circFOXO3 Induced by KLF16 Modulates Clear Cell Renal Cell Carcinoma Growth and Natural Killer Cell Cytotoxic Activity through Sponging miR-29a-3p and miR-122-5p

Renal cell carcinoma (RCC) is one of the most common urological malignancies with high incidence and metastatic relapse. Clear cell RCC (ccRCC) comprises nearly 70% of all RCC cases and is responsible for the majority of morbidity and mortality of RCC. Due to the poor diagnosis strategy and unsatisfactory clinical intervention, ccRCC causes a huge economic burden and poor patient quality of life; therefore, novel diagnostic or therapeutic targets for ccRCC are urgently needed. This study investigated the biological role of circFOXO3 in ccRCC development, showing that circFOXO3 is highly expressed in RCC cells and tissues and inhibits the viability of ccRCC cells. circFOXO3 dysregulation regulates NK cell cytotoxicity towards RCC cells by directly sponging miR-29a-3p and miR-122-5p. Overexpression of miR-29a-3p or miR-122-5p attenuated NK cell toxicity towards RCC cells and the transcriptional factor Kruppel-Like Factor 16 (KLF16) regulates circFOXO3 expression in RCC cells. In conclusion, this study has partially elucidated the function of circFOXO3 in ccRCC development, providing potential novel therapeutic targets for ccRCC.


Introduction
Renal cell carcinoma (RCC), which originates from the renal epithelium, is a common urological tumor accounting for approximately 90% of kidney cancers [1]. RCC is well known for its high incidence and metastatic relapse [2] with clear cell RCC (ccRCC) responsible for 70% of RCC cases, as well as the primary morbidity and mortality of RCC [3,4]. Surgical resection remains the primary clinical intervention of ccRCC because it is resistant to chemotherapy or radiotherapy; however, approximately 30% of ccRCC patients eventually develop metastasis [5,6]. The molecular mechanisms that contribute to ccRCC initiation or progression have been widely investigated but the mechanisms of ccRCC development remain poorly understood.
Circular RNAs (circRNAs) are a newly identified class of noncoding RNAs spliced from exons or introns and feature a covalently closed loop structure [7,8]. The specific structure without terminal 5 ′ caps and 3 ′ poly-A tails means that cir-cRNAs are abundant, conserved, and endogenous RNAs in mammalian cells [9]. With the technological innovation of high-throughput sequencing, the molecular characterization and biological functions of circRNAs have been studied in depth, including cancer initiation and development [10,11], for example, the role of circRNAs in hepatocellular carcinoma, breast cancer [12], head and neck carcinoma, and gastric cancer [13][14][15][16]. In the past five years, several circRNAs have been studied in ccRCC development. Wang et al. demonstrated that cir-cHIAT1 functions in ccRCC development through miR-195-5p/29a-3p/29c-3p/CDC42 signalling [17], while Xue et al. elucidated that circ-AKT3 suppresses ccRCC progression by altering the miR-296-3p/E-cadherin pathway [18]. Han et al. explored the aggravative effect of circATP2B1 in ccRCC progression [19], and Lv et al. revealed that circAGAP1 promotes ccRCC tumorigenesis via acting as a sponge for miR-15-5p [20]. Thus, there is increasing evidence that circRNAs play vital roles in ccRCC progression, so they are a promising direction for ccRCC basic research.
This study hypothesized that circFOXO3 plays a role in the development of ccRCC and showed that circFOXO3 was highly expressed in ccRCC tumor tissues and cells. Furthermore, circFOXO3 inhibited ccRCC cell proliferation and regulated NK cell-mediated cytotoxicity towards ccRCC cells by directly targeting miR-29a-3p and miR-122-5p. Moreover, circFOXO3 expression in ccRCC cells was transcriptionally mediated by KLF16. Our study identified a novel KLF16/cir-cFOXO3/miR-29a-3p/miR-122-5p signalling pathway in ccRCC progression which might be a new direction for ccRCC basic research.

Materials and Methods
2.1. Human Tissue Samples. Thirty pairs of ccRCC tumor tissues and their adjacent normal tissues were collected from patients who had not undergone radiotherapy or chemotherapy at the Affiliated Hospital of Youjiang Medical University for Nationalities. All patients provided informed consent, and the study was approved by the ethics committee of the Affiliated Hospital of Youjiang Medical University for Nationalities (YYFY-LL-2021-35). After surgery, all specimens were immediately stored at -80°C for further analysis and all tissue samples were diagnosed and confirmed by three pathologists independently.
2.2. Cell Culture and Transfection. Human RCC cell lines (ACHN, A498, 786-O, 769-P, and Caki-1) and the normal renal cell HK-2 were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in highglucose DMEM (Gibco, CA, USA) with 10% FBS (Gibco) at 37°C and 5% CO 2 . The short hairpin RNA (shRNA) was applied to silence circFOXO3 levels using the Pglvu6/Puro vector, with the full length of the circFOXO3 coding sequence amplified and cloned into a pcDNA3.1 vector for overexpression. miR-29a-3p and miR-122-5p mimics, small interfering RNA targeting KLF16, and the DNA fragment encoding the mutant circFOXO3 were synthesized and purchased from VectorBuilder (Guangzhou, China). A Lipofectamine 3000 kit (Invitrogen, CA, USA) was used to perform transfections following the manufacturer's protocol.

Fluorescence In Situ Hybridization (FISH) Assay.
Fluor 488-labelled probes to detect circFOXO3 were designed and synthesized by RiboBio. Cells were subjected to prehybridization buffer for fixation, and the probes were hybridized with the cells hybridized for 120 min at 50°C. DAPI was used to stain the nuclei, and a FISH Kit (RiboBio, Guangzhou, China) was used to detect probe signals, with the images visualized and captured by a Leica SP8 laser scanning confocal microscope.

Cell
Proliferation. The proliferation of ACHN and Caki-1 cells was quantified by ethynyldeoxyuridine (EdU) incorporation using a Cell-Light EdU DNA Cell Proliferation Kit (RiboBio, Guangzhou, China) according to the manufacturer's protocol. First, cells were cultured at 37°C, 5% CO 2 for two days. Subsequently, the cells were incubated with 50 mM EdU solution for 120 min, fixed with 4% paraformaldehyde, and stained with Apollo Dye Solution and DAPI. The results were analyzed by ImageJ software (National Institutes of Health, AZ, USA).
2.6. Apoptosis. The apoptosis rate of ACHN and Caki-1 cells was assessed using an Annexin V-PI apoptosis kit (AccuRef Scientific). Briefly, cells were incubated with EPI for 24 h and fixed with Annexin V-PI. After double staining, the apoptotic cells were detected using the FACSAria II flow cytometer (BD Biosciences) and calculated by FlowJo software (version 7; FlowJo LLC).   Disease Markers 4°C and then washed three times with wash buffer before qRT-PCR to detect the retrieved RNAs.
2.8. Luciferase Reporter Assay. circFoxo3 and the 3 ′ -untranslated region (3 ′ -UTR) wild or mutant fragments of miRNA plasmids or KLF16 and FOXO3 mutant region mimic fragments of the 3 ′ -untranslated region (3 ′ -UTR) wild or mutant plasmids were cotransfected into cells, respectively, using Lipofectamine 3000. After 48 h, a Dual-Luciferase Reporter Assay System (Promega) was used to measure the luciferase activities of both firefly and renilla luciferase.
2.9. NK Cell Purification and Expansion. K562 aAPC was incubated with human peripheral blood mononuclear cells purified from healthy donors in a 1 : 2 ratio after 100 Gy radiation. Subsequently, expanded NK cells were cultured for 2~3 weeks for further study. The experimental procedure was as previously described [26].
2.10. Calcein Release Assay. Cells were stained with 30 μM calcein-AM (Dojindo) for 30 min at room temperature and cultured with NK cells at 20, 40, or 60 E/T ratios for 3 h before the supernatant was collected for fluorescent detection at 490 nm excitation. Subsequently, the spontaneous release value (SRV) and maximum release value (MRV) were measured to calculate ½ðtest release value -SRVÞ/ðMRV -SRVÞ.
2.11. Perforin Polarization Assay. RCC cells and NK cells were cocultured (1 : 1) for 30 min and then seeded onto poly-D-lysine-coated slides in a 12-well plate for 1 h at room temperature. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 in PBS before incubation with the primary antibody (antiperforin) and secondary antibody (Alexa Fluor 568-conjugated secondary antibody). The results were visualized by a confocal microscope, and the polarization was recorded.  Caki-1 Relative expression of circFOXO3      2.13. Statistical Analysis. All data were analyzed using SPSS 21.0 (IBM, IL), and the results are presented as mean ± standard deviation (SD). Experiments were repeated at least three times. Two-way analysis of variance (ANOVA) or Student t-tests were used to calculate the significance among groups. P < 0:05 was considered statistically significant; * P < 0:05, * * P < 0:01, and * * * P < 0:001.

Expression and Characterization of circFOXO3 in ccRCC.
To investigate whether circFOXO3 plays a role in ccRCC progression, the expression of circFOXO3 was examined in ccRCC cell lines and HK-2 cells, showing that circFOXO3 was generally upregulated in ccRCC cells (Figure 1(a)). To confirm the circular RNA characteristics of circFOXO3 in ccRCC cells, circFOXO3 and its linear form FOXO3 were measured in ACHN or Caki-1 cells after actinomycin D treatment, showing that circFOXO3 was more stable than its linear form after actinomycin D treatment (Figures 1(b) and 1(c)). Next, RNase R treatment was applied to digest RNAs with cir-cFOXO3 being significantly more resistant to RNase R digestion compared to its linear form (Figures 1(d) and 1(e)). The intracellular distribution assay (Figures 1(f) and 1(g)) and RNA-FISH assay (Figure 1(h)) revealed that circFOXO3 was mainly located in the cytoplasm of ccRCC cells. Furthermore, circFOXO3 expression was upregulated in the thirty pairs of ccRCC tumor tissues compared to their adjacent normal tissues (Figure 1(i)) indicating that circFOXO3 might exert a biological function in ccRCC progression.

Dysregulation of circFOXO3
Influences the Susceptibility of ccRCC Cells to NK Cells. As an antitumor innate immune factor, NK cells are an essential tumor suppressor in tumorigenesis, including ccRCC [27,28], but whether circFOXO3 influences the susceptibility of ccRCC cells towards NK cells is unknown. circFOXO3 knockdown or overexpression cell models were constructed (Figures 2(a)-2(d)), showing that circFOXO3 overexpression inhibited the proliferation and promoted cell apoptosis of ccRCC cells, which would be reversed when circFOXO3 was downregulated (Figures 2(e)-2(h)). Interestingly, the calcein release assay (Figures 2(i)-2(l)),     (Figures 2(q)-2(t)) revealed that circFOXO3 overexpression significantly promoted cell death, whereas circFOXO3 downregulation markedly inhibited cell death, indicating that circFOXO3 aggravates the cytotoxic activity of NK cells to ccRCC cells.
3.6. circFOXO3 Expression Is Transcriptionally Regulated by KLF16. circRNA expression is regulated by transcription factors [29,30], so the JASPAR tool was used to predict four upstream transcription regulators of the circFOXO3 promoter. Subsequently, circFOXO3 expression was shown to be reduced by si-KLF16 in ACHN (Figure 6(a)) or Caki-1 (Figure 6(b)) cells suggesting that KLF16 might be the upstream regulator of circFOXO3. Next, the predicted binding region of KLF16 and the circFOXO3 promoter was obtained from the JASPAR database (Figure 6(c)). The P1 and P2      (Figure 6(e)) cells, indicating that KLF16 might interact with the P1 and P2 regions of the circFOXO3 promoter. The possible interaction was confirmed by a dual-luciferase reporter assay, showing that KLF16 knockdown markedly reduced the luciferase activity of the WT circFOXO3 promoter, which was alleviated when the circFOXO3 promoter was mutated (P1 or P2 Mut) in the P1 and P2 regions (Figures 6(f) and 6(g)). KLF16 expression in ccRCC tumor tissues was upregulated compared to that in adjacent normal tissues ( Figure 6(h)) suggesting that circFOXO3 expression in ccRCC is transcriptionally upregulated by KLF16.

Discussion
ccRCC is one of the most lethal cancer types characterized by high incidence, recurrence, and mortality rates [31]. Furthermore, nearly one-third of ccRCC patients are diagnosed at a localized or distant metastatic status [32,33], and despite advancements in medication management or surgical intervention, the five-year survival rate of ccRCC is still less than 10% [34]. For surgical intervention, nearly 40% of patients who undergo partial or radical nephrectomy experienced cancer recurrence and only 20% survived for five years [35]. Moreover, ccRCC patients are resistant to chemotherapy or radiotherapy [36]. Immunotherapy for RCC is rapidly expanding globally and has significantly improved clinical outcomes for RCC patients, but novel therapeutic targets for ccRCC clinical intervention are urgently needed [37].
This study investigated the role of circFOXO3 in RCC, confirming that circFOXO3 is abundantly expressed in ccRCC tissues and cells, predominately located in the cell cytoplasm, and thus may be related to ccRCC progression. Next, we verified that circFOXO3 inhibited the viability of ccRCC cells. NK cells are a well-known antitumor innate immune factor and an essential tumor suppressor in various cancers including ccRCC [27,28]. Although the function of circFOXO3 in multiple tumors has been elucidated, whether circFOXO3 influences NK cell susceptibility remains unknown. Of interest, we explored the biological function of circFOXO3 in the interaction of ccRCC cells and NK cells, showing that dysregulation of circFOXO3 modulates NK cell toxicity towards ccRCC cells. It has been well documented that NK cells play a crucial role in the human immune system, such as secreting cytokines, killing cancer cells directly, and suppressing tumor cell growth or metastasis [38,39]. Hence, our result might enrich the prognostic value of circFOXO3 in ccRCC. Subsequent molecular analysis revealed that circFOXO3 sponges miR-29a-3p and miR-122-5p and that overexpression of miR-29a-3p or miR-122-5p attenuated NK cell toxicity to ccRCC cells.
The expression of circRNAs can be regulated by transcription factors; for example, circSEPT9 expression in triple-negative breast cancer cells is mediated by E2F1 and EIF4A3 [40]. In glioma stem cells, circRNA ARF1 is transcriptionally regulated by U2AF2 [41] and circRNF121 in osteoarthritis is regulated by LEF1 [42]. Herein, the JASPAR dataset was utilized to show that circFOXO3 expression was transcriptionally regulated by KLF16 in ccRCC cells.

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The present study has partially elucidated the biological function of circFOXO3 in ccRCC progression, but a more in-depth investigation using clinical samples is required to confirm the results, as well as to further investigate the KLF16/circFOXO3/miR-29a-3p/miR-122-5p axis. Nonetheless, this novel KLF16/circFOXO3/miR-29a-3p/miR-122-5p axis in ccRCC progression may help to identify novel diagnostic or therapeutic targets for ccRCC in the future.

Data Availability
All data are available from the corresponding author upon reasonable request.

Ethical Approval
The study was approved by the ethics committee of the Affiliated Hospital of Youjiang Medical University for Nationalities (YYFY-LL-2021-35).

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