Circular RNA CSPP1 motivates renal cell carcinoma carcinogenesis and the Warburg effect by targeting RAC1 through microRNA-493-5p

Circular RNAs (circRNAs) take on regulatory roles in renal cell carcinoma (RCC). The research’s goal was to figure out circ-CSPP1’s role and molecular mechanism in RCC. The results clarified that circ-CSPP1 expression was enhanced in RCC. Down-regulating circ-CSPP1 refrained the proliferation, migration, invasion, and Warburg effect (aerobic glycolysis), but accelerated apoptosis of RCC cells. The luciferase activity assay exhibited that circ-CSPP1 could perform as an endogenous sponge for miR-493-5p. Elevating miR-493-5p repressed RCC progression. The bioinformatics website starBase confirmed that ras-related C3 botulinum toxin substrate 1 (RAC1) was a target gene of miR-493-5p. Circ-CSPP1 up-regulated RAC1 by sponging miR-493-5p, and elevating RAC1 could turn around the effect of down-regulating circ-CSPP1 on RCC cells. Taken together, circ-CSPP1 is identified as a novel RCC-promoting RNA that could serve as a latent therapeutic target for RCC therapy.


INTRODUCTION
Renal cell carcinoma (RCC) is a class of diversified malignant neoplasms of the renal cortex with diverse clinical, morphological, and genetic features (Rysz et al., 2021).Though clinical interventions like chemotherapy, surgery, and radiotherapy have made great progress, RCC patients' prognosis is still unpleasing.Therefore, it is imperative to develop new therapeutic strategies considering the biological characteristics of RCC cells.It is well known that metabolic reprogramming, a recognized hallmark of cancer, achieves tumor cell proliferation and survival by changing bioenergetic and biosynthetic pathways.Tumor cells having glycolysis high rates (Warburg effect) is a phenomenon that has been extensively studied in RCC (Emberley et al., 2021).Therefore, figuring out the regulatory mechanism of the Warburg effect is likely to be beneficial for the development of effective RCC treatments.
Circular RNAs (circRNAs), a class of covalently closed endogenous RNAs without a 5' cap or 3' poly(A) tail, are cell-specific, tissue-specific, and developmental stagespecific (Huang et al., 2020).For a long time, circRNAs have been regarded as waste products of splicing errors.Recent studies have discovered circRNAs can perform as gene regulators in diversified physiological and pathological processes and can regulate tumorigenesis and cancer progression (Li et al., 2020).Meanwhile, circRNAs have also been clarified to exert biological functions by performing as miRNA sponges or protein sponges (van Zonneveld et al., 2021).Therefore, circRNAs have been identified as latent biomarkers for tumor diagnosis (Zhu et al., 2021).Circ-CSPP1 is a newly identified circRNA that has been confirmed to take on a momentous role in tumorigenesis (Xue, 2020;Sun et al., 2020).However, its role in RCC and its latent mechanisms remain uncertain.
MicroRNAs (miRNAs) are a class of single-stranded non-coding RNAs that post-transcriptionally control gene expression (Bushati et al., 2007).miRNA dysregulation is the key to the pathogenesis of diversified diseases, including cancer (Ali Syeda et al., 2020) and can function as latent oncogenes or tumor suppressor genes (Iorio et al., 2012).For instance, miR-133b impacts cell proliferation, chemosensitivity, and invasion of RCC cells by refraining the ERK signaling pathway (Xu et al., 2020).A former study clarified that miR-493-5p expression is reduced in hepatocellular carcinoma (HCC) (Yang et al., 2021).However, miR-493-5p's functions in RCC and its interaction with circ-CSPP1 remain ambiguous.
Ras-related C3 botulinum toxin substrate 1 (RAC1) is also commonly present in mammalian tissues and cells as a multiplex regulator of epithelial differentiation and is involved in apoptosis-related pathways through the production of reactive oxygen species (Lee et al., 2012).RAC1 has been implicated in multiple cellular processes, such as cell proliferation, tumor metastasis, and epithelial-mesenchymal transition (Yoon et al., 2017).The same pathways shared by circRNAs, miRNAs, and RAC1 suggest that they might function coordinately to orchestrate tumorigenesis.RAC1 has been reported to contribute to the tumorigenesis of RCC (Zhang, 2022;Shan et al., 2017).Thus, RAC1 could be the target gene of miRNAs to regulate the tumorigenesis of RCC.
This study was conducted to figure out the biological characteristics and latent mechanisms of circ-CSPP1 in RCC progression and Warburg effect.Circ-CSPP1 motivated RCC progression and the Warburg effect by targeting miR-493-5p to control RAC1, suggesting that circ-CSPP1 may offer a novel biomarker and latent therapeutic target for RCC.

Clinical sample collection
The research was approved by the Ethics Committee of The Second Affiliated Hospital of Xi'an Jiaotong University (Approval number.201803XAHT),and informed consent was obtained from each subject before sample collection.Forty pairs of RCC tumor tissues and adjacent normal tissues were harvested from patients in the urology department at The Second Affiliated Hospital of Xi'an Jiaotong University, and two pathologists independently made a definite pathological diagnosis.Adjacent normal tissue was harvested at least 3 cm from the tumor site.Tissues were kept in liquid nitrogen.Clinical data including clinical stage, age, gender, tumor stage, lymph node metastasis, and distant metastasis were recorded.

Colony formation assay
Transfected cells were seeded in 6-well plates and cultivated for colony formation assay.The colonies were fixed with 4% formaldehyde (Macklin, Shanghai, China), and then staining with 0.3% crystal violet was implemented.Counting and analyzing visible colonies were done by Image J software 1.8.0 (Wang et al., 2020).

Flow cytometry
Apoptosis was tested by Annexin V-fluorescein isothiocyanate/propidium iodide (PI) apoptosis detection kit (Invitrogen).Briefly, double-staining was done with Annexin V and PI, and then the apoptosis rate was examined via FACSCalibur (Becton Dickinson) and FlowJo 10.0 software (Lin et al., 2020).

Transwell assays
For cell invasion assay, transfected cells were seeded into 24-well transwell chambers with Matrigel.After incubation, non-invasive cells were wiped off, invasive cells were fixed with paraformaldehyde, and then staining with crystal violet was implemented.Counting and photographing stained cells in each well were conducted under an inverted microscope (Olympus, Japan).As for the migration experiment, cells were seeded into the upper chamber without Matrigel, and other steps were similar to the cell invasion experiments.

Lactate production, glucose uptake, and ATP
Quantification of glucose levels was done using a glucose assay kit (Sigma-Aldrich, St. Louis, MO, USA).Measurement of lactate levels was implemented using a lactate assay kit (BioVision, Mountain View, CA, USA).ATP levels were tested by the CellTiter-Glo fluorescent cell viability assay (Promega, Madison, MI, USA).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from tissues or cells using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol.cDNA was generated using the Promega reverse transcription kit.RT-qPCR was carried out in the ABI7500 system using the SYBR Green method.Relative gene expression was determined by the 2 -ΔΔct method.The primers were clarified in Table 1 (Cao et al., 2020).

Luciferase activity assay
After predicting the binding sites of circ-CSPP1 and RAC1 3'UTR in miR-493-5p, pmirGLO reporter vector (Promega) was utilized to construct circ-CSPP1 and RAC1 3'UTR wild-type (WT) and Mutant (MUT) reporter vectors.A549 cells were incubated with miR-545-5p mimic or mimic NC with the reporter vectors.Then cell luciferase activity was tested by the dual luciferase reporter gene detection kit (Promega) (Yuan et al., 2021).

Statistics
All data were processed by SPSS 21.0 statistical software (SPSS, Inc, Chicago, IL, USA).Measurement data were shown as mean ± standard deviation (S.D.).For the measurement data obeyed a normal distribution, a ttest was used for comparison between the two groups, and a one-way analysis of variance and the Bonferroni post-hoc test were used for comparison among multiple groups.To evaluate the relationship between circ-CSPP1 and the survival prognosis of RCC patients, the Kaplan-Meier method was used for survival analysis, and a log-rank test was used to determine that the difference between high and low expression curves was statistically significant.Pearson correlation analysis in clinical samples was conducted.P<0.05 emphasized obvious statistical meaning.

Circ-CSPP1 is elevated in RCC and is associated with poor prognosis
Circ-CSPP1 was up-regulated in RCC tissues (Fig. 1A).Endogenous circ-CSPP1 was elevated in RCC cell lines, and highest in ACHN cells (Fig. 1B).Therefore, ACHN cells were chosen for subsequent experiments.The 40 patients were divided into two groups according to circ-CSPP1 expression, and circ-CSPP1 was associated with distant metastasis, clinical stage, and tumor stage (Table 2).Meanwhile, it came out that clinical stage, tumor stage, distant metastasis, and circ-CSPP1 were independent predictors of overall survival (OS) of RCC patients (Table 3).Kaplan-Meier survival analysis exhibited that RCC patients with higher circ-CSPP1 expression had poor OS (Fig. 1C).These results suggested  that circ-CSPP1 is elevated in RCC and is associated with poor prognosis.

Circ-CSPP1 accelerates RCC progression and glycolysis through the miR-493-5p/RAC1 axis
After verifying that RAC1 is the target of miR-493-5p, we investigated whether circ-CSPP1 accelerates the development of RCC by controlling the miR-493-5p/ RAC1 axis.It was found that upregulation of circ-CSPP1 elevated RAC1 expression, and repression of RAC1 restored RAC1 expression following upregulation of circ-CSPP1 (Fig. 6A).It turned out that repression of RAC1 abolished the motivation of cell proliferation by elevation of circ-CSPP1 (Fig. 6B, C).Likewise, the repressive effect of up-regulated circ-CSPP1 on apoptosis was abolished by down-regulated RAC1 (Fig. 6D).Moreover, RAC1 repression attenuated the promoting effect of circ-CSPP1 upregulation on cell invasion and migration (Fig. 6E, F).Also, repression of RAC1 could turn around the motivation of cellular glycolysis by up-regulation of circ-CSPP1 (Fig. 6G-I).Taken together, these data suggested that circ-CSPP1 motivated RCC progression and glycolysis through the miR-493-5p/RAC1 axis.

DISCUSSION
CircRNAs are extensively studied cancer markers recently (Yan et al., 2021).More studies have shown that circRNA takes on a crucial role in the genesis and progression of diversified tumors.For example, hsa_ circ_0065217 is elevated in RCC and motivates RCC growth and metastasis via controlling the miR-214-3p-ALPK2 axis (Zhang et al., 2021).Circ_001842 is elevated in RCC and exerts an oncogenic role in RCC via a miR-502-5p-dependent SLC39A14 mechanism (Zeng et al., 2020).Our research revealed that circ-CSPP1 was upregulated in RCC tissues, and RCC patients with high circ-CSPP1 expression had a poor prognosis, suggesting that circ-CSPP1 may be a prognostic biomarker for RCC patients.Meanwhile, circ-CSPP1 silencing restrained RCC cell progression in vitro, while elevation of circ-CSPP1 showed opposite results.These data suggested that circ-CSPP1 motivated the progression of RCC.
It is well known that mammalian cells generate energy through oxidative phosphorylation under aerobic conditions, and during tumorigenesis, cancer cells must rewire their metabolism to maintain survival and reproduction (Urbano et al., 2021).Most cancer cells utilize the Warburg effect to support anabolic growth and tumorigenesis, namely aerobic glycolysis, a metabolic mode that is favored even in the presence of oxygen (Lundø et al., 2020).Warburg effect was found to be beneficial for cancer cell progression and was involved in immune responses and drug resistance (Samec, 2020;Icard et al., 2018).Recent studies have shown that circRNAs can modulate tumor phenotypes, such as metastasis and the Warburg effect.For example, circFOXP1 motivates RCC progression and Warburg effect by sponging miR-423-5p to upregulate U2AF2 (Fang et al., 2021).This study identified a novel circRNA as a regulator of the Warburg effect.The results clarified that downregulation of circ-CSPP1 suppressed the Warburg effect in RCC cells.
Several studies have shown that circ-CSPP1 performs as an oncogene in diversified human cancers and exerts its biological functions by adsorbing miRNAs.For example, circ-CSPP1 is elevated in cervical cancer (CC), and circ-CSPP1 knockdown suppresses CC tumor growth and migration via downregulating ITGB1, PI3K and Akt expression by sponging miR-361-5p (Yang et al., 2020).Circ-CSPP1 is upregulated in ovarian cancer (OC), and silencing circ-CSPP1 represses OC cell biological functions via upregulating miR-1236-3p (Li et al., 2019).Here, it was confirmed that circ-CSPP1 was a decoy of miR-493-5p.Furthermore, repressing miR-493-5p turned around the effect of circ-CSPP1 silencing on RCC cell progression and Warburg effect, while elevating miR-493-5p turned around the effect of upregulating circ-CSPP1 on RCC progression and Warburg effect in HCC.These results suggested that circ-CSPP1 controlled RCC progression and the Warburg effect via performing as a sponge for miR-493-5p.
MiRNAs control gene expression by combining with mRNAs' 3'-UTR, thereby regulating diversified aspects of cellular activity (Huang et al., 2011).Here, RAC1 was a target of miR-493-5p.RAC1 is a highly conserved rho family small GTPase that is essential for diversified cellular and developmental processes (Banka et al., 2022).RAC1 is elevated in different types of tumors, including RCC, and motivates tumorigenesis by participating in tumor cell biological behaviors (Liang et al., 2021;Goka et al., 2020).This study also confirmed that RAC1 was elevated in RCC tissues, and its expression was up-regulated by elevation of circ-CSPP1 but refrained by upreg- ulation of miR-493-5p.Moreover, repression of RAC1 clearly turned around the effect of circ-CSPP1 elevation on RCC cell progression and the Warburg effect.These results suggested that circ-CSPP1 motivated RCC progression and glycolysis through miR-493-5p/RAC1 axis.
However, the research still takes on several limitations.Although the role of circ-CSPP1 in RCC progression has been partially elucidated, the study had a small sample size, and more tissue samples are still needed to further examine circ-CSPP1 expression trend in RCC.Also, further animal experiments are required to validate the effect of the circ-CSPP1/miR-493-5p/RAC1 axis on RCC tumor growth in vivo.Moreover, the Crabtree effect (inhibitory effect of high glucose on mitochondrial oxidative phosphorylation) may also exist in cancer cells.In other words, under the condition of high glucose concentration, glycolytic metabolism is dominated in cells while aerobic respiration is inhibited, thus generating lactic acid (Diaz et al., 2011;Redman et al., 2013).The Crabtree effect is similar to the Warburg effect, the only difference being that the Crabtree effect is caused by high concentrations of glucose (Hammad et al., 2016), therefore, in future studies, exogenous glucose concentration will be a parameter that needs to be designed more precisely to exclude the interference of Crabtree effect as much as possible.

CONCLUSION
All in all, circ-CSPP1 motivates RCC progression and the Warburg effect by acting as a miR-493-5p sponge to upregulate RAC1.The study findings suggest that circ-CSPP1 may serve as a hopeful diagnostic and therapeutic target for RCC.

Figure 1 .
Figure 1.Circ-CSPP1 is elevated in RCC and is linked with poor prognosis (A, B) RT-qPCR to test circ-CSPP1 in RCC tissues and adjacent tissues (n=40), and RCC cell lines; (C) Kaplan-Meier survival analysis.Measurement data were shown as mean ± S.D.