR expression we analyzed the differentially expressed mRNA (DEmRNAs) from TCGA for GO, KEGG, and GSEA enrichment analysis. Then, these DEmRNAs were also jointly analyzed with the DEmRNAs from GSE4290 to construct a lncRNA-miRNA-mRNA triple regulatory network. Furthermore, survival analysis, nuclear-cytoplasmic localization study, and correlation analysis of RNAs from triple regulatory networks showed a critical ceRNA network (HELLPAR-hsa-let-7i-5p-RRM2).Finally, the diagnostic and prognostic values of RRM2 in gliomas were determined using expression analysis, survival analysis, and cox regression analysis.

Gliomas is the most common malignancy of the CNS, and more than half of gliomas are detected at an advanced stage, accompanied by rapid malignant progression, resulting in poor prognosis and high mortality [49, 50]. Early diagnosis and effective treatment of gliomas patients can improve their prognosis and survival. As a result, early detection and identification of glioma growth, as well as the discovery of new treatment targets and the development of new therapeutic techniques, are critical [51, 52]. The rapid development of sequencing and omics technologies in recent years has given researchers greater opportunity to learn more about the pathophysiology of gliomas and to investigate diagnostic and therapeutic targets [53‐55]. Previous research has linked high HMMR expression to a bad prognosis in a variety of malignancies, such as breast, colorectal, gastric, endometrial, prostate, and multiple myeloma [56‐61]. Furthermore, these investigations imply that HMMR is linked to tumor growth and metastasis. Several human malignancies, including lung cancer, breast cancer, and hepatocellular carcinoma, have been revealed to contain ceRNA regulatory networks implicated in their genesis and progression [62‐64]. However, the prognostic usefulness of HMMR in gliomas has not been thoroughly examined, and just a few research have focused on the integrated ceRNA regulation network as a means of predicting glioma prognosis.

In this study, first, we comprehensively analyzed the expression of HMMR in pan-cancer using bioinformatics and multiple databases, and found that HMMR is abnormally expressed in most cancers including gliomas, which is consistent with current literature reports [14, 65, 66]. The expression of HMMR was significantly increased in gliomas and was significantly associated with high-grade gliomas, advanced age, wild-type IDH and non-codeletion of 1p19q suggesting that HMMR might associated with gliomas disease progression. Meanwhile, HMMR was a useful glioma diagnostic marker, and its AUC was 0.937. HMMR was found to be an independent risk factor for glioma prognosis in both univariate and multivariate Cox regression analyses. Because HMMR expression was a powerful predictive predictor, we created a nomogram that combined HMMR expression and clinical data, and the nomogram predicted 1-, 3-, and 5-year OS in glioma patients more correctly. In addition, an increasing number of studies have discovered that immune cells play a significant function in the tumor microenvironment and play a role in tumor formation and development [67‐69]. By performing immune infiltration analysis by the ssGSEA method, we discovered a link between HMMR expression and immune infiltration in gliomas, which supports the findings of the current study [70‐73]. We investigated DEmRNAs in high and low HMMR expressing gliomas samples and performed functional enrichment analysis to fully understand the function of HMMR in gliomas. It was found that HMMR may be involved in glioma through cell cycle pathways, nuclear division, and other pathway. The result of GSEA revealed that HMMR may be related to the extracellular matrix organization pathway, m-phase pathway, and cell cycle checkpoints pathway. HMMR has also been implicated in a variety of biological functions, including cell proliferation, cycle control, migration, and invasion, according to previous research [74‐77]. To characterize the level of immune infiltration in gliomas, we assessed the association between HMMR and immune cell populations based on transcriptomic data. The results showed that the expression of HMMR was closely related to immune infiltration, with the most positive correlation with Th2 cells, macrophages, and aDC, and the most negative correlation with pDC cells, NK CD56bright cells, and TFH. These findings suggested that HMMR might play an important role in regulating immune functions in gliomas.

Considering the important role of HMMR in predicting gliomas prognosis and the potentially important role of ceRNA network in gliomas disease progression. Then, we constructed a HMMR-related ceRNA network. Combined analysis of DEmRNA from HMMR expression group and GSE4290 resulted in 41 overlapped of DEmRNAs. The enrichment analysis of these 41 DEmRNAs found that the cell cycle and mitotic pathways were enriched which consisted with previous results. Potential interaction miRNAs and lncRNAs were predicted by miRWalk and StarBase, respectively. After analyzed with DEmiRNAs and DElncRNAs, a total 4 mRNAs, 2 miRNAs, and 14 lncRNAs were selected to construct triple regulatory network. This triple regulatory network was also subjected to a survival analysis. We also performed subcellular localization analysis of the lncRNAs in the network because the interactions in the ceRNA network exist solely in the cytoplasm. Finally, combined the results of correlation analysis, the HELLPAR-hsa-let-7i-5p-RRM2 overexpressed ceRNA network was obtained. Further analysis indicated RRM2 expression was shown to be considerably higher in giomas and high RRM2 and HELLPAR expression were linked to a poor outcome in survival analysis..

Ribonucleotide reductase (RNR) is a key enzyme in DNA synthesis and has two subunits, ribonucleotide reductase subunits M1 and 2 (RRM1, RRM2) [78]. In proliferating cells, the RRM1-RRM2 holoenzyme supplies deoxyribonucleoside triphosphates (dNTPs) for nuclear DNA replication and repair in S-phase, as well as dNTPs for mitochondrial DNA replication and repair [79, 80]. RRM2 is commonly expressed in cancer, and it is thought to be an oncogene and a potential cancer treatment target [81, 82]. Gandhi et al. found the YBX1-RRM2-TYMS-TK1 axis, which governed nucleotide metabolism, could be modulated by lincNMR to control tumor cell growth [83]. Xie et al. reported miR-520a could inhibit lung cancer progression through suppressing RRM2 expression and Wnt signaling pathway activation [84]. In glioma disease, RRM2 can promote human glioblastoma cell proliferation, migration and invasion and RRM2 expression controlled by BRCA1 can protect glioblastoma cells from endogenous replication stress while also increasing tumorigenicity [85, 86]. At the same time, through the functional enrichment analysis of RRM2-related genes, we found that cell cycle, nuclear division, and mitosis pathway were significantly enriched, suggesting that RRM2 may be involved in glioma tumorigenesis and progression by regulating cell proliferation, which is consistent with the current RRM2 function. Given that this result was consistent with previous enrichment analyses, we speculated that the HMMR and RRM2 were involved in glioma disease progression by regulating cell cycle and proliferation.

MicroRNAs can control gene expression by recognizing homologous sequences and interfering with transcription, translation, and epigenetic processes. The regulatory role of miRNAs on tumors has been confirmed by many studies [87, 88]. Yang et al. found the HDAC6-hsa-let-7i-5p-TSP1 pathway can regulate neoplastic and antiphagocytic behaviors of hepatocellular carcinoma [89]. Liu et al. performed bioinformatics analysis and concluded that hsa-let-7i-5p functions as a tumor promoter in clear cell renal cell carcinoma and facilitates cell proliferation, migration and invasion by targeting HABP4 [90]. And GALE can be regulated by hsa-let-7i-5p to inhibit human glioblastoma growth [91]. However, whether hsa-let-7i-5p participates in the development of gliomas by regulating RRM2 still needs further research to confirm.

Although studies have shown that HELLPAR affects the transition of the extravillous trophoblast (EVT) from a proliferative to an invasive state, the role of HELLPAR in glioma has not been reported and further experiments are needed to verify its specific function [92, 93].

This study also has several limitations. First, most of the data used for the analysis were mined from public databases and validated in in vitro experiments; however, some results may require further validation in future studies. Second, limited by the difficulty of obtaining normal brain tissue, the GSE4290 dataset analyzed the brain tissue of epilepsy patients as a control group, and the immunohistochemical images of normal cerebral cortex tissue were obtained from HPA, which may have an impact on the results. Third, the binding affinity of lncRNAs, miRNAs, and mRNAs retrieved from the database should be studied further in the lab. Finally, we need to further study the function and mechanism of HMMR and theHELLPAR/ RRM2 axis in gliomas by experiments.

In conclusion, this study provides multi-layered and multifaceted evidence for the importance of HMMR in gliomas and establishes a HMMR related ceRNA (HEELPAR-hsa-let-7i-5p-RRM2) overexpressed network of gliomas, which is better for understanding the link among lncRNA-miRNA-mRNA. These findings suggest potential targets for gliomas treatment.