CircPSD3 Aggravates Tumor Progression by Maintaining TCA Cycle and Mitochondrial Function via Regulating SUCLG2 in Thyroid Carcinoma

doi:10.21203/rs.3.rs-4659931/v1

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CircPSD3 Aggravates Tumor Progression by Maintaining TCA Cycle and Mitochondrial Function via Regulating SUCLG2 in Thyroid Carcinoma

https://doi.org/10.21203/rs.3.rs-4659931/v1

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In recent years, there has been a rapid increase in the incidence of thyroid carcinoma (TC). Our study focuses on the regulatory effect of circular RNAs on metabolism of TC, aiming to provide new insights into the mechanisms of progression and a potential therapeutic target for TC. In this study, we identified high expression levels of circPSD3 in TC tissues through RNA sequencing. Papillary thyroid cancer tissue cohorts verified the circPSD3 expression level was positively correlated with larger tumor size. circPSD3 promoted the proliferation of TC cells and reduced apoptosis both in vitro and vivo. Proteomics and metabolomics suggested that circPSD3 might play a crucial role in regulating the tricarboxylic acid (TCA) cycle. Specifically, circPSD3 acted as a miR-338-5p sponge to upregulate SUCLG2, an enzyme of the TCA cycle, accelerates the conversion of α-ketoglutarate (α-KG) to succinate. Knockdown of circPSD3 disrupts the TCA cycle and impairs mitochondrial function, resulting in decreased membrane potential and aerobic respiration rate. The reduction in mitochondrial function resulted in the inhibition of proliferation and initiation of mitochondria-mediated apoptosis.

Biological sciences/Cancer/Endocrine cancer/Thyroid cancer

Biological sciences/Cell biology/Cell death/Apoptosis

circPSD3

thyroid carcinoma

tricarboxylic acid cycle

SUCLG2

Thyroid carcinoma (TC) is the most prevalent endocrine malignancy.¹ Most thyroid carcinomas originate from follicular cells, with papillary thyroid carcinoma (PTC) being the most common subtype, constituting 85% of TC cases. Despite the generally favorable prognosis of PTC, clinicopathological features such as lymph node metastasis (37%), extrathyroidal extension (24.8%), and distant metastasis (4.1%) significantly affect TC outcomes.² Notably, anaplastic thyroid carcinoma (ATC) is recognized as the most aggressive subtype, with a median survival time of six months.³ Therefore, a deeper understanding of TC progression mechanisms is essential for developing novel treatments and improving patient prognosis.

Metabolic reprogramming is widely recognized as a hallmark of malignancy. A major metabolic characteristic of tumor cells is their heightened glycolytic activity, even under aerobic conditions, known as the “Warburg effect”.⁴ However, the tricarboxylic acid (TCA) cycle, serving as a central pathway for metabolism and oxidative phosphorylation, also plays a pivotal role in cancer development and progression. For instance, elevated carbon flux through the TCA cycle has been observed in non-small cell lung cancer tissues compared to benign lung tissue in vivo using intraoperative ¹³C-glucose infusions.⁵ Additionally, lung metastatic tumors of triple-negative breast cancer exhibited higher TCA flux compared to both primary tumors and healthy tissues.⁶ These findings suggest that the TCA cycle contributes to cancer development and progression. As cancer cells typically utilize pyruvate from glycolysis for lactate production, alternative pathways such as glutamine and fatty acid metabolism that fuel the TCA cycle exhibit overactivation, resulting in characteristic metabolic and epigenetic changes correlated with tumor progression.⁷ In glioma, the proto-oncogene MYC, a critical regulator of glutaminolysis, upregulates both glutamine transporters and glutaminase to activate glutamine metabolism and sustain the TCA cycle.⁸ Collectively, these findings highlight the active and crucial role of the TCA cycle in tumor progression across several cancer types, although its role in TC remains to be determined.

Over the past decade, circular RNAs (circRNAs) have emerged as a large class of primarily non-coding RNA molecules, characterized by covalently closed loops produced by back-splicing of pre-mRNA.⁹ CircRNAs may function through diverse mechanisms, such as microRNA sponging, protein interactions, and protein translation.¹⁰ Previous studies have shown that circNDUFB2 functions as a protein scaffold to activate antitumor immunity¹¹, while circZNF609 enhances cell proliferation, metastasis, and stemness by sponging miR-15a-5p/15b-5p¹². Additionally, circRNAs have been shown to regulate the cell cycle, induce apoptosis and autophagy, promote angiogenesis, and reprogram metabolism.^13,14 In hepatocellular carcinoma (HCC), circMAT2B activated the circMAT2B/miR-338-3p/PKM2 axis to promote cell progression by enhancing glycolysis under hypoxia.¹⁵ CircMBOAT2 promoted cholangiocarcinoma progression by stabilizing PTBP1 to facilitate FASN mRNA cytoplasmic export, thereby altering the lipid metabolic profile.¹⁶ Thus far, the function and mechanism of circRNAs in regulating TC metabolism require further investigation.

Our study demonstrates the upregulation of circPSD3 in TC and its promotion of cell proliferation both in vitro and in vivo. We elucidate that circPSD3 can reprogram the TCA cycle in TC by sponging miR-338-5p to upregulate SUCLG2. Our findings suggest that circPSD3 may serve as a potential therapeutic target aimed at modulating the TCA cycle in TC.

Clinical samples

All PTC and adjacent normal tissues used in this study were obtained from the First Affiliated Hospital of Sun Yat-sen University between 2018 and 2020. The diagnosis of PTC was confirmed by postoperative histopathological examination confirmed. The biospecimen collection procedure and research protocol were approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University.

Cell lines and cell culture

The cell lines utilized in this study included normal human thyroid epithelial cells (Nthy-ori 3 − 1), papillary thyroid carcinoma (PTC) cell lines (BCPAP, TPC-1), anaplastic thyroid carcinoma (ATC) cell line (8305C), and a utility cell line (HEK293T). Nthy-ori 3 − 1 and BCPAP were obtained from Guangdong Provincial People's Hospital. TPC-1, 8305C and HEK293T were obtained from the American Type Culture Collection (ATCC). These cells were authenticated and tested for mycoplasma contamination. All the cells were cultured with specific media according to ATCC instructions.

Antibodies and sequences

The primary antibodies used in western blotting and RNA immunoprecipitation were listed in Table 1. shRNAs were synthesized by IGE Biotechnology Co., Ltd. (Guangzhou, China). siRNA and miR Inhibitor used in intratumoral injection and probes targeting the junction area of circPSD3 were synthesized by RiboBio Technology Co., Ltd. (Guangzhou, China). Sequences of shRNAs and siRNAs are listed in Table 2. Sequences of miR inhibitors and probes are listed in Table 3.

Construction of stable cell strains

We constructed two plasmids containing a tetracycline-inducible promoter and two different specific short hairpin RNAs (shRNAs) targeting the junction area of circPSD3. To harvest the lentivirus solution, HEK-293T cells were transfected with the PAX2 packaging plasmid, PMD2G envelope plasmid, and Tet-sh-circPSD3 using PEI. After 48 hours, the supernatant containing the lentivirus was collected and filtered through a 0.4 µm pore size filter. TPC-1 and 8305C cells were infected with the lentivirus solution containing polybrene (8 µg/mL). After 72 hours of incubation, the solution was replaced with complete medium containing puromycin (2–4 mg/mL) for selection. The shRNA targeted the junction area of circPSD3 48 hours after doxycycline treatment.

CircRNA sequencing

The circRNA sequencing for PTC and adjacent tissues was conducted by Liebing Biomedical Technology Co., Ltd. (Shanghai, China). Differential expression analysis of circular RNAs was performed using the edgeR package.

RNase R treatment

Total RNA was extracted and mixed with ribonuclease R (RNase R) at 37°C for 20–30 minutes to digest linear RNA. The RNase R digestion system consisted of 2 µg RNA, 2 U RNase R, 2 µL 10× buffer, and DEPC water adjusted to a final volume of 20 µL. The control group did not include RNase R, while all other components remained the same. After digestion, samples were subjected to 85°C for 10 minutes to inactivate the enzyme. RT-qPCR was conducted to assess the levels of linear and circular RNA, respectively.

Nuclear and cytoplasmic RNA extraction

The nuclear/cytoplasmic RNA fraction was isolated using the PARISTM Kit (Thermo Fisher, AM1921) following the manufacturer's instructions. Briefly, 1×10⁷ adherent cells were collected and washed once with ice-cold PBS. The cells were then incubated with 300 µL separation buffer for 10 minutes. After centrifugation, the supernatant was collected as the cytoplasmic fraction, while the pellet was retained as the nuclear fraction for subsequent RNA extraction. An equal volume of lysis buffer and 100% ethanol was sequentially added to each fraction, followed by filtration through the provided column to capture RNA on the filter core. After washing the column with wash buffer three times, RNA was eluted by adding elution buffer to the center of the filter core. The obtained nuclear/cytoplasmic RNA was directly used for subsequent RT-qPCR analysis. GAPDH and U3 were employed as internal controls for cytoplasmic and nuclear fractions, respectively, serving as standards for assessing the degree of nuclear-cytoplasmic separation. Primer sequences are listed in Table 4.

RNA fluorescence in situ hybridization (FISH)

Cells were seeded onto dishes for confocal microscopy in advance. After washing with PBS, cells were fixed with 4% paraformaldehyde for 10 minutes, followed by permeabilization for 5 minutes. Pre-hybridization buffer was applied for 30 minutes at 37°C for blocking, and hybridization solution was then prepared by diluting probes to 0.5 µM and incubating overnight at 37°C. On the following day, the hybridization solution was discarded, and sequential washes with hybridization wash buffer were performed to remove unhybridized probes. DAPI staining solution was used to stain the cell nuclei after washing. Samples were stored at 4°C with an anti-fading agent for further observation.

Cell proliferation assays (EdU Assay & CCK-8 Assay)

Cell proliferation rate and cell viability were detected using the Cell-Light EdU Kit (RiboBio, C10310-1) and the Cell Counting Kit-8 (Ape×Bio, K1018). For the EdU assay, treated cells were seeded into a 96-well plate overnight. On the next day, cells were incubated in complete medium containing EdU reagent (1:500) for 2 hours, then subjected to Apollo staining and Hoechst 33342 staining according to the manufacturer’s instructions. Fluorescence images were taken under a 5x objective lens using a fluorescence microscope. EdU positive cells (%) were calculated as (EdU-positive cells / DAPI-positive cells) × 100%. For the CCK-8 assay, cells were treated and seeded into four 96-well plates. After cells were incubated in complete medium containing CCK-8 reagent (10%) for 2 hours, the absorbance at 450 nm was measured to represent cell viability at 0, 24, 48, and 72 hours. The procedure was carried out for four days at 24-hour intervals.

Cell apoptosis assays

The apoptosis rate was measured using the Annexin V-FITC/PI Apoptosis Kit (Elabscience, E-CK-A211). Cells were seeded in a 6-well plate and treated for 72 hours before harvesting. Both cells and cell debris in the supernatant were collected in the same tube, washed once, and then resuspended in 200 µL binding buffer. Cells were stained with Annexin V/PI following the manufacturer's instructions and then analyzed using fluorescence-activated cell sorting (FACS). FACS was performed using a NovoCyte Flow Cytometer (Agilent Technologies, USA) and analyzed with NovoExpress software. The apoptosis rate was calculated as the proportion of Annexin V+/PI + and Annexin V+/PI- cells, representing the sum of cells in early and late apoptosis.

Animal experiments

The study was approved by the Institutional Animal Care and Use Committee (IACUC), Sun Yat-Sen University (SYSU-IACUC-2023-000896). Cells were harvested, washed, resuspended in PBS, and then mixed with Matrigel (Corning, 356234) at a ratio of 1:1. Half male and half female BALB/c nude mice at four weeks of age were randomly divided into three or four groups, each group with 4 mice. Each mouse was injected subcutaneously in the back with 10⁷ cells in 100 µL cell suspension. After tumor formation, mice were randomly assigned to different groups. The siRNA and miRNA inhibitors were administered into established tumors by multi-point injection every three days at a dosage of 2 nmol per mouse. Tumor volume was measured before each administration using callipers, and the volume was estimated according to the formula: 0.5 × length × width². According to the animal use protocol approved by IACUC, mice were euthanized by cervical dislocation before the maximal tumor size reached 2000 mm³ or maximum tumor diameter reached 2 cm. Tumors were dissected, photographed, and weighed. Tumors were fixed in 4% paraformaldehyde for subsequent embedding in paraffin and sectioning.

Proteomics

Proteomic sequencing was carried out after knocking down circPSD3 in TPC-1 and 8305C cell lines, with three replicates in each group. Twelve protein samples were subjected to protein mass spectrometry analysis by Novogene Co., Ltd. (Beijing, China). Differential expression analysis of proteins was conducted using the limma package, followed by KEGG pathway enrichment analysis using the clusterProfiler package.

Metabolomics

Cells were seeded onto 6 cm dishes in advance. Metabolomic sequencing was carried out after knocking down circPSD3 in TPC-1 and 8305C cell lines, with four replicates in each group. After washing twice with cold PBS, cells were rapidly frozen in liquid nitrogen. Subsequently, samples were fixed in 800 µL methanol at -80°C for 30 minutes. Next, the mixture was scraped, transferred to EP tubes, and mixed with an equal volume of acetonitrile. After centrifugation at 4°C, the supernatant was collected and immediately subjected to LC-MS analysis using the 1290 Infinity II liquid chromatography-mass spectrometry system (Agilent Technologies, USA). Metabolites were standardized based on internal standards, and integrated analysis was performed using MassHunter quantitative software.

Oxygen consumption rate measurement

The oxygen consumption rate (OCR) was measured using the Seahorse XF96 Analyzer (Agilent Technologies, USA). Cells were seeded in Seahorse XF Cell Culture Microplates overnight to ensure a cell density of 90–100% for the experiments (usually 15-20k per well). The complete medium was discarded, and the cells were incubated in 180 µL assay medium for 45 minutes in a non-CO2, 37°C incubator. Meanwhile, the reagents were diluted with assay medium (the final concentration of FCCP in this study was 0.5 µM), and Oligomycin, FCCP, and Rotenone were loaded into cartridge ports A, B, and C, respectively. The assay was run according to the template. All reagents and supplies required were purchased from Agilent Technologies, Inc. (USA).

Mitochondrial membrane potential detection

Mitochondrial membrane potential (MMP) was measured using the MitoProbe™ JC-1 Kit (Thermo Fisher, M34152). JC-1 exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~ 529 nm) to red (~ 590 nm). Mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. After the cells were digested and gently washed with warm PBS, 10⁶ cells were collected and incubated at 60°C for 1 minute as a positive control group. Cells from the positive control group and other treated groups were incubated with JC-1 solution or blank solution at 37°C for 30 minutes. JC-1 was added in 1 mL complete medium at a 1:1000 proportion. After incubation, the cells were washed twice with warm PBS and resuspended in the same solution for FACS. MMP was determined by the polymer/monomer ratio.

NAD+/NADH measurement

The NAD+/NADH ratio was measured using the NAD+/NADH Ratio Assay Kit (Beyotime, S0175) according to the manufacturer’s manual. Briefly, the intracellular NAD+/NADH fraction was extracted and divided into two groups: half of the sample was used to determine the total concentration of NAD + and NADH, and the other half (treated at 60°C for 30 minutes to resolve the NAD + fraction) was used to detect the concentration of NADH. The absorbance at 450 nm was measured to calculate the concentration and NAD+/NADH ratio according to the standard curve.

Metabolite supplementation

Metabolite rescue was conducted by adding α-ketoglutarate (Sigma, K1128) and succinate (Sigma, PHR1418) to complete medium at concentrations of 4 mM and 2 mM, respectively. The conditional medium was adjusted to a neutral pH.

RNA immunoprecipitation

Cells were transfected with HA-tagged Ago2 plasmid prior to RNA immunoprecipitation. The cells were harvested, centrifuged, and re-suspended in 100 µL IP lysis buffer supplemented with protease and RNase inhibitors for 5 minutes. Subsequently, the lysate was centrifuged, and the supernatant was transferred to a new EP tube. Ten percent of the supernatant was reserved as an input control and stored at -80°C. The remaining supernatant was divided into two equal parts: one part was incubated with IgG control antibody, and the other part with HA antibody, both at 4°C for 2 hours. After this incubation, protein A/G agarose beads were added and incubated overnight. The following day, the beads were washed with PBST on a magnetic rack to remove unbound complexes. The immunoprecipitated RNAs were extracted using the TRIzol method for subsequent RT-qPCR analysis.

RNA pull-down

Cells were transfected with the circPSD3-overexpression plasmid in advance. Forty-eight hours later, the cells were harvested and lysed in IP lysis buffer for 5 minutes, followed by centrifugation at 4°C at 13,000 rpm for 10 minutes. The supernatant was transferred to a new EP tube. Next, NC probe/biotin-labelled circPSD3 probe/biotin-labelled miR-338-5p probes were mixed with the supernatants separately and incubated overnight at 4°C with rotation. Streptavidin magnetic beads were then added and incubated at room temperature for 2 hours with rotation. Afterward, the beads were washed with PBST on a magnetic rack to remove unbound RNA complexes. RNA was extracted using the TRIzol method for subsequent RT-qPCR analysis.

Dual-luciferase reporter assay

Wild-type and mutant fragments of the SUCLG2 3’UTR were constructed into the pmirGLO vector. The constructed reporter plasmids and miR-338-5p mimics or NC were then co-transfected into 293T cells. After 48 hours of incubation, the luciferase activity was quantified using the Dual-Luciferase® Reporter Assay System (Promega, E1910). The firefly to Renilla luciferase ratios were calculated. MiR-338-5p mimics and miR-NC were synthesized by RiboBio Technology Co., Ltd. (Guangzhou, China).

HE, IHC and RNA in situ hybridization

PTC tissues and mouse xenograft tumors were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. All paraffin sections were deparaffinized and hydrated using xylene and ethanol. For IHC staining, after immersion in boiling EDTA unmasking solution (Solarbio, C1034) for antigen retrieval and subsequent blocking in 20% goat serum, the slides were incubated at 4°C overnight with a primary antibody against Ki67. The following day, the slices were incubated with a secondary antibody, and the result was detected using a 3,3′-diaminobenzidine (DAB) peroxidase substrate kit (Dako, K5007). For HE staining, sections were stained with eosin and hematoxylin solution only. After dehydration and clearing, the slices were sealed with neutral resin and prepared for subsequent observation under a microscope. RNA in situ hybridization was conducted using an enhanced sensitive in situ hybridization detection kit (BOSTER, MK1030), following the manufacturer’s instructions. CircPSD3 junction site-targeting/non-targeting labelled probes conjugated to digoxin were synthesized by Geneseed Biotech, Co., Ltd. (Guangzhou, China). The slides were hybridized with probes (0.5 µg/mL) at 40°C overnight. To estimate the level of circPSD3, three areas from each slide were randomly selected, and the positive-staining area (1–4) and positive-staining intensity (0–3) were scored. The total staining score was calculated by multiplying the staining area by the staining intensity. The scoring process was performed independently by two researchers.

Statistical analysis

All experimental data were obtained from at least three independent trials, analyzed using t-tests (two-sided) in GraphPad Prism 8.0 software, and presented as mean ± SD. A P value of 0.05 was considered statistically significant.

circPSD3 is up-regulated in thyroid carcinoma

RNA sequencing was conducted on five pairs of PTC tissues and adjacent normal thyroid tissues. In the analysis of circRNA expression profiles, 177 upregulated circRNAs and 149 downregulated circRNAs were identified, as illustrated in the volcano plot (Fig. 1A). Five upregulated circRNAs from RNA sequencing were identified by re-analyzing the public database of thyroid cancer (GSE93522) to screen for the most representative circRNAs in thyroid cancer (Fig. 1B, upper). Based on the expression levels of these five circRNAs in the normal thyroid cell line Nthy-ori 3 − 1 and three TC cell lines, circPSD3 (Fig. 1B, lower) was found to be the most significantly upregulated circRNA, consistent with the sequencing results. CircPSD3 is generated by back splicing of exons 5–8 of PSD3 (Fig. 1C) and was upregulated in PTC tissues relative to adjacent normal thyroid tissues in our clinical cohort (Fig. 1D). Then, we investigated the relationship between circPSD3 expression and clinicopathological features in our cohort. The data showed that circPSD3 expression level was positively correlated with tumor size (Supplementary Fig. 1A). Furthermore, there was a probable positive trend between lymph node metastasis status and circPSD3 expression level, while gender, age, multifocality, and extrathyroidal extension appeared to have no correlation with circPSD3 level (Supplementary Fig. 1B, C). To clarify this, we applied another cohort for RNA hybridization in situ to detect the expression of circPSD3. The result indicated that tumors with lymph node metastasis exhibited a higher expression level of circPSD3 (Fig. 1E, F). In contrast to its linear isoform, circPSD3 exhibited resistance to RNase R digestion in TC cells 8305C and TPC-1 (Fig. 1G). The Sanger sequence and DNA gel electrophoresis demonstrated that the distinct junction of circPSD3 was amplified using divergent primers (Fig. 1H). In accordance with these results, the properties of circPSD3 as a circular RNA were validated. Additionally, nucleoplasm separation followed by qPCR and FISH analyses of circPSD3 localization revealed that circPSD3 was predominantly localized in the cytoplasm, with a minor presence in the nucleoli (Fig. 1I and 1J).

circPSD3 promotes cell proliferation and inhibits cell apoptosis in thyroid carcinoma

To investigate the function of circPSD3 in TC, we selected the TPC-1 and 8505C cell lines, which exhibited the highest expression levels. We employed two short hairpin RNAs to create stable knockdown of circPSD3. The sh-circPSD3 groups demonstrated a notable decrease in circPSD3 expression compared to the NC group (Fig. 2A and Supplementary Fig. 2A). The downregulation of circPSD3 was correlated with reduced cell viability, detected by the CCK8 assay (Fig. 2B and Supplementary Fig. 2B) and the EdU assay (Fig. 2C, D and Supplementary Fig. 2C, D). Furthermore, flow cytometry analysis revealed the rates of cell apoptosis increased following the suppression of circPSD3 (Fig. 2E, F and Supplementary Fig. 2E, F). Moreover, the tumor growth was significantly inhibited in the sh-circPSD3 groups in vivo compared to the NC groups (Fig. 2G-I and Supplementary Fig. 2G-I). In the circPSD3 shRNA group, the expression level of circPSD3 in xenograft tumors was significantly decreased, and showed negative relation with Ki67 expression rate (Fig. 2J-L and Supplementary Fig. 2J-L). These results indicated that circPSD3 promoted the proliferation of TC cells and reduced apoptosis both in vitro and vivo.

circPSD3 regulates the TCA cycle and mitochondrial function via SUCLG2

Proteomics was employed to evaluate the differentially expressed proteins in TPC-1 and 8305C cells after knocking-down circPSD3 to investigate the potential targets and signaling pathways of circPSD3(Fig. 3A, B and Supplementary Fig. 3A). KEGG analysis indicated that circPSD3 played a pivotal role in multiple metabolic pathways, in which the TCA cycle emerged as the most prominent pathway (Fig. 3C). The differential expressed protein profile from proteomics revealed a significant downregulation of key enzymes in the tricarboxylic acid (TCA) cycle, including ACO2, OGDH, SUCLG2, and FH, with SUCLG2 being significantly downregulated in both cell lines.

Metabolomics analysis was also performed to detect changes of intermediate metabolites in both the sh-circPSD3 and NC groups. The results showed a significant decrease in succinate, citrate, and isocitrate levels in the sh-circPSD3 group in 8305C cells, while succinate and oxaloacetate levels were reduced in the sh-circPSD3 group in TPC-1 cells (Fig. 3D, E). Integrating the findings from proteomics and metabolomics suggested that circPSD3 might play a crucial role in regulating the conversion of α-ketoglutarate (α-KG) to succinate. Consequently, decreased expression of SUCLG2, which facilitates the transformation of α-KG into succinate within the TCA cycle, was observed in the sh-circPSD3 group compared to the NC group in both TPC-1 and 8305C cells (Fig. 3F).

Another characteristic of the disrupted TCA cycle was found in an increased NAD+/NADH ratio (Fig. 3G and Supplementary Fig. 3B). Given the significant influence of the TCA cycle on mitochondrial function, our subsequent investigation focused on examining mitochondrial aerobic respiration and mitochondrial membrane potential (MMP). Our study indicated that the downregulation of circPSD3 impeded mitochondrial aerobic respiration, as evaluated by the Seahorse test (Fig. 3H, I and Supplementary Fig. 3C, D). Additionally, decreased circPSD3 expression resulted in reduced MMP levels, as observed through JC1 staining (Fig. 3J, K and Supplementary Fig. 3E, F).

The reduction in mitochondrial function triggered the activation of caspase-3, resulting in the initiation of mitochondria-mediated apoptosis (Supplementary Fig. 3G). Overall, these findings suggested that inhibiting circPSD3 could reduce the protein expression of SUCLG2, thereby impeding the production of succinate in the tricarboxylic acid (TCA) cycle and suppressing mitochondrial activity, ultimately leading to apoptosis.

circPSD3 promotes cell proliferation by regulating TCA cycle in thyroid carcinoma

To confirm the role of circPSD3 in cell growth and its impact on the TCA cycle, we supplemented the sh-circPSD3 group with additional α-ketoglutarate and succinate, as these metabolites are up- or down-stream of SUCLG2. Flow cytometry analysis revealed that the decrease in mitochondrial membrane potential (Fig. 4A, B and Supplementary Fig. 4A, B) and the increase in cell apoptosis caused by circPSD3 knockdown were restored with the addition of α-ketoglutarate and succinate (Fig. 4C, D and Supplementary Fig. 4C, D). This rescued effect was consistent with the results of the EdU assay (Fig. 4E, F and Supplementary Fig. 4E, F). Additionally, the reduced oxygen consumption rate (OCR) and ATP synthesis resulting from circPSD3 knockdown were also rescued after supplementation with α-ketoglutarate and succinate (Fig. 4G, H and Supplementary Fig. 4G, H). These results indicated that the suppression of circPSD3 had a detrimental effect on mitochondrial function, impeding cell proliferation through the disruption of the TCA cycle.

circPSD3 functions as a sponge of miR-338-5p

As circPSD3 was predominantly located in the cytoplasm, we hypothesized that it functioned as a miRNA sponge to regulate the expression of SUCLG2. Initially, the RNA immunoprecipitation (RIP) assay was conducted to assess its ability as a miRNA sponge. The anti-HA antibody showed a significant enrichment of circPSD3 compared to the anti-IgG antibody, indicating a binding interaction between AGO2 and circPSD3 (Fig. 5A). Potential target miRNAs of both circPSD3 and SUCLG2 were predicted using the online bioinformatics tool (https://circinteractome.nia.nih.gov/) (Fig. 5B), and six potential miRNAs were identified.

The RNA pull-down assay, using a specific anti-circPSD3 probe, revealed that only miR-338-5p was enriched in both TPC-1 and 8305C cell lines (Fig. 5C and D). Meanwhile, circPSD3 was significantly enriched using the miR-338-5p probe (Fig. 5E and F). The direct interaction between circPSD3 and miR-338-5p was further confirmed by a dual-luciferase experiment (Fig. 5G and H). Western blot analysis showed that inhibition of miR-338-5p effectively restored the downregulation of SUCLG2 caused by the knocking-down of circPSD3 (Fig. 5I).

To verify SUCLG2 as the downstream target of circPSD3, we performed rescue experiments by overexpressing SUCLG2 after circPSD3 knockdown. The efficiency of SUCLG2 overexpression was confirmed by western blot analysis (Supplementary Fig. 5A). Upregulation of SUCLG2 mitigated the decrease in MMP (Fig. 5J, K and Supplementary Fig. 5B, C) and cell proliferation (Fig. 5L, M and Supplementary Fig. 5D, E) resulting from circPSD3 knockdown. Therefore, it could be inferred that circPSD3 served as a miR-338-5p sponge, influencing the malignant characteristics of thyroid cancer through the modulation of SUCLG2 expression.

circPSD3 regulates TCA cycle and promotes proliferation by sponging miR-338-5p

To investigate circPSD3's role as a sponge for miR-338-5p in regulating the TCA cycle, we displayed rescue experiments using a miR-338-5p inhibitor. Flow cytometry analysis validated the restored effects of the miR-338-5p inhibitor on mitochondrial damage (Fig. 6A, B and Supplementary Fig. 6A, B) and cell apoptosis (Fig. 6C, D and Supplementary Fig. 6C, D) caused by circPSD3 knockdown. The miR-338-5p inhibitor significantly enhanced mitochondrial function and inhibited cell apoptosis induced by circPSD3 knockdown. Additionally, the EdU assay results showed that the miR-338-5p inhibitor effectively countered the inhibitory impact of sh-circPSD3 on cell proliferation (Fig. 6E, F and Supplementary Fig. 6E, F). The Seahorse assay yielded similar findings in terms of OCR and ATP generation rate (Fig. 6G, H and Supplementary Fig. 6G, H). Consistent outcomes were observed in nude mice regarding xenograft tumor formation, indicating that the in vivo growth inhibition of subcutaneous tumors was effectively counteracted by the miR-338-5p inhibitor (Fig. 6I-K and Supplementary Fig. 6I-K). Overall, these results suggested that circPSD3 acted as a sponge for miR-338-5p, thereby facilitating the development of malignant characteristics in thyroid cancer (Fig. 7).

Circular RNAs (circRNAs), a class of predominantly non-coding RNA molecules, have been found to exhibit varying expression levels and play significant roles in the development and progression of cancer through various mechanisms.¹⁴ PSD3 (pleckstrin and sec7 domain-containing 3) is an oncogene highly expressed in thyroid cancer (TC), generating multiple circRNAs from distinct exons. Our study focused on circPSD3 derived from exons 5–8 (circBase ID: hsa_circ_0004458).¹⁷ A previous study reported upregulation of hsa_circ_0004458 in gastric cancer and papillary thyroid carcinoma (PTC), which aligns with our findings showing significant upregulation in TC tissues and cells.^18,19 Additionally, our study highlighted a positive correlation between its expression level and tumor size as well as lymph node metastatic status, indicating an oncogenic role for circPSD3 in TC by enhancing cancer cell proliferation and suppressing apoptosis.

Proteomics analysis suggests that circPSD3 regulates the tricarboxylic acid (TCA) cycle as one of its downstream pathways. The TCA cycle comprises a series of biochemical reactions in the mitochondrial matrix, providing energy, macromolecules, and redox balance to the cell.⁷ Despite the Warburg effect's assertion that cancer cells primarily generate ATP via glycolysis, evidence supports the notion that some cancer cells utilize oxidative phosphorylation (OXPHOS) as the major means, relying on TCA cycle flux.^20,21 TC exhibits a very active TCA cycle.²² Overactivated pyruvate gluconeogenesis and glutamine metabolism replenish intermediate metabolites and fuel the TCA cycle in TC.^23,24 Besides its catabolic role, the TCA cycle also serves an anabolic function, providing precursors for other biosynthetic processes, including gluconeogenesis, fatty acid, steroids, and amino acids biosynthesis.²⁵ Our study confirmed that disrupted TCA cycle suppressed NADH and ATP generation via OXPHOS. Furthermore, we observed mitochondrial damage associated with circPSD3 knockdown, characterized by a loss in mitochondrial membrane potential and decreased oxidative respiration rate.

Previous studies have shown that abnormal TCA enzymes in cancer cells lead to impaired mitochondrial function. Mitochondrial membrane potential (MMP) is a critical bioenergetic parameter reflecting mitochondrial condition.^26,27 Decreased MMP contributes to an early and irreversible step in a cascade of events, including the release of cytochrome c and subsequent activation of caspase-3, ultimately leading to apoptosis via the intrinsic mitochondrial pathway.^28,29 Our study detected a decrease in MMP and downstream activation of caspase-3, resulting in increased mitochondria-mediated apoptosis associated with circPSD3 knockdown.

Subsequently, we demonstrated that circPSD3 promotes the conversion of α-ketoglutarate (α-KG) to succinate by upregulating SUCLG2. SUCLG2 overexpression and succinate accumulation have been shown to promote tumorigenesis and progression in various malignancies.²⁹ Additionally, succinate accumulation in tumors stabilizes HIF-1α and activates related oncogenes by inhibiting the activity of prolyl hydroxylases (PHDs).⁷ A recent study indicated that the deletion of SUCLG2 upregulates succinylation levels of mitochondrial proteins and inhibits the function of key metabolic enzymes, impairing mitochondrial function in lung adenocarcinoma cells.³⁰ Our study observed an overall metabolic disorder in the TCA cycle related to the downregulation of circPSD3 and SUCLG2, characterized by succinate deficiency. The findings from the aforementioned study may provide insights into the overall metabolic disorder observed in our research. These findings suggest that SUCLG2 acts as a potent oncoprotein through several pathways, and targeting the TCA cycle via SUCLG2 may represent a promising therapeutic strategy.

Building on our previous findings that circPSD3 is predominantly located in the cytoplasm and is formed by exons, we have confirmed the circPSD3/miR-338-5p/SUCLG2 regulatory pathway in the progression of thyroid cancer (TC). Studies have found that circRNAs function by binding to miRNAs in TC.^31,32

In summary, our study identified that circPSD3 is upregulated and positively correlates with tumor size in TC. CircPSD3 acts as a regulator in the TCA cycle through the circPSD3-miR-338-5p-SUCLG2 axis, maintaining aerobic respiration and stabilizing mitochondrial function, thereby enabling proliferation and preventing mitochondria-mediated apoptosis in TC. Our present study provides insights into the mechanisms of progression and a promising therapeutic target for TC.

In this study, we identified a high expression level of circPSD3 in TC. Knockdown of circPSD3 disrupted the TCA cycle and caused mitochondrial dysfunction, thereby inhibiting proliferation and inducing apoptosis in TC. Mechanistically, circPSD3 acted as a miR-338-5p sponge to upregulate SUCLG2, an enzyme of the TCA cycle, thereby sustaining the TCA cycle and mitochondrial function.

Conflict of Interest

Authors declare no competing financial interests.

Data Availability

All datasets generated and analyzed during the current study are not publicly available but are available from the corresponding author upon reasonable request.

Acknowledgements

This work was supported by the General Program of the National Natural Science Foundation of China (No.82072956 and No.82002823), the Youth Program of the National Natural Science Foundation of China (No.81802677), and the program from Guangdong Basic and Applied Basic Research Foundation (2022A1515010168). The normal thyroid follicular epithelial cell line Nthy-ori 3-1, and the human PTC cell lines BCPAP were generously provided by Professor Haixia Guan from the Department of Endocrinology, Guangdong Provincial People's Hospital.

Author Contributions

S.H. and X.L. conceived and designed the study. Y.S. and B.H. performed the experiments and bioinformatic analysis. J.G. and X.D. provided support in bioinformatic analysis. Z.H., Y.Z. and H.W. provided experimental support. B.L. collected the clinical tissues. J.L., S.Y., Y.L. and H.X. gave critical comments. Y.S. analyzed the data and drafted the manuscript. S.H. and X.L. supervised the study and revised the manuscript. All authors read and approved the final paper.

Ethical approval and consent to participate

The research protocol has been approved by IEC for Clinical Research and Animal Trials of the First Affiliated Hospital of Sun Yat-sen University (approval number: [2021]109). The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Sun Yat-Sen University (approval number: SYSU-IACUC-2023-000896).

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Tables 1 to 4 are available in the Supplementary Files section

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You are reading this latest preprint version

CircPSD3 Aggravates Tumor Progression by Maintaining TCA Cycle and Mitochondrial Function via Regulating SUCLG2 in Thyroid Carcinoma

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Clinical samples

Cell lines and cell culture

Antibodies and sequences

Construction of stable cell strains

CircRNA sequencing

RNase R treatment

Nuclear and cytoplasmic RNA extraction

RNA fluorescence in situ hybridization (FISH)

Cell proliferation assays (EdU Assay & CCK-8 Assay)

Cell apoptosis assays

Animal experiments

Proteomics

Metabolomics

Oxygen consumption rate measurement

Mitochondrial membrane potential detection

NAD+/NADH measurement

Metabolite supplementation

RNA immunoprecipitation

RNA pull-down

Dual-luciferase reporter assay

HE, IHC and RNA in situ hybridization

Statistical analysis

Results

circPSD3 is up-regulated in thyroid carcinoma

circPSD3 promotes cell proliferation and inhibits cell apoptosis in thyroid carcinoma

circPSD3 regulates the TCA cycle and mitochondrial function via SUCLG2

circPSD3 promotes cell proliferation by regulating TCA cycle in thyroid carcinoma

circPSD3 functions as a sponge of miR-338-5p

circPSD3 regulates TCA cycle and promotes proliferation by sponging miR-338-5p

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1