Circ-PGPEP1 augments renal cell carcinoma proliferation, Warburg effect, and distant metastasis

Circular RNAs (circRNAs) contribute to the malignant phenotype and progression of several types of human cancers, including renal cell carcinoma (RCC). This study probed the molecular mechanism of circPGPEP1 regulating RCC proliferation, Warburg effect, and distant metastasis by targeting the miR-378a-3p/JPT1 axis. Here identified higher circPGPEP1 expression in RCC tissues and cells by RT-qPCR, and high levels of circPGPEP1 were positively correlated with high histological grade and distant metastasis in RCC patients. Furthermore, patients with high levels of circPGPEP1 had a worse survival prognosis. Functional assays presented that knockdown of circPGPEP1 inhibited RCC proliferation, invasion, migration, EMT, and Warburg effect. Dual-luciferase reporter assay, RNA immunoprecipitation, nucleoplasmic RNA isolation, and functional rescue experiments confirmed that circPGPEP1 induced JPT1 expression by sponging miR-378a-3p, thereby promoting RCC malignant phenotype. Xenograft assays and metastasis models further demonstrated that down-regulation of circPGPEP1 effectively inhibited tumor growth and distant metastasis of RCC. Taken together, circPGPEP1, a prognostic circRNA in RCC, acts through the miR-378a-3p/JPT1 axis to regulate RCC progression.


INTRODUCTION
Renal cell carcinoma (RCC) is the second leading cause of death in patients with urinary system tumors, accounting for about 3% of all adult malignant tumors (Scelo & Larose, 2018).Although partial and radical nephrectomy is the most effective treatment for early or localized RCC, approximately one-third of RCC patients are primarily diagnosed with advanced disease, and despite aggressive treatment, RCC at this stage has a low overall survival rate (Li et al., 2020).In addition, recurrence and metastasis occur in approximately 30% of RCC patients (Choueiri & Motzer, 2017;Lara & Evans, 2019).RCC proliferation is a complex network involving multiple carcinogens and diverse genetic backgrounds, resulting in alterations in tumor suppressors or oncogenes (Moch et al., 2014).There is a need to identify molecular mechanisms of RCC progression.
Circular RNAs (circRNAs) can be formed through back splicing events, in which upstream splice acceptor sites join with downstream splice donor sites, resulting in exon circularization (Yu et al., 2020;Zhang et al., 2014).Due to the stability and abundance of circRNAs, an increasing number of circRNAs have been identified to be aberrantly expressed in RCC (Zhou et al., 2022).Mechanistically, these circRNAs act as sponges for microRNAs (miRNAs), thereby protecting downstream mRNAs from miRNAs-mediated degradation (Han et al., 2018).For example, circ_0005875 knockdown suppresses RCC progression by regulating the miR-502-5p/ETS1 axis (Luo et al., 2022).circNUP98, a potential biomarker, acts as an oncogene in RCC through the miR-567/PRDX3 axis (Yu et al., 2020).circ_PGPEP1 is a sponge for miR-1297 and is involved in gastric carcinogenesis (Wang et al., 2021).However, the role and mechanism of circPG-PEP1 in RCC development remain to be explored.
Most malignancies are characterized by the Warburg effect (aerobic glycolysis), a unique mode of cellular metabolism in cancer cells that exhibits increased rates of glucose uptake and lactic acid fermentation in an aerobic environment (Cao et al., 2020).Warburg effect has been intensively studied in the cellular progression of cancer cells over the past decade (Liberti & Locasale, 2016).circRNAs have been identified that can modulate the Warburg effect in human cancers (Li et al., 2021), such as circRNA-FOXP1 (Fang et al., 2021) and circ_0091579 (Chen et al., 2021).
This study focused on the biological role of circPG-PEP1 in RCC progression, and the Warburg effect, in combination with the regulatory network of circPG-PEP1/miR-378a-3p/JPT1 involved in RCC.Taken together, these findings may provide new insights into the treatment of RCC.

Clinical samples
Specimens were RCC tissue and adjacent normal renal tissue (>5 cm from cancer tissue) collected by radical or partial nephrectomy from 48 cases of RCC patients (no radiation or chemotherapy or other tumors) between 2013 and 2015 at Affiliated Hospital and Clinical Medical College of Chengdu University.Histological features of the specimens were confirmed by 2 pathologists.Dis-P.R. Wang and others tant metastases of tumor cells were evaluated by examining the lungs, liver, bone, intestine, and pancreas of patients with RCC.Samples were rapidly frozen at -80°C after enucleation for subsequent studies.This work was approved by the Ethics Committee of Affiliated Hospital and Clinical Medical College of Chengdu University and all patients gave written informed consent.

RT-qPCR
Total RNA acquisition was done with TRIzol ® Reagent (Invitrogen, Thermo Fisher Scientific).RNA reverse transcription was performed at 37°C for 60 min using M-MLV buffers, dNTP and random primers, and Moloney Mouse leukemia virus RT kits (all from Promega, USA).Next, on a Bio-Rad CFX96 system (BioRad), PCR was done with SYBR Green Real-time PCR Master Mix (Solarbio, Beijing, China).Relative gene expression was determined using the 2 -ΔΔct method.The primer sequences are listed in Table 1.GAPDH and U6 were used as endogenous controls for genes.

RNase R and Actinomycin D treatment
Extracted RNA (2 μg) from ACHN cells was treated with 3 U/μg RNase R (Epicenter Technologies).For the RNA stability assay, RNA extracted from ACHN cells was incubated with actinomycin D (Sigma-Aldrich) at 5 μg/ml for the indicated times.circPGPEP1 RNA expression levels were detected by RT-qPCR.

Cell transfection
To overexpress circPGPEP1 and JPT1, the overexpression plasmids of circPGPEP1 and JPT1 were constructed using the pcDNA 3.1 vector (Green Seed Biotech).siRNAs targeting circPGPEP1 and JPT1 were synthesized by Genepharm, while mimic/inhibitor (oligonucleotides) of miRNA and negative control were by RiboBio.The transfection reagent was Lipofectamine 2000 (Invitrogen).

Colony formation assay
Cells were maintained in DMEM containing 10% FBS for 2 weeks and those fixed in methanol were dyed with 1% crystal violet and counted under a microscope (Olympus).

Proliferation assay
A certain amount of 5-ethynyl-2'-deoxyuridine (EdU) solution (RiboBio) was added to each well containing 1×10 4 cells and incubated for 2 h.After being fixed with 4% paraformaldehyde, cells were reacted with glycine, 0.5% Triton X-100, and Apollo reaction solution until Hoechst 33342 staining and imaging under a fluorescence microscope.

Extracellular acidification rate (ECAR)
ECAR (mpH/min) was determined by Seahorse Extracellular Flux Analyzer XF96 (Seahorse Bioscience) to reflect glycolytic activity in cells.2×10 4 cells were grown overnight in 96-well plates and treated accordingly.Data were analyzed using Seahorse XF-96 Wave software.

Transwell
Cell migration and invasion were detected by transwell chambers with or without Matrigel (Corning Life Sciences, USA) (Zhu et al., 2021).First, cells were added to 100 ml serum-free medium and seeded into the upper chamber, then 500 ml of DMEM with 10% serum were seeded into the lower chamber.Cells attached to the lower surface were then fixed with 4% paraformaldehyde after 24 h of incubation.After crystal violet staining, the samples were observed under a microscope and analyzed statistically.

Nucleoplasmic RNA isolation
A nucleoplasmic RNA purification kit (Norgen Biotek Corp) was adopted to locate circPGPEP1 in cells.Afterward, concentrations of nuclear and cytoplasmic circPG-PEP1 RNA were determined by RT-qPCR.

RNA immunoprecipitation (RIP)
RIP assays were implemented using the EZ Magna RIP kit (Millipore).Cell lysates were mixed with anti-Ago2 or anti-IgG (Millipore) before protein A/G (Thermo Fisher Scientific)-conjugated magnetic beads were added.Before the RT-qPCR assay, RNA purification was done with proteinase K.

Nude mouse xenograft experiments
The animal experiments were carried out after obtaining the approval of the Animal Ethics Committee of the Affiliated Hospital and Clinical Medical College of Chengdu University.ACHN cells (3×10 6 cells) stably transfected with si-NC or si-circPGPEP1 were inoculated subcutaneously into the right upper back of BALB/c mice (female, 6-week-old; n=5/treatment).The width and length of the tumor were measured at an interval of 1 week to calculate tumor volume as (length×width 2 )/2.Tumor weight was assessed when the tumor was excised after 5 weeks.Gene expression was analyzed by IHC (Wang, Deng, et al., 2020) or Western blot.
ACHN cells (2.5×10 6 ) stably knocked down circPG-PEP1 were injected into the tail vein of nude mice.Livers and lungs were excised 7 weeks later to carry out HE staining to assess the number of metastatic nodules (Liu et al., 2020).

Data analysis
Statistical analysis was performed using GraphPad Prism 9.0.Data are presented as mean ± standard deviation (S.D.).Chi-square testing assessed the association of circPGPEP1 with clinicopathological features, while the Kaplan-Meier method evaluated that of circPGPEP1 with RCC patients' survival.Statistical analysis between two groups was performed by Student's t-test, and that of multiple groups was by one-way analysis of variance (ANOVA).All experiments were performed with at least three biological replicates.P<0.05 was considered statistically significant.

High circPGPEP1 level is associated with poor prognosis in RCC patients
To investigate whether circPGPEP1 has a biological function in RCC, circPGPEP1 expression in RCC tissues was evaluated by RT-qPCR.As measured, circPGPEP1 expression was up-regulated in patients' tumor tissues compared with normal tissues (Fig. 1A).circPGPEP1 in RCC cell lines was subsequently evaluated.The results showed that circPGPEP1 expression in the five RCC cell lines was higher than in HK-2 cells, and circPGPEP1 expression was the highest in ACHN cells (Fig. 1B).
In accordant with the bioinformatics website circbase, circPGPEP1 is located on chromosome 19p13.11with a length of 557 bp (Fig. 1C).RNase R and actinomycin D treatment identified the ring structure of circPGPEP1.As reflected in Fig. 1D, E. RNase R and actinomycin D treatments decreased the expression and half-life of the linear RNA GAPDH mRNA, respectively, but not circPGPEP1.
Subsequently, RCC patients were divided into a high circPGPEP1 expression group and a low circPGPEP1 expression group according to the median expression of circPGPEP1 in RCC patients.The association of circPGPEP1 with clinicopathologic features in patients with RCC was evaluated.As listed in Table 2, high levels of circPGPEP1 were positively correlated with high histological grade and distant metastasis in RCC patients.The relationship between circPGPEP1 and survival outcomes in patients with RCC was evaluated by Kaplan-Meier.Patients with high levels of circPGPEP1 had a worse survival prognosis (Fig. 1F).Briefly, aberrant ex- Knockdown of circPGPEP1 inhibits RCC cell proliferation, invasion, migration, EMT, and Warburg effect Next, the biological function of circPGPEP1 in RCC cells was explored.CircPGPEP1-targeting siRNA was transfected into ACHN cells to knock down circPG-PEP1 (Fig. 2A).The proliferation ability of cells was first evaluated by colony formation assay and EdU assay.The clonogenic ability of cells decreased and the ratio of EdU-positive cells decreased after the down-regulation of circPGPEP1 (Fig. 2B, C).The invasive and migratory abilities were subsequently assessed by Transwell, and it was found that knocking down circPGPEP1 reduced the number of cells invaded and migrated (Fig. 2D).Since EMT is a key process in the distant metastasis of cancer  cells, EMT-related proteins were evaluated by immunoblot.It was presented that knockdown of circPGPEP1 suppressed N-cadherin and Snail and promoted E-cadherin expression (Fig. 2E).Warburg effect is an important way for cancer cells to acquire capacity through aerobic glycolysis (Vaupel & Multhoff, 2021).Therefore, changes in the Warburg effect were observed in RCC cells after interfered with circPGPEP1.As reported in Fig. 2F-H, ECAR, glucose uptake, and lactate production were reduced after the knockdown of circPGPEP1 (Fig. 2F-H).The expression of proteins associated with aerobic glycolysis was then evaluated.Knockdown of circPGPEP1 inhibited the expression of HK2 and PKM2 proteins (Fig. 2I).

Competitive adsorption of miR-378a-3p by circPGPEP1
Our research focus was shifted to the downstream molecules of circPGPEP1.The subcellular localization of circPGPEP1 was first assessed, finding that circPG-    PEP1 was mainly localized in the cytoplasm of ACHN cells (Fig. 3A), which suggested that circPGPEP1 mediates gene expression mainly by targeting and adsorbing miRNAs.The bioinformatics website https://starbase.sysu.edu.cnpredicted 10 miRNAs with potential binding sites for circPGPEP1, which were screened by RIP experiments to find that miR-378a-3p and circPG-PEP1 were enriched in the Ago2 precipitation complex (Fig. 3B, C).Their targeting relationship was further examined by dual-luciferase, showing that when miR-378a-3p was overexpressed, the luciferase activity decreased in circPGPEP1 WT (Fig. 3D).Subsequently, the expression of miR-378a-3p in RCC was evaluated.Both RCC tissues and RCC cell lines expressed miR-378a-3p at a low level (Fig. 3E, F).Interestingly, miR-378a-3p expression was effectively restored in ACHN cells after knockdown of circPGPEP1 (Fig. 3G).These data suggest that circPGPEP1 binds to miR-378-3p in RCC and regulates miR-378a-3p expression.
miR-378a-3p contributes to a reversal of the effects of si-circPGPEP1 on RCC miR-378a-3p inhibitor was simultaneously transfected in ACHN cells transfected with si-circPGPEP1 to determine whether circPGPEP1 affects RCC development by regulating miR-378a-3p.The promoting effect of si-circPGPEP1 on miR-378a-3p expression was restrained by miR-378a-3p inhibitor (Fig. 4A).Furthermore, functional experiments observed that the preventive effects of si-circPGPEP1 on ACHN cell proliferation, invasion and migration, EMT, and aerobic glycolysis were all blocked after co-transfection of miR-378a-3p inhibitor (Fig. 4B-I).These data suggest that circPGPEP1 affects the biological behavior of RCC by regulating the expression of miR-378a-3p.

miR-378a-3p targets JPT1 expression
Next, the downstream target genes of miR-378a-3p were explored.The bioinformatics website https://starbase.sysu.edu.cnfound 10 mRNAs with potential binding sites for miR-378a-3p.Among them, JPT1 was found to be significantly enriched with miR-378a-3p in the Ago2 precipitation complex in ACHN cells (Fig. 5A, B).Dual luciferase reporter experiments showed that co-transfection of JPT1 WT with miR-378a-3p mimic mediated a decrease in the luciferase activity (Fig. 5C).Subsequently, the expression of JPT1 in RCC was explored by immunoblot.JPT1 protein expression was higher in RCC tissues and cells than in normal tissues or cells (Fig. 5D, E).In addition, decreased or increased expression of JPT1 was detected in ACHN cells overexpressing or knocking down miR-378a-3p, respectively (Fig. 5F).These data suggest that targeting miR-378a-3p regulates JPT1 expression.

circPGPEP1 promotes RCC tumor growth and distant metastasis in vivo
In vivo experiments were included to further support the above in vitro results.Tumor growth and distant metastasis were assessed by subcutaneous inoculation or tail vein injection of ACHN cells stably knocking down circPGPEP1 into nude mice.Depleting circPG-PEP1 suppressed tumor volume and weight (Fig. 7A-C).IHC staining showed that knockdown of circPGPEP1 inhibited JPT1 and Ki-67 positive cell ratio in tumors (Fig. 7D).Immunoblot found that depleting circPG-PEP1 reduced N-cadherin, Snail, HK2, and PKM2 protein levels, and elevated E-cadherin protein levels (Fig. 7E).HE staining exhibited that silencing circPG-PEP1 reduced the number of lung and liver metastatic nodules (Fig. 7F).These data suggest that down-regulating circPGPEP1 effectively inhibits tumor growth and distal metastasis of RCC.

DISCUSSION
RCC is a malignancy in the urinary system that has a poor prognosis despite improved treatments (15).The plight of RCC treatment makes it necessary to further explore its mechanism and find effective RCC therapeutic targets.Recently, circRNAs have been intensively studied as a promising direction for gene-targeted therapy (Wang et al., 2020).In line with this, the circPG-PEP1-oriented mechanism in RCC was probed through miR-378a-3p/JPT1.
CircPGPEP1 was stably and highly expressed in human RCC tissues and cells, and high levels of circPG-PEP1 were associated with poor clinicopathological features and prognosis in RCC patients.Recently, a large number of studies have proposed that circRNAs are involved in the malignant progression in RCC, such as circNUP98 (Yu et al., 2020), Circ_0005875 (Luo et al., 2022), Circ-EGLN3 (Zhang et al., 2021), etc., which are highly expressed in RCC cells and are closely related to the growth and metastasis of RCC.Wang et al., disclosed that circPGPEP1 silencing hampers pro-apoptosis, migration, and invasion of cancer cells, and reduces tumor growth in vivo (Wang et al., 2021).In the present study, circPGPEP1 silencing led to a reduction of cell proliferation, invasion, and migration in RCC cells.Influences of circPGPEP1 on EMT-related proteins were studied since EMT is a key process in the distant metastasis of cancer cells (Piva et al., 2016), which showed that circPGPEP1 silencing inhibited EMT progression.Tumor cells obtain energy through the Warburg effect to maintain tumor development (Liberti and Locasale 2016) and circRNAs enable themselves to mediate the Warburg effect in RCC (Li et al., 2021).Here, our study confirmed that downregulated circPGPEP1 inhibited the Warburg effect in RCC cells.Additionally, inhibiting circPGPEP1 could effectively inhibit the tumor growth and distant metastasis of RCC, further reflecting the tumor-promoting action of circPGPEP1 in RCC.Recently, it has been gradually discovered that circRNAs in exosomes act in intercellular communication, tumor immune response, tumor cell migration, and invasion (Liu et al., 2021).Therefore, RCC cell exosomes can be a study target in subsequent studies.
CircRNAs have been widely demonstrated to act as ceRNAs to counteract the effects of miRNAs (Jorgensen and Ro 2022).Accumulated miRNAs have conferred regulatory influences on RCC metastasis, implicated in a variety of cellular processes, including proliferation, migration, invasion, EMT, and Warburg effect (Abozed et al., 2017).It is known that miR-378a-3p expresses differentially in cancers, including hepatocellular carcinoma (Li et al., 2022), prostate cancer (Cannistraci et al., 2022), breast cancer (Niu et al., 2021), etc. Notably, miR-378a-5p is considered a tumor suppressor in RCC and correlates with a good prognosis for patients (Pan et al., 2019).The present study focused on miR-378a-3p and revealed that miR-378a-3p was down-regulated in RCC.miR-376a-3p could be targeted and regulated by circR-NA and then involved in cancer progression (Mao et al., 2020).More importantly, the present study confirmed that circPGPEP1 mainly mediates gene expression by absorbing miRNAs, and circPGPEP1 affects RCC development by regulating miR-378a-3p.In detail, the preventive influences of silencing circPGPEP1 on RCC cells were all hampered when miR-378a-3p was inhibited.
miRNAs are widely involved in cancer promotion and repression by regulating cancer-related genes (Abozed et al., 2017).JPT1 is a ~16 kDa protein (Q9UK76, uniprot.org) that was originally identified as an abundant transcript in mouse hematopoietic and brain cells (Bateman et al., 2020;Tang et al., 1997).Notably, JPT1 is abnormally high expressed in cancers.For example, JPT1 expression is 3 times higher in ovarian cancer cells than in normal ovarian epithelial cells (Lu et al., 2004).In glioma, lowering JPT1 reduces tumor growth (Laughlin et al., 2009).In addition, JPT1 can interact with STMN1 to promote the growth and metastasis of thyroid anaplastic carcinoma (Pan et al., 2021).Likewise, the present study noted that JPT1 was upregulated in RCC and JPT1 acted as an oncogene in RCC.Knockdown of JPT1 reversed the promotion effect of circPGPEP1 overexpression on proliferation, invasion, migration, EMT, and glycolysis of RCC cells.This suggests that circPGPEP1 plays a role in promoting cancer by up-regulating JPT1 expression.
Although the circPGPEP1/miR-378a-3p/JPT1 axisrelated mechanism in RCC progression has been partially elucidated, further investigation is still required.For example, it is necessary to further determine circPGPEP1 pattern in the serum of RCC patients and to determine the potential of circEHD2 as a diagnostic biomarker for RCC.Furthermore, the downstream pathways of JPT1 in RCC cell progression need to be further elucidated.

CONCLUSION
Our findings suggest that circPGPEP1 is an "oncogene" in RCC.Highly circPGPEP1 promotes cell proliferation and metastasis through miR-378a-3p-mediated JPT1.Our findings not only explain the mechanism of circPGPEP1 in regulating RCC cell progression but also provide potential therapeutic targets for RCC.

Figure 1 .
Figure 1.High circPGPEP1 level is associated with poor prognosis in RCC patients (A) RT-qPCR detection of circPGPEP1 expression in RCC tissues and normal tissues; (B) RT-qPCR detection of circPGPEP1 expression in RCC cell lines and HK-2 cells; (C) circPGPEP1 gene information on circbase; (D) RNase R experiment tested circPGPEP1 ring structure; (E) Actinomycin D treatment analyzed the stability of circPGPEP1; (F) circPGPEP1 expression and survival prognosis of RCC patients; data representation = mean ± S.D. (N=3); P<0.05.

Figure 7 .
Figure 7. circPGPEP1 promotes RCC tumor growth and distant metastasis in vivo ACHN cells with stable knockdown of circPGPEP1 were injected subcutaneously or intravenously into nude mice to evaluate tumor growth and distant metastasis.(A) representative image of tumor; (B-C) tumor volume and weight: (D) IHC staining evaluated tumor JPT1 and Ki-67 expression; (E) Immunoblot assayed E-cadherin, N-cadherin, Snail, HK2, and PKM2 proteins level in tumors; (F) HE staining assessed the ability of the tumor to metastasize to lung and liver; data representation = mean ± S.D. (n=5); P<0.05.