Involvement of the circular RNA/microRNA/glucose-6-phosphate dehydrogenase axis in the pathological mechanism of hepatocellular carcinoma

Ying Wng ,# , Xin-Yi Zhou ,# , Xing-Yun Lu , Ke-D Chen , Hng-Ping Yo ,*

a Shulan International Medical College, Zhejiang Shuren University, Hangzhou 310015, China

b State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310 0 03, China

Keywords:Hepatocellular carcinoma Glucose-6-phosphate dehydrogenase Non-coding RNA

ABSTRACT Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide with high mortality. The incidence of HCC is increasing in China. Abnormal activation of glucose-6-phosphate dehydrogenase (G6PD) exists in all malignant tumors, including HCC, and is closely related to the development of HCC. In addition, the differential expression of non-coding RNAs is closely related to the development of HCC. This systematic review focuses on the relationship between G6PD, HCC, and noncoding RNA, which form the basis for the circRNA/miRNA/G6PD axis in HCC. The circular RNA (circRNA)/microRNA (miRNA)/G6PD axis is involved in development of HCC. We proposed that non-coding RNA molecules of the circRNA/miRNA/G6PD axis may be novel biomarkers for the pathological diagnosis,prognosis, and targeted therapy of HCC.

Introduction

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. The incidence of HCC is only inferior to that of lung cancer and gastric cancer. It is reported that about 50% HCCs in the world are related to hepatitis B virus (HBV)infection. China is one of the countries with a high incidence of hepatitis B, accounting for more than half of cases worldwide. The incidence and mortality of HCC in China are increasing [ 1-3 ]. Since Warburg proposed the Warburg phenomenon in tumor cells in 1956 [4] , many studies have found that alteration of the pentose phosphate pathway (PPP) is an important metabolic characteristic of tumor cells [ 5-7 ]. Glucose-6-phosphate dehydrogenase (G6PD)is the rate-limiting enzyme of the PPP and is often aberrantly activated in malignant tumors, including HCC [ 8-10 ]. G6PD plays a key role in cell proliferation and carcinogenesis through a reprogramming signaling pathway. Therefore, it is necessary to explore the causes of abnormal G6PD activation in HCC. In recent years, noncoding RNAs have provided new pathways for aberrant G6PD activation. Numerous studies [ 11 , 12 ] have proven that a large number of non-coding RNAs are associated with HCC. Among them, circular RNAs (circRNAs) are involved in cellular autophagy, apoptosis, proliferation, and regulation of tumor cell growth. With their advantages of highly stable expression and maintenance in tissues and blood, circRNAs have gradually emerged as a new class of potential biomarkers.

In this review, we discuss the relationship between the circRNA/microRNA (miRNA)/G6PD axis and HCC, and the mechanism by which the circRNA in the circRNA/miRNA/G6PD axis acts as a competitive endogenous RNA. According to the method of complementary base pairing, the key circRNAs were further screened to identify those function in the circRNA/miRNA/G6PD axis as diagnostic markers of HCC. The prediction and mining of circRNAs can not only provide a theoretical basis for further study of the influence of abnormal activation of G6PD on the development of HCC but also have important practical significance for the early diagnosis and targeted treatment of HCC.

HCC and G6PD

The commonly used clinical diagnosis and treatment methods have low accuracy and poor curative effect. Like many other tumors, the development of HCC is a multi-step process, from the development of underlying liver disease leading to ongoing damage to the liver, which in turn undergoes metabolic changes to sustain the more rapid proliferation of HCC. The early stages of HCC are clinically latent and most cases are already in the middle to late stages when presented at the hospital. The lack of early diagnostic markers with high sensitivity and high specificity hinders the early diagnosis of HCC, and early diagnosis and treatment of HCC are essential in this field.

G6PDexpression is elevated in many cancers, including HCC [ 13-16 ]. G6PD is a key enzyme in the PPP, which produces phospho-5-ribose (R5P) and reduced coenzyme II (NADPH), both of which are important raw materials for tumor cell proliferation and division activities, promoting tumor growth. G6PD is a ratelimiting enzyme of the PPP that is closely associated with cancer development [ 13 , 17 , 18 ]. G6PD initiates different metabolic flux patterns in response to different metabolic requirements of cells.In HCC cells, G6PD promotes tumor development by increasing its own expression through HBV [19] , and HBV upregulatesG6PDexpression by HBx-mediated activation of Nrf2. During the development of HCC, tumor cells are in an over-proliferative state and require more energy, which is provided by the enhanced activity and elevated expression ofG6PDcompared with those in paraneoplastic tissues. Lu et al. [20] found that elevatedG6PDexpression is significantly associated with HCC metastasis and poor prognosis, and that knockdown ofG6PDinhibits cell migration and invasion in HCC cell lines. In addition,G6PDinduced epithelial-mesenchymal transition through activation of signal transducer and activator of transcription 3 (STAT3) pathway, eventually aggravating the development of HCC. Tumor protein P73 (TP73) is a structural homology of pre-eminent tumor suppressor p53. However, unlike p53, TAp73 enhances tumor growth via enhancing the expression of G6PD [21] .In the human hepatoma cell line PLC/PRF/5, knockdown ofG6PDinhibited the ability of the cell line to proliferate and grow [22] .

It is now generally accepted that abnormal activation ofG6PDis closely associated with the development of HCC; therefore, it is necessary to explore the causes of the abnormal activation ofG6PDin HCC.

Non-coding RNA and HCC

With the in-depth study of liver cancer transcriptomics, the potential involvement of non-coding RNAs in the biological function,regulation, and clinical diagnosis of HCC has gradually been accepted. Long non-coding RNAs (lncRNA), such asUCA1,HULC9, andDANCR10, can be used as biological markers to diagnose HCC. The sensitivity ofUCA1for predicting HCC is as high as 90%, and the specificity is as high as 82% [ 23 , 24 ].

HCC cells show differential expression of a large number of miRNAs, a type of non-coding RNA that regulates genes at the transcriptional and post-transcriptional levels, and their expression influences the pathogenesis and progression of cancer [25] .For example, miR-296-5p was able to downregulate the expression of biomarkers, and then inhibited liver cancer cells through the Brg1/Sall4 axis [26] . The new evidence indicated that the relationship between tumor and stroma was closely related to the development of HCC. A study reported that miRNA-21 stimulated tumor progression via activation of PDK1/Akt signal pathway [27] .

Nearly 40 years ago, electron microscopy revealed the existence of covalently closed circRNAs in eukaryotic cells [28] . Compared to lncRNAs and miRNAs, circRNAs have the advantages of tissue specificity and stable presence in the blood and are increasingly being used as a biomarker for tumors [29] . circRNAs have important regulatory functions, such as participating in transcriptional regulation in the nucleus, competitive cleavage of mRNA precursors during transcription, competing with miRNAs in the cytoplasm for the targeted binding site of mRNA, and having ribosome entry sites.circRNAs can promote the translation and expression of proteins, in which the circRNA acts as a "molecular sponge" by binding to miRNAs, effectively relieving miRNA-based repression of downstream target gene mRNA translation. This might be one of the important ways in which circRNAs participate in tumor metabolic reprogramming [ 30-34 ]. A large number of differentially expressed circRNAs have been identified in HCC. circRNA_10720 can sponge a miRNA targetingVIM(vimentin) to promote the development of HCC [35] .circRNA_0016788 sponges miR-486, which promotes the expression of cyclin-dependent kinase 4 (CDK4) and has carcinogenetic effect on HCC [36] . Moreover, the expression level of circ-ITCH in HCC tissues is significantly lower than that of adjacent tissues, and HCC patients with high circ-ITCH expression have a better prognosis [37] . A similar study found that circRNA-5692 expression was downregulated in HCC tissues, and that miR-328-5p promoted the HCC cell growth. circRNA-5692 is deemed to be a miRNA sponge,absorbing miR-328-5p and enhancing DOC-2/DAB2 interactive protein (DAB2IP) expression to inhibit the progression of HCC [38] .

These results suggest that non-coding RNAs are closely related to the development of HCC and could be used as biomarkers for the susceptibility and prognosis of patients with HCC. However,many unknown non-coding RNAs still remain to be explored in HCC.

Non-coding RNA and G6PD

miRNA can reduce the stability of the mRNA or inhibit the translation of the mRNA by targeting its 3’ untranslated region(UTR). Abnormal expression of miRNAs might lead to altered expression of target genes, includingG6PD, and promote the development of cancer. A previous study has reported that in cervical cancer, miRNA-1 targets theG6PD3 ′ -UTR seed region and suppresses endogenousG6PDexpression, inhibits its translation,prohibits proliferation and promotes apoptosis in cervical cancer cells [39] . Among a large number of differentially expressed miRNAs in HCC, some of them can inhibit the expression ofG6PDby interacting with the 3 ′ UTR ofG6PDmRNA. Overexpression ofG6PDsignificantly attenuated the miR-206 mimic-mediated suppression of cell proliferation while miR-206 can directly bind toG6PDmRNA 3’ UTR and downregulateG6PDexpression [40] .miRNA-1 and/or miRNA-122 can affect the expression ofG6PDand thus the development of HCC by inhibiting the PPP. Further studies revealed that miRNA-122-mediated inhibition ofG6PDexpression is essential for maintaining the metabolic homeostasis of cells [ 41 , 42 ].

Now, the research of circRNA and HCC has gradually become a new hot spot. In the past, it was generally believed that low expression of circRNA might inhibit the growth of tumor cells [43] . One study reported that knocking out hsa_circ_0 0 01955 inhibited the proliferation, migration, and invasion of HCC [44] .circRNA-100338 functions as an endogenous miRNA sponge for miR-141-3p, and activated mTOR signaling pathway by circRNA-100338/miR-141-3p/RHEB axis shows high correlation with poor prognosis [ 45 , 46 ]. This suggests that circRNAs are potentially highly sensitive biomarkers and therapeutic targets. Differential expression of non-coding RNA often affects the development of HCC,which suggests that circRNA may directly or indirectly regulate the expression ofG6PD, but there is still a lack of studies in this field. Therefore, it is necessary to study the mechanism of the circRNA/miRNA/G6PD axis on the oncogenesis of HCC.

CircRNA/miRNA/mRNA network in HCC

Fig. 1. The circRNA/miRNA/G6PD axis in HCC. Schematic representation of circRNAs acting as a miRNA sponge. circRNA: circular RNA; miRNA: microRNA; G6PD: glucose-6-phosphate dehydrogenase; HCC: hepatocellular carcinoma.

In recent years, with the development of high-throughput sequencing technology, circRNAs have received increasing attention in HCC research. Competing endogenous RNAs (ceRNAs) are transcripts that act as miRNA sponges, regulating at post-transcription level [47] . Increasing studies [ 48-50 ] have revealed that many circRNAs are involved in HCC initiation and progression by the ceRNA mechanism. For example, circ-BIRC6 is deemed to be a miRNA sponge, promoting HCC progression by targeting the miR-3918/Bcl2 axis [48] . circRNA/miRNA/mRNA networks link the function of protein-coding mRNAs with that of non-coding RNAs such as miRNA, lncRNA, and circRNA. By employing a comprehensive method of big data mining and computational biology,Xiong et al. [49] have demonstrated that hsa_circRNA_100291 and hsa_circRNA_104515 may function as ceRNAs to exert roles in HCC. Lin and Chen [50] have demonstrated that hsa_circ_0078279,hsa_circ_0 0 07456, and hsa_circ_0 0 04913 related circRNA-miRNAmRNA networks were associated with the carcinogenesis of HCC.

The study of G6PD began with antimalarial drugs that cause hemolytic reaction due to G6PD deficiency [51] . Recently, numerous studies have shown the importance of G6PD in cell growth.Studies found that G6PD activity is closely related to the transformation and metastasis of cancer cells, including HCC [ 52 , 53 ]. By investigating tissue samples from HCC patients and HCC cell lines,a study found thatG6PDexpression was significantly associated with HCC metastasis and poor prognosis. Therefore, G6PD is a major contributor to invasion and migration in HCC [21] .

It should be noted that gene changes will affect downstream signaling pathways, resulting in hepatocarcinogenesis. For example, phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway is one of the most common altered signaling pathways in human cancer. PI3K/Akt pathway promotes the Warburg effect by increasing the expression of glucose transporter, membrane transporter,and the activities of glycolysis-related enzymes. Some studies have shown that PI3K activation can promote the PPP branch pathway,activate Akt and support the growth of tumor cells by inhibitingubiquitin mediated G6PD degradation [ 54 , 55 ]. Therefore, PI3K/Akt controls the activity of G6PD. G6PD regulates the oxidation arm of PPP and determines the rate of glycolysis intermediates entering PPP [54] .

Therefore, the circRNA/miRNA/G6PD axis is likely to affect the expression level ofG6PD, which then affects the oncogenesis of HCC. The possible mechanism is shown in Fig. 1 . The involvement of G6PD in cancer can be mediated by factors influencing the cell cycle. Analyzing the mechanism of action of circRNA/miRNA/G6PD axis and revealing the interaction network of related non-coding RNA molecules can provide a theoretical basis for the development of HCC diagnostic markers and targeted therapy based on circRNA/miRNA/G6PD axis.

Conclusion and future perspective

In clinical practice, patients with HCC often have no obvious symptoms in the early stage, and once symptoms appear, most of them have reached the late stage. Detection of serological markers is helpful for early diagnosis, early treatment, and follow-up,recurrence or prognosis evaluation of HCC. There are many serum markers in HCC, but no ideal index with good specificity and sensitivity. Alpha-fetoprotein (AFP) is commonly used as a marker for the detection of HCC, but clinical practice shows that the sensitivity of AFP is not satisfactory. Also, AFP is sometimes elevated in patients with chronic hepatitis and cirrhosis, which can be confusing in the differential diagnosis. Therefore, further research on the mechanism of action of the circRNA/miRNA/G6PD axis and the discovery of its biomarkers is particularly important.

G6PD, as a key enzyme in the PPP, plays an important role in normal cell metabolism. The abnormal activation of G6PD is closely related to the growth, proliferation, and metastasis of tumors; however, the mechanism of its activation requires further exploration. As a new type of non-coding RNA, circRNAs rely on their unique circular structures, which are more stable than other non-coding RNAs, with high specificity and conserved sequence,causing much hot research [ 56 , 57 ]. At the same time, with the development of high-throughput sequencing technology and bioinformatics, many studies [ 55 , 58 ] reported that circRNAs play an important role in regulating the pathophysiology of cancer, which might provide a novel perspective to study the abnormal expression of theG6PDgene in HCC.

Due to circular structure and tissue specificity, circRNAs are widely expressed in different organs and tissues [59] . circRNAs also can be secreted into the extracellular environment and can be detected in blood, urine, tissues, and secretions by non-invasive means [60] . Real-time quantitative PCR (qPCR) andinsituhybridization (FISH) were specific and sensitive for circRNAs detection [57] . In recent years, some circRNAs are shown to be involved in the development of HCC, which shows that these circRNAs may be biomarkers and disease prognostic indicators of HCC [ 61 , 62 ].Compared with other ceRNAs, circRNAs show a much better binding ability to miRNAs by sponge-specific adsorption. Then circRNAs regulate gene expression through circRNAs/miRNAs/mRNA axis and further regulate the expression of functional proteins. In the past, we used a high-throughput bioinformatic analysis method to screen for non-coding RNA molecules related to the specific expression ofG6PDin HCC, performed association analysis on its downstream target genes, and predicted its possible mode of action. The analysis of functional pathways can describe and explore the possible biological effects of non-coding RNAs from different perspectives, which greatly improves the specificity and accuracy of screening and prediction.

As mentioned above, these circRNAs can become promising therapeutic vectors in the treatment of HCC, demonstrating potential therapeutic value. In circRNA treatment strategies, we can utilize CRISPR/Cas9 or siRNA technology to reduce the expression of circRNAs and use nanoscale delivery systems to increase the expression of circRNAs [ 63 , 64 ]. However, this treatment can only target circRNAs present in the cytoplasm because nanoparticles cannot enter the nucleus. A study indicates that circRNAs can influence the chemotherapy sensitivity and drug resistance of tumors [65] . Gao et al. [66] found that the expression of hsa_circ_0 0 06528 in acellular dermal matrix (ADM) resistant human breast cancer tissues and cell lines was significantly higher than that in ADM sensitive group, suggesting that has_circ_0 0 06528 has the potential to predict chemotherapy resistance of breast cancer. But Ma et al. [67] found that silencing circRACGAP1 can regulate autophagy by targeting miR-3657 and ATG7, making gastric cancer cells more sensitive to lapatinib.Therefore, regulating autophagy of HCC and enhancing the radiotherapy and chemotherapy sensitivity of HCC by targeted therapy of circRNAs are imaginative therapeutic strategies. The significance and value of circRNAs in tumor therapy are obvious, and it is urgent to conduct more detailed and in-depth studies on circRNAs.

Acknowledgments

None.

CRediT authorship contribution statement

Ying Wang: Formal analysis, Methodology, Writing - original draft. Xin-Yi Zhou: Data curation, Methodology, Writing - original draft. Xiang-Yun Lu: Data curation, Methodology. Ke-Da Chen:Conceptualization, Writing - review & editing. Hang-Ping Yao: Supervision, Funding acquisition, Writing - review & editing.

Funding

This study was supported in part by grants from the National Natural Sciences Foundation of China (81872883), 2019 High-level Pre-research Project of Zhejiang Shuren University (KXJ1218607).

Ethical approval

Not needed.

Competing interest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.