IDH2-siRNA nanoparticles induce apoptosis in HeLa cell by regulating the miR204/mitophagy pathway

IDH2-siRNA nanoparticles induce apoptosis in HeLa cell by regulating the miR204/mitophagy pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article IDH2-siRNA nanoparticles induce apoptosis in HeLa cell by regulating the miR204/mitophagy pathway Seonhee Kim, Young-Rae Kim, Shuyu Piao, Ikjun Lee, Dong Woon Kim, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6222927/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Mitochondrial dysfunction plays an important role in modulation of cancer and considered as a therapeutic target for anticancer treatment. Mutation of mitochondrial protein isocitrate dehydrogenase 2 (IDH2) has been identified in various cancers. However, the role of wild-type IDH2 in cancer proliferation and migration, particularly in cervical cancer, remains poorly understood. Methods In the present study, we examined the antitumor mechanism of IDH2 deficiency in HeLa cervical cancer cells using both in vitro assay and in vivo xenograft mouse model. Poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles encapsulating IDH2-specific siRNA (siIDH2-NP) were used IDH2 downregulation. Results IDH2 siRNA. IDH2 deficiency suppressed cell proliferation, migration, as well as mitochondrial homeostasis in HeLa cells. IDH2 deficiency markedly inhibited cell proliferation and migration while disrupting mitochondrial homeostasis in HeLa cells. The mitochondrial dysfunction led to the upregulation of endoplasmic reticulum (ER) stress and mitochondrial unfolded protein response (UPRmt) markers, accompanied by a significant reduction in PINK1 accumulation and impaired Parkin translocation to mitochondria suppressing mitophagy. Additionally, STAT3 de-phosphorylation in IDH2 deficient cells increased miR204 expression, which inhibited PINK1 expression and suppressed mitophagy. Consistent findings were obtained in vivo , with significantly reduced tumor volume in treatment of siIDH2-NP. Conclusions These findings suggest that IDH2 deficiency induces apoptotic cell death by disrupting mitochondrial homeostasis and suppressing mitophagy. This study highlights IDH2 as a potential therapeutic target and miR204 as a novel mediator in cervical cancer, providing a promising avenue for the development of anticancer strategies. PLGA nanoparticles cervical cancer IDH2 miR204 PINK1 mitophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Cervical cancer is the second-leading factor of cancer-related death in women worldwide and it is still increasing in the past decades 1 . In addition to human papillomavirus (HPV) infection, various genetic and epigenetic factors contribute to the formation and progression of cervical cancer. Treatments of cervical cancer, including radiation therapy, surgery, and systemic therapy such as chemotherapy, targeted therapy, virus-eliminated therapy, immune therapy or any combination of these approaches have been applied in cervical cancer patients 2 . However, identification of new therapeutic agents and biomarkers for cervical cancer remains a significant challenge. Therefore, it is necessary to the development of safe and effective drug delivery systems. Nanoparticles (NPs) provide a platform for the targeted delivery of drugs to treat various diseases 3 , 4 . Poly (lactic- co -glycolic acid) (PLGA)-based NPs are biodegradable polymeric particles approved by the United States Food and Drug Administration. PLGA particles are suitable for delivering small interfering RNA (siRNA), as they are small enough to penetrate the cell membrane 5 – 7 . NPs deliver insoluble drugs to both local and distant tumor sites, reducing systemic side effects associated with conventional drug therapies. Furthermore, PLGA NPs are biocompatible, non-immunologic, and non-toxic, offering conventional drug delivery system 8 . Mitochondria have emerged as a critical therapeutic target in cancer treatment, with several anticancer strategies focusing on mitochondrial metabolism 9 . The proliferation and metastasis of cancer cells can be inhibited by inducing mitochondrial dysfunction through disruption of the Tricarboxylic acid cycle (TCA) and inhibition of the Oxidative phosphorylation (OXPHOS) complex 10 . NADP + -dependent isocitrate dehydrogenase 2 (IDH2) is a mitochondrial enzyme that converts isocitrate to α-ketoglutarate (α-KG), an essential metabolite for adenosine triphosphate (ATP) production through the TCA cycle. IDH2 deficiency has been reported to impair the function of OXPHOS complex, induces oxidative stress, and mitochondrial dysfunction in endothelial cells 11 . Therefore, IDH2 is a potential therapeutic target for cancer treatment. Mitophagy, a selective autophagy process that removes damaged mitochondria, is regulated by key proteins such as PINK1 and Parkin. PINK1 accumulates on the outer mitochondrial membrane during mitochondrial damage, where it recruits Parkin and downstream autophagy-related proteins to initiate mitophagy. Dysregulation of mitophagy can lead to the accumulation of dysfunctional mitochondria, ultimately triggering apoptotic pathways. In IDH2-deficient cells, mitophagy is suppressed due to decreased PINK1 expression and impaired Parkin translocation to the mitochondria. MicroRNAs (miRNAs) are small non-coding RNAs that inhibit target gene mRNA expression and translation via binding to a target seed sequence in its mRNA. miRNAs are expressed in various tissue and diseases, including many cancers 12 , 13 . miR204 has been identified as a tumor suppressor in various cancers, including colorectal cancer, glioma, and thyroid carcinoma 14 . In cervical cancer, however, the role of miR204 in regulating mitophagy and apoptosis remains unclear. Here, we investigated the role of IDH2 in HeLa cell apoptosis by using PLGA-based NP encapsulating IDH2-siRNA. Our findings suggest that IDH2-siRNA NPs regulate the miR204/mitophagy pathway to induce apoptosis, offering a therapeutic strategy for cervical cancer. 2 Materials and methods 2.1 PLGA (Poly Lactic- co -Glycolic Acid) nanoparticles preparation IDH2 siRNA (human siRNA sequence: sense, 5'-CAGUAUGCCAUCCAGAAGA-3’ and antisense, 5'-UCU UCU GGA UGG CAU ACU G-3') or negative control siRNA-encapsulating PLGA NPs were purchased from Nanoglia (Daejeon, Korea). PLGA NPs with 1:1 ratio of lactic and glycolic acid carrying IDH2 siRNA or scrambled siRNA were prepared using an emulsification/solvent evaporation method 5 , 6 , 43 and their physical characteristics were analyzed with the Zetasizer Nano ZS90 (Malvern Instruments, UK). The NP synthesis and characterization are shown in the supplementary Fig. 1. 2.2 Cell culture, transfection, and cell growth Human cervical epithelial cell (HCEC), SiHa, C33A and HeLa cells were purchased from Clonetics (San Diego, CA, USA) and cultured in DMEM/HIGH glucose media (HyClone Laboratories Inc., South Logan, UT, USA) including 10% Fetal bovine serum (HyClone Laboratories Inc., South Logan, UT, USA) and 1% antibiotic-antimycotic (gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C with 5% CO 2 according to the manufacturer’s instructions. HeLa cells were transfected with siIDH2-NP and siCON-NP treatment, which with miR204 mimic, an inhibitor of miR204 (5′-AGG ATG ACA AAG GGA-3′; miR204-I) (Bioneer, Daejeon, South Korea) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. These transfected cells were incubated for 2 days at 37°C in a 5% CO 2 incubator. Cell counting were performed using ADAM MC Automatic Cell Counter (Bulldog Bio, Portsmouth, NH, USA) according to the manufacturer’s recommendations. 2.3 Mouse Xenograft models All experiments were approved and conducted at Chungnam National University (CNUH-021-A0029) following the guidelines of the Institutional Animal Care and Use Committee. BALB/c nude Mice (5 weeks, male) were purchased at OrientBio inc (Gyeonggi-do, KOREA). Mice were maintained in a controlled environment (ambient temperature 22–24°C; humidity 50–60%; 12-h light/dark cycle). For tumor establishment, mice were anesthetized using Avertin (250 mg/kg), and HeLa cells (1 × 10 7 cells/mouse) mixed with Matrigel (Corning, 356230) were injected subcutaneously into the flanks of the mice. For cancer assessment, cancer length and width were measured daily with digital calipers, and the volumes were calculated using the following formula: (length ∗ width 2 /2). Following the establishment of subcutaneous HeLa cell xenograft in mice 24 and 28 days after tumor cell injection, mice with the same cancer volume (mm 3 ) were divided into two groups (siIDH2 or siCON) and injected 100 µl of PLGA-Nanoparticle (siIDH2, siCON) to tumor directly. Following the establishment of subcutaneous HeLa cell xenograft in mice 24 days after tumor cell injection, an inhibitor of miR204 (5′-AGG ATG ACA AAG GGA-3′; miR204-I) (Bioneer, Daejeon, South Korea) and miR204-mimic were injected 100 µl (100 µM) to tail vein using Invivofectamine 3.0 Reagent (Cat. IVF3001, ThermoFisher, USA), after which the mice were sacrificed by an overdose of Avertin and the tumors were dissected for further experiments. 2.4 Immunoblotting Cultured HeLa cells and cancer tissues were harvested and lysed in 100 µL RIPA buffer (1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.05 M Tris-HCl pH 8), containing 1× Halt protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min on ice. After clearing by centrifugation at 13,000 rpm for 10 min, protein concentration of cell lysate was measured using a bicinchoninic acid (BCA) protein assay kit (iNtRON, cat. 21071). Equal amounts of protein per well were resolved via 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was then washed with Tris-buffered saline (10 mM Tris, 150 mM NaCl) containing 0.1% Tween 20 (TBST) and blocked in TBST containing 5% bovine serum albumin Fraction V (Roche, Basel, Switzerland). Membranes were then incubated with the following antibodies: anti-IDH2 (ab129180), anti-Parkin (ab77924) and anti-PINK1 (ab23707), (Abcam, Cambridge, UK); anti-P-STAT3 (9145S), anti-STAT3 (4904S), anti-cleaved Cas9 (9509S), anti-cleaved Cas3 (9664S), anti-PARP (9542S) (Cell Signaling Technology, Danvers, MA, USA); anti-TOM20 (sc-17764) and anti-GAPDH (sc-47724) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoblotting of 30 µg of whole-cell lysate or tissue homogenate was performed similarly using appropriate primary and secondary antibodies. The membranes were treated with an appropriate peroxidase-conjugated secondary antibody, and the chemiluminescent signal was developed using Super Signal West Pico or Femto Substrate (Pierce Biotechnology, Rockford, IL, USA). Values were normalized to GAPDH and TOM20 as loading controls. 2.5 RNA extraction and quantitative real-time PCR (RT-qPCR) Total RNA was isolated using TRIzol Reagent (Invitrogen) based on the acid guanidinium thiocyanate–phenol–chloroform method. Total RNA concentration was determined using a SmartSpec 3000 spectrophotometer (Bio-Rad, Hercules, CA, USA). Complementary DNA was prepared from total RNA using the Maxime RT Premix kit (information). Quantitative real-time PCR was performed using the Prism7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with the Super Script III Platinum SYBR GreenOne-Step qRT-PCR Kit (Invitrogen). The primers used for human PINK1 were sense 5’-CAA GAG AGG TCC CAA GCA AC-3’ and antisense 5’-GGC AGC ACA TCA GGG TAG TC-3’; human Parkin were sense 5’-CGC CGC CAT GAT AGT CTT TGT T-3’ and antisense 5’-TCC TGC AGC GTC AAG TCG TT-3’; human GRP78 were sense 5’-GAA AGG ATG GTT AAT GAT GCT GAG-3’ and antisense 5’-GTC TTC AAT GTC CGC ATC CTG-3’; human eIF2a were sense 5’-CAA TGG CAA AAT CTC ACT GC-3’ and antisense 5’-AAC CTC ATC TCT ATT AAA AAC ACC AAA-3’; human ATF4 were sense 5’-GAG CTT CCT GAA CAG CGA AGT G-3’ and antisense 5’- TGG CCA CCT CCA GAT AGT CAT C-3’; human ATF6 were sense 5’-TTG ACA TTT TTG GTC TTG TGG-3’ and antisense 5’-GCA GAA GGG GAG ACA CAT TT-3’; human XBP1 were sense 5’-CAA CCA GGA GTT AAG AAC ACG-3’ and antisense 5’-AGG CAA CAG TGT CAG AGT CC-3’; human XBP1 spliced were 5’-CTG AGT CCG AAT CAG GTG CAG-3’ and antisense 5’-GTC CAT GGG AAG ATG TTC TGG-3’; human CHOP were sense 5’-TAT CTC ATC CCC AGG AAA CG-3’ and antisense 5’-GGG CAC TGA CCA CTC TGT TT-3’; human ClpP were sense-5’- GCC AAG CAC ACC AAA CAG A -3’ and antisense-5’- GGA CCA GAA CCT TGT CTA AG -3’; human HSPD1 were sense 5’-GAT ATG GCT ATT GCT ACT GGT GGT GC-3’ and antisense 5’-CCT AAG TCA TGA GCT TGA ACA TCT TC-3’; human Htra2 were sense 5’-CTC CCC GGA GTC AGT ACA ACT-3’ and antisense 5’-AGG ATC TCG ATA TAG ACC ACG G-3’; human LonP1 were sense 5’-AGC CTT ATG TCG GCG TCT TTC-3’ and antisense 5’-CGT CCC CGT GTG GTA GAT TTC-3’. The primers for human glyceraldehyde 3-phosphate dehydrogenase, used as the internal control, were as follows: sense 5’-ATGACATCAAGAAGGTGGTG-3’ and antisense 5’-CATACCAGGAAAATGAGCTTG-3’. Dissociation curves were monitored to check the aberrant formation of primer-dimers. The fold change in the interest gene expression was calculated by using the 2 −ΔΔCt method. 2.6 miRNA isolation and qPCR Total RNA was isolated using TRIzol Reagent (Invitrogen) based on the acid guanidinium thiocyanate–phenol–chloroform method. Total RNA concentration was determined using a SmartSpec 3000 spectrophotometer (Bio-Rad, Hercules, CA, USA). miRNA isolation and cDNA synthesis were synthesized from total RNA used to miScript II RT Kit (Qiagen, cat.218161). Quantitative real-time PCR was performed using the Prism7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with miScript SYBR Green PCR kit (Qiagen, cat.218073). The primers used for miR204-5p were sense-5’-CGC TTC CCT TTG TCA TCC TA-3’ and antisense-universal primer at miScript SYBR Green PCR kit (Qiagen, cat.218073). The primers for U6 snRNA (RNU6), used as the internal control, were as follows: sense-5’-GCA AAT TCG TGA AGC GTT CC-3’ and antisense-universal primer at miScript SYBR Green PCR kit (Qiagen, cat.218073). The fold change in the target gene expression was calculated by using the 2 −ΔΔCt method. 2.7 Histological analysis After washing with phosphate-buffered saline, tumor tissues were fixed with 4% (w/v) paraformaldehyde and then embedded in paraffin. Paraffin sections were deparaffinized and rehydrated according to standard protocols and stained with hematoxylin-eosin (H&E). For immunohistochemistry staining, tumor tissues were stained with primary antibodies anti-MMP9 (diluted in 1:100; Cat.MA5-15886, ThermoFisher), anti-CD31 (diluted in 1:100; Cat.550274, BD science) and anti-PCNA (diluted in 1:100; PC 10; Sigma-Aldrich) overnight at 4°C. HRP-conjugated anti-rabbit and anti-mouse IgG was treated for 60 min at room temperature. Color was developed for 30 sec by incubation with 3,3’-diaminobenzidine (DAB). Sections were counterstained with hematoxylin and examined using microscope (Motic, Richmond, BC, Canada) at 100X magnification. 2.8 CCK-8 cell proliferation assay Cells were transfected with siCON or siIDH2 (50 µm) for 48 h. Cell proliferation was then measured using CCK-8 kit (Dojindo, Japan) according to the manufacturer’s instructions. Briefly, cells were washed with PBS and resuspended in growth media including CCK-8 reagent added at 1/50 the media volume. These cells were then incubated at 37°C for 1 h in the dark. Cell proliferation was measured using an absorbance detector, with measurements performed at 450 nm. 2.9 Cell scratch assay A wound healing assay was used to assess cell migration. Cells were transfected as described previously with siCON or siIDH2 (50 µm) in 6 well tissue culture plates for 24 h, after which a sterile 200 µL pipette tip was used to detach the cells from the monolayer across the center of the well. Floating cells were flushed out by gently rinsing twice with PBS and replaced with serum-free medium (to rule out cell proliferation as the cause of wound closure) followed by incubation for another 24 h. The total incubation time post transfection was therefore 48 h. Cell movement was monitored using microscopy. Photographs were taken immediately and at 24 h after scratching. The relative wound area was quantitatively evaluated using ImageJ software (NIH, Bethesda, MD, USA). 2.10 Transwell assay Transwell assay was employed to detect cell migration. Cells were transfected with siCON or siIDH2 (50 µm) in 6-well tissue culture plates for 24 h, followed by transfer of 5 X 10 5 /ml cells in the upper transwell chamber (24-well plate; Corning, New York, USA) and culture with FBS free medium. Complete growth medium with 10% FBS was added to the lower chamber and incubated for another 24 h. Then, cells on the upper side (nonmigrating cells) were removed and migrated cells on the lower face were washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI and counted on 5 random high-power fields (200x magnification) under a microscope and averaged. 2.11 Measurement of α-ketoglutarate Cells were transfected siCON or siIDH2 (50 µm) for 48 h. After 2 days transfection, cells were lysed in assay buffer. The α-KG concentration was determined using an Alpha-Ketoglutarate Colorimetric/Fluorometric Assay Kit (Cat. #K677-100; Biovision, Milpitas, CA, USA) according to the manufacturer’s instructions. 2.12 Measurement of glutamine Cells were transfected siCON or siIDH2 (50 µm) for 48 h. After 2 days transfection, cells were lysed in assay buffer. The glutamine concentration was determined using a Glutamine Colorimetric Assay Kit (Cat. #K556-100; Biovision, Milpitas, CA, USA) according to the manufacturer’s instructions. 2.13 TUNEL (detecting DNA fragmentation) assay and Flow cytometer TUNEL assay is used to detect DNA fragmentation, such as apoptosis. Cells were transfected siCON or siIDH2 (50 µm) for 48 h. After 2 days transfection, cells were washed twice with PBS and detached from plate using trypsin/EDTA and collected 15 ml tube. These cells were fixed in 100% Ethanol for overnight at 4°C. TUNEL was measured using TUNEL Assay Kit - FITC (Abcam, cat.ab66108) according to the manufacturer’s instructions. Stained cells were analyzed by flow cytometry for FITC using a NovoCyte flow cytometer, as indicated by the manufacturer (ACEA Biosciences, San Diego, CA, USA). Flow cytometry data were analyzed using NovoExpress software. 2.14 Oncomine data collection Microarray datasets were downloaded from public websites or provided by the authors upon request. The web addresses to download particular datasets are listed at ONCOMINE ( www.oncomine.org ). All data that were available from the authors were included in processing and analysis. Raw data was uploaded DYRAD. DYRAD address is written in Supporting Information 2.15 Statistical analysis Statistical analysis was performed using Prism 8 software (GraphPad Software Inc., La Jolla, CA, USA). Data are means ± standard error of the mean (SEM). Differences between two groups were evaluated using t-tests. For multiple comparisons, one-way analysis of variance (ANOVA) was performed followed by a Tukey’s multiple comparison test. p-Values < 0.05 were considered indicative of statistical significance. Data are representative of at least three independent experiments. 3 Results 3.1 IDH2 is overexpressed in cervical cancer cell lines and tumors Deletion of IDH2 in various cell types has been shown to reduce oxidative capacity and triggered mitochondrial dysfunction 11 , 15 . Analysis of the Oncomine Database revealed a significant upregulation of IDH2 mRNA expression in cervical cancer patients compared to normal tissue (Fig. 1 A). Similarly, IDH2 expression was markedly elevated in cervical cancer cell lines including C33A [HPV negative], SiHa [HPV 16 + ], and HeLa [HPV 18 + ] cells, relative to normal human cervical epithelial cells (HCECs). Notably, HeLa cells exhibited the highest levels of IDH2 mRNA and protein expression among the cervical cell lines examined (Fig. 1 B, C). These findings provided a strong rationale for using HeLa cells as a model system to elucidate the functional consequences of IDH2 deficiency and its role in apoptosis induction via regulation of the miR204/mitophagy pathway. 3.2 Downregulation of IDH2 inhibits proliferation and migration by inducing apoptosis in HeLa cells To deliver IDH2 siRNA into HeLa and cancer cells, we used poly-PLGA copolymers, a biodegradable and biocompatible material. The synthesis and characterization of NP are detailed in Supplementary Fig. 1A-C. Treatment of siIDH2-encapsulated NPs reduced the expression of IDH2 in HeLa cells within 48 hours (Fig. 2 A). Hereafter, siCON-NPs and siIDH2-NPs are referred to as siCON and siIDH2, respectively. Silencing of IDH2 significantly reduced the cell doubling time (Fig. 2 B) and decreased cell proliferation, as confirmed by the CCK-assay (Fig. 2 C). In transwell migration assays, siIDH2-treated cells showed a significant reduction in migration, as evident from the decreased number of cells in the lower chamber. Migrated cells were stained with DAPI and quantified under a microscope (Fig. 2 D). Similarly, a cell scratch-wound healing assay showed a marked reduction in wound closure rates in siIDH2-treated cells compared to siCON-treated cells (Fig. 2 E). Collectively, these results indicate that IDH2 knockdown impairs HeLa cell proliferation and migration. To investigate the mechanism of the results, we performed immunoblotting for apoptosis-related proteins. IDH2 knockdown led to a significant increase in cleaved caspase-9, which subsequently activated caspase-3 cleavage. The activated caspase-3 induced PARP cleavage, thereby triggering the apoptotic cascade (Fig. 2 F). DNA fragmentation, a hallmark of apoptosis, was confirmed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Fig. 2 G), which demonstrated an increased presence of fragmented DNA in siIDH2-treated cells. Collectively, these findings indicate that IDH2 deficiency promotes apoptosis, contributing to the inhibition of proliferation and migration in HeLa cells. 3.3 IDH2 knockdown suppresses tumor growth in a HeLa cells xenograft To evaluate the effect of IDH2 knockdown on tumor growth in vivo, HeLa cell xenografts were established in nude mice. After 24 and 28 days of tumor injection, the mice were divided into groups with the same tumor volume (mm 3 ) and the tumors were directly injected with siIDH2 PLGA-NPs or siCON PLGA-NPs. NP injection was well tolerated, with no obvious side-effects such as weight loss or behavioral changes. siIDH2 NPs selectively reduced IDH2 expression in cancer tissues without affecting adjacent or distant tissues, including heart, lung, spleen (Fig. 3 A). Over a 33-day observation period, the volume of tumor (width × width × length ¸ 2) in mice treated with siCON PLGA-NPs increased from 600 to 1400 mm 3 . In contrast, siIDH2 PLGA-NPs treated tumors significantly slower growth, with a mean tumor volume of 800 mm 3 (Fig. 3 B, C). H&E staining of paraffin-embedded tumor sections revealed distinct morphological differences. Tumors from siCON-treated mice displayed a compact epithelial cell structure, while those from siIDH2-treated mice exhibited a looser epithelial cell organization (Fig. 3 D). To further investigate the effects of IDH2 knockdown on tumor proliferation, neovascularization, and metastasis, immunohistochemical staining was performed. Tumor sections stained with anti-PCNA antibody, a marker of cell proliferation, showed significantly reduced proliferation in siIDH2-treated mice compared to siCON (Fig. 3 D). Additionally, siIDH2-treated tumor sections displayed markedly lower neovascularization, as evidenced by reduced CD31 staining, and a diminished metastatic index, as indicated by decreased MMP-9 expression (Fig. 3 D). These findings demonstrate that IDH2 deficiency effectively suppresses tumor proliferation, neovascularization, and metastatic potential in vivo, 3.4 IDH2 deficiency disrupts mitochondrial homeostasis via UPR activation and mitophagy suppression Mitochondrial dysfunction, a critical trigger for apoptosis, often activates the mitochondrial UPRmt and mitophagy 16 , 17 . Reactive oxygen species (ROS) generated by IDH2 deficiency disrupt mitochondrial homeostasis by inhibiting TCA cycle progression 15 . Damaged mitochondria activate the UPRmt, upregulating stress-response proteins such as CHOP, Hsp60, Htra2, ClpP, and LONP to restore function (Fig. 4 A). CHOP, a key transcription factor, binds to the promoter regions of repair genes under mitochondrial stress, promoting mitochondrial recovery 18 . In addition, IDH2 deficiency impairs mitophagy by reducing PINK1 protein accumulation on the outer mitochondrial membrane, a critical step for mitophagy activation, despite unchanged PINK1 and Parkin mRNA levels (Fig. 4 B). This disruption prevents Parkin translocation to the mitochondria, further compromising mitophagy (Fig. 4 C). These results suggest that IDH2 deficiency-induced mitochondrial dysfunction activates stress responses, promoting apoptosis in HeLa cells. 3.5 IDH2 deficiency induces miR204 expression by reducing the mitochondrial glutamine level Cancer stem cells in colorectal, brain, breast, and cervical cancers rely on both mitochondrial oxidative metabolism and glycolysis for energy production 19 , 20 . In IDH2-deleted cells, the mitochondrial α-KG concentration was markedly decreased (Fig. 4 D). Glutamine, a key regulator of cancer cell proliferation, migration, and antioxidant defense, is converted into glutamate and transported into mitochondria to replenish α-KG. IDH2 deficiency significantly reduced mitochondrial glutamine levels compared to cytosolic levels (Fig. 4 E). Glutamine is metabolized to α-KG via glutaminolysis, a process involving glutaminase (GLS) and glutamate dehydrogenase 1 (GDH1) 21 , 22 . Solute-linked carrier family 1 member A5 (SLCA15) is a transporter of glutamine in rapidly growing epithelial and tumor cells 23 . In IDH2-deficient cells, GLS and GDH1 mRNA levels were upregulated, while solute carrier family 1 member A5 (SLCA15), a glutamine transporter, remained unchanged (Fig. 4 F). This result suggests intracellular glutamine is consumed to compensate for the loss of α-KG, depleting mitochondrial glutamine stores (Fig. 4 G). Glutamine also regulates STAT3 phosphorylation, which suppresses miR204 by binding to the TRPM3 promoter region 24 , 25 , 26 . STAT3 phosphorylation was significantly decreased in IDH2 deficient cells miR204 expression was increased in IDH2-deficient HeLa cells without glutamine (Fig. 4 H). Reduced glutamine levels in IDH2-deficient cells led to decreased STAT3 phosphorylation, thereby de-repressing miR204 expression (Fig. 4 I, J). Elevated miR204 expression was observed in IDH2-deficient cells under glutamine-deprived conditions (Fig. 4 K). Furthermore, overexpression of dominant-negative STAT3 (DN-STAT3) inhibited miR204 repression (Fig. 4 L, M) These findings demonstrate that IDH2 deficiency reduces mitochondrial glutamine levels, which decreases STAT3 phosphorylation and upregulates miR204 expression. 3.6 miR204 directly inhibits PINK1 expression miRNAs regulate target gene expression by binding to target mRNA with the seed region (positions 2–7 from the miRNA 5´-end) being critical for this interaction. To confirm that miRNA204 directly targets PINK1, we analyzed the PINK1 mRNA coding sequence and identified a conserved miR204-binding site within the coding region (Fig. 5 A). The binding site includes a complementary sequence to the miR204 seed region, suggesting a post-transcriptional regulatory mechanism. To confirm the interaction, we co-transfected miR204 mimics and plasmids expressing wild-type (WT) or mutant PINK1 fused to a Myc-tag. The mutant PINK1 was designed to disrupt the miR204 seed-binding site. Overexpression of miR204 significantly inhibited Myc-PINK1 expression in cells transfected with the WT construct, while no inhibition was observed in cells transfected with the mutant construct (Fig. 5 B, C). These results demonstrate that miR204 directly binds to the PINK1 coding region, suppressing the PINK1 expression at the post-transcriptional level. 3.7 Inhibition of miR204 restores mitophagy and reduces apoptosis PINK1 expression rescued IDH2 siRNA and miR204 inhibitor (miR204-I) co-transfected HeLa cells but did not affect Parkin expression (Fig. 5 D). Treatment with an miR204 inhibitor (miR204-I) restored PINK1 levels and facilitated Parkin translocation from the cytosol to mitochondria, effectively rescuing mitophagy (Fig. 5 E, F). Immunofluorescence analysis confirmed enhanced Parkin localization to damaged mitochondria in miR204-I-treated cells (Fig. 5 F). Levels of cleaved caspase-8, -3, and PARP, key markers of apoptosis, were significantly decreased following miR204 inhibition (Fig. 5 G). Similarly, TUNEL assays showed reduced DNA fragmentation in cells treated with miR204-I, indicating suppression of apoptosis (Fig. 5 H). These results confirm that miR204 downregulation restores mitochondrial quality control mechanisms and mitigates apoptosis by enhancing PINK1 expression and Parkin recruitment to mitochondria. 3.8 miR204 suppressed cancer growth in HeLa cell xenografts To evaluate the role of miR204 in tumor growth, siIDH2 nanoparticles (NPs) were injected directly into tumors on days 24 and 28, with tail-vein injections of either an miR204 mimic or inhibitor (miR204-I) administered on day 24 using Invivofectamine™ 3.0. After 33 days, tumor volumes in the control and siIDH2 + miR204-I groups increased from 1200 mm³ to 1500 mm³. In contrast, the tumor volumes in the miR204 mimic group exhibited significantly slower growth, with an average tumor volume of 600 mm³ (Fig. 6 A). Representative images of tumor size confirmed the inhibitory effect of the miR204 mimic compared to controls (Fig. 6 B). miR204-I treatment suppressed miR204 expression in siIDH2-treated mice (Fig. 6 C). The siIDH2 + miR204-I group showed tumor growth similar to that of the control group (Fig. 6 A). H&E staining of tumor paraffin sections revealed that tumors from the miR204 mimic group exhibited loose epithelial organization, whereas tumors from the siIDH2 + miR204-I and control groups displayed compact epithelial structures (Fig. 6 D). Immunohistochemical staining showed significantly reduced levels of PCNA, CD31, and MMP9 in the miR204 mimic group compared to the other groups (Fig. 6 D). The PINK1 expression significantly decreased in the miR204 mimic group, while PINK1 levels was not changed in siIDH2 + miR204-I group (Fig. 6 E). These findings suggest that the anticancer effect of IDH2 deficiency is mediated by miR204 upregulation, which suppresses tumor proliferation, neovascularization, and metastasis by targeting PINK1. 4 Discussion Cervical cancer remains a leading cause of cancer-related mortality in women, with poor survival rates and frequent relapses, particularly in younger patients 27 , 28 . It is most commonly associated with high-risk oncogenic HPV types, typically HPV16 and HPV18 29 . In addition, mitochondria participate in carcinogenesis through energy production, macromolecular synthesis, and the regulation of cell survival. Malignant tumors selectively retain the mitochondrial genome and OXPHOS complex function, whereas tumors with pathogenic mitochondrial DNA mutations are benign, indicating the importance of mitochondria for cancer progression. Moreover, mutant TCA cycle enzymes, such as IDH, produce oncometabolites that promote tumorigenesis 30 . IDH2 mutation is found in several cancer types and has been associated with a poor prognosis 31 . However, the effect of WT IDH2 or IDH2 overexpression in cervical cancer is controversial. Previous studies have shown that IDH2 deficiency induces mitochondrial dysfunction, oxidative stress, inflammation, and endothelium-dependent vasomotor function in endothelial cells 11 , 15 . To achieve efficient and targeted IDH2 knockdown, we used PLGA-NPs for the delivery of siIDH2. PLGA is a biocompatible and biodegradable polymer approved by the FDA for clinical use, making it a vehicle for siRNA delivery 32 . In our study, siIDH2-loaded nanoparticles demonstrated IDH2 silencing in HeLa cells and tumor tissues without affecting non-cancerous tissues such as the heart, lung, or spleen. The targeted delivery minimized off-target effects and systemic toxicity (Fig. 3 A). In this study, our findings demonstrate that IDH2 deficiency disrupts mitochondrial homeostasis, resulting in impaired cellular function and increased apoptosis. Specifically, IDH2 knockdown in HPV18 + HeLa cells suppressed cancer cell proliferation and migration both in vitro and in vivo. These results suggest that the potential therapeutic implications of targeting IDH2 in cervical cancer. Mitochondrial dysfunction plays a critical role in cancer cell survival and apoptosis. Cancer cells are typically resistant to mitochondrial stress, as mitophagy eliminates dysfunctional organelles and prevents cellular damage 33 , 34 . However, apoptosis can be triggered if there is excessive mitochondrial damage 35 . Our study has shown that IDH2 deficiency induces upregulation of UPRmt markers such as GRP78, ATF4, CHOP, HSPD1, and ClpP and ER stress These markers indicate attempts to restore mitochondrial function under stressful conditions. Despite the activation of UPRmt, IDH2 deficiency inhibits PINK1 accumulation and blocks parkin translocation to mitochondria, thus damaging mitochondrial dysfunction (Fig. 4 , Fig. 5 ). These findings suggest that IDH2 deficiency disrupts both mitochondrial repair mechanisms and quality control pathways, leading to apoptosis. In addition, IDH2 deficiency significantly altered mitochondrial metabolism, reducing α-KG and glutamine levels. Glutamine is a critical substrate for the TCA cycle and supports cancer cell survival by replenishing mitochondrial intermediates through glutaminolysis. Our results demonstrated that IDH2 deficiency depleted mitochondrial glutamine levels while upregulating glutaminase (GLS) and glutamate dehydrogenase 1 (GDH1), key enzymes involved in glutaminolysis. These changes suggest that intracellular glutamine is rapidly consumed to compensate for the loss of α-KG, leading to mitochondrial glutamine depletion and metabolic dysregulation (Fig. 4 ). In particular, glutamine levels directly regulate STAT3 phosphorylation, which is a key transcription factor involved in cancer progression 36 . Reduced mitochondrial glutamine levels in IDH2-deficient cells decreased STAT3 phosphorylation, leading to the upregulation of miR204. Elevated miR204 levels were observed under glutamine-deprived conditions, whereas glutamine supplementation had minimal impact on its expression. Furthermore, dominant-negative STAT3 (DN-STAT3) overexpression inhibited STAT3 phosphorylation and its DNA-binding activity, confirming that STAT3 inactivation mediates miR204 upregulation (Fig. 4 ). These findings link mitochondrial glutamine metabolism to transcriptional regulation through the STAT3/miR204 axis. IDH2 deficiency induces miR204 expression in hepatocytes 37 . miR204 has been reported to regulate various physiological and pathological processes, including cardiovascular diseases, tumor suppression, and β-cell proliferation3 38–43 . Here, we demonstrate that miR204 directly suppresses PINK1 expression by binding to its coding region sequence (CDS). This inhibition of PINK1 translation impairs mitophagy and further promotes apoptosis by preventing Parkin translocation to mitochondria. In particular, inhibition of miR204 restored PINK1 expression, rescued Parkin-mediated mitophagy, and mitigated apoptosis in IDH2-deficient cells (Fig. 6 ). These results suggest that the critical role of miR204 in mediating the effects of IDH2 deficiency on mitochondrial quality control and apoptosis. 5 Conclusions Collectively, our findings reveal that IDH2 deficiency suppresses cervical cancer progression by disrupting mitochondrial homeostasis through the inhibition of mitophagy. The subsequent upregulation of miR204 contributes to mitophagy suppression and apoptosis by targeting PINK1. These results suggest that the IDH2/miR204/PINK1 axis as a potential therapeutic target in cervical cancer and other malignancies characterized by mitochondrial dysfunction. Future studies should explore the translational potential of targeting this pathway in combination with existing therapies to overcome tumor resistance and improve patient outcomes. Declarations Acknowledgements The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/DwAPeK Author contributions S.K and C.S.K initiated and designed this study. S.K performed and analysed the majority of experiments. S.K., Y.R.K., and C.S.K wrote the manuscript. S.P, Y.R.K., I.L and H.N performed and analysed individual experiments. I.L, S.P and S.C performed data curation. C.S.K, D.W.K, B.H.J and K.I supervised the study. S.K and Y.R.K contributed to equal this work. C.S.K and K.I were corresponding author in this work. Funding This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A6A1029617) and by the research fund of Chungnam National University and by the research fund of Chungnam National University Hospital. Data availability The datasets supporting the conclusions of this article are available from the corresponding author upon reasonable request. Ethics approval and consent to participate All experiments were approved and conducted at Chungnam National University (CNUH-021-A0029) following the guidelines of the Institutional Animal Care and Use Committee. Informed consent was obtained from each participate. Competing interests The authors declare no conflict of interest. References Olusola, P., Banerjee, H. N., Philley, J. V. & Dasgupta, S. Human Papilloma Virus-Associated Cervical Cancer and Health Disparities. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6222927","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444217826,"identity":"4056e40a-d2c5-45dd-ba00-aed3a28fb7eb","order_by":0,"name":"Seonhee Kim","email":"","orcid":"","institution":"Chungnam National University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Seonhee","middleName":"","lastName":"Kim","suffix":""},{"id":444217828,"identity":"bf044c3f-ffce-461a-adc3-9a93e43a615d","order_by":1,"name":"Young-Rae Kim","email":"","orcid":"","institution":"University of Iowa Carver college of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Young-Rae","middleName":"","lastName":"Kim","suffix":""},{"id":444217829,"identity":"f0fdbaad-77d2-4d6a-9a2f-ae9cf9139fcb","order_by":2,"name":"Shuyu Piao","email":"","orcid":"","institution":"Chungnam National University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuyu","middleName":"","lastName":"Piao","suffix":""},{"id":444217830,"identity":"e676f658-6955-4658-9d81-47dfacd31d71","order_by":3,"name":"Ikjun Lee","email":"","orcid":"","institution":"Chungnam National University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ikjun","middleName":"","lastName":"Lee","suffix":""},{"id":444217831,"identity":"89540a9e-8935-4e54-891d-ea794b8acab9","order_by":4,"name":"Dong Woon Kim","email":"","orcid":"","institution":"Chungnam National University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"Woon","lastName":"Kim","suffix":""},{"id":444217832,"identity":"498e2c0f-f49c-4b4b-ba80-4eea6184fc76","order_by":5,"name":"Byeong Hwa Jeon","email":"","orcid":"","institution":"Chungnam National University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Byeong","middleName":"Hwa","lastName":"Jeon","suffix":""},{"id":444217833,"identity":"52a793e6-076e-4036-b9c2-6901a88d3a5c","order_by":6,"name":"Kaikobad Irani","email":"","orcid":"","institution":"Department of veterans affairs","correspondingAuthor":false,"prefix":"","firstName":"Kaikobad","middleName":"","lastName":"Irani","suffix":""},{"id":444217836,"identity":"0671647b-a0ad-48b1-afad-3ec4cc28b7fb","order_by":7,"name":"Cuk-Seong Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYFACNoYDDBUwzgGitZwhVQsDYxspWvj7jyUeujnPLs/gAPPDDwxn7hHWInEj7cDh3G3JxQYH2IwlGG4UE9ZiIMHeANRyIHHDAQYzBoYPCURo4T8O1DIHpIX9G5FaGEAOawBp4QHacoMILUC/JBzOOZacOPMwT7FEwhkitABDzPhzTo1dYt/x9o0fPhwjQgsCMAMxSRpGwSgYBaNgFOAGAN8+PhYE4UCCAAAAAElFTkSuQmCC","orcid":"","institution":"Chungnam National University College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Cuk-Seong","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-03-14 00:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6222927/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6222927/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81144855,"identity":"d6c4d6b8-2126-4985-9c91-2a7b1bd46974","added_by":"auto","created_at":"2025-04-22 17:41:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":214821,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIDH2 si overexpressed in cervical cancer cell lines and patients regardless of stage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) IDH2 mRNA expression was analyzed in cervical cancer patient cohorts (Biewenga Cervix and Scotto Cervix) using the Oncomine microarray database. (B-C) IDH2 mRNA and protein expression levels were evaluated in normal human cervical epithelial cells (HCECs) and cervical cancer cell lines, including C33A (HPV-negative), SiHa (HPV 16+), and HeLa (HPV 18+). GAPDH was used as a loading control. Protein expression levels were quantified by densitometric analysis (bottom panels). All experiments are representative of three independent experiments. These data are presented as means ± SEM. *p \u0026lt; 0.05 vs. HCEC.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6222927/v1/d3faa6c3d13027e61f06f90d.png"},{"id":81145058,"identity":"b4cdded6-c61c-4cd5-9651-a1a637207a69","added_by":"auto","created_at":"2025-04-22 17:49:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":924460,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIDH2 deficiency induces apoptosis and inhibits proliferation and migration in HeLa cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa cells were treated with siCON-NP or siIDH2-NP for 48 h. (A) IDH2 expression was measured using immunoblotting. GAPDH was used as the internal control. (B) Cell number was measured using an ADAM-MC cell counting machine during 7 days of transfection. (C) Cell proliferation assay was performed using a CCK-8 kit. (D) Transwell assay was conducted to determine cell migration. Scale bar; 50 μm. (E) Wound healing assays were conducted, and images were acquired at 24 and 48 hours using a light microscope. (F) Immunoblotting analysis of apoptosis-related proteins in IDH2-deleted HeLa cells. GAPDH was used as an internal control. The protein levels were qualified by densitometric analysis (right panel). (G) The apoptotic rare in HeLa cells was measured by TUNEL staining after siIDH2 transfection. All experiments are representative of three independent experiments. These data are presented as means ± SEM. *p \u0026lt; 0.05 vs. siCON.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6222927/v1/c51501f87a6b90b4664b31f3.png"},{"id":81144860,"identity":"13d57da0-7654-48fd-b396-4e0cb64a8da7","added_by":"auto","created_at":"2025-04-22 17:41:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1657189,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIDH2 deficiency suppresses tumor growth in HeLa Xenograft models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa cells were subcutaneously xenografted into nude mice, followed by a 24-day stabilization period. siCON- or siIDH2-loaded nanoparticles were directly injected into the tumor tissues on days 24 and 28.\u003cbr\u003e\n(A) IDH2 protein expression in tumor, spleen, lung, and heart tissues was evaluated by immunoblotting. GAPDH or α-tubulin served as internal controls. Quantification of IDH2 expression in tumor and non-tumor tissues is shown on the right. (B) Tumor volumes were measured over a 9-day period post-injection. Tumor growth was significantly suppressed in siIDH2-treated mice compared to siCON-treated controls. (C) Representative images of excised tumors from the siCON- and siIDH2-treated groups. (D) Histological and immunohistochemical analysis of tumor sections. H\u0026amp;E staining was used to assess tissue morphology, and immunostaining for PCNA (proliferation marker), CD31 (neovascularization marker), and MMP9 (metastasis marker) was performed. Quantitative analysis of positively stained cells is shown in the lower panel. Scale bar: 50 μm. All experiments are representative of three independent experiments. These data are presented as means ± SEM. *p \u0026lt; 0.05 vs. siCON or siCON injected HeLa xenograft.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6222927/v1/b12142f05196b68a2f639062.png"},{"id":81144859,"identity":"603af46f-2f16-468f-9174-6142a7435a38","added_by":"auto","created_at":"2025-04-22 17:41:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":722657,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIDH2 deficiency suppress mitophagy and induces miR204 expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa cells were treated with siCON-NP or siIDH2-NP for 48 h. (A) mRNA expression levels of ER stress markers (GRP78, ATF4, ATF6, XBP1, and CHOP) and mitochondrial UPR markers (ClpP, HSPD1, HTRA2, and LONP1) were measured using qPCR. (B) mRNA levels of PINK1 and Parkin were quantified in siCON- and siIDH2-treated cells. (C) Immunoblotting analysis of PINK1 and Parkin protein levels in siCON- and siIDH2-treated cells. GAPDH was used as the internal control. Densitometric analysis is shown right. (D) Subcellular fractionation showing α-ketoglutaratelevels (α-KG) in mitochondrial and cytosolic fractions in siCON- and siIDH2-treated cells. (E) Mitochondrial glutamine (Gln) levels in siCON- and siIDH2-treated cells were quantified. (F) mRNA expression levels of GLS, GDH1, and SLC1A5, genes related to glutamine metabolism, were measured using qPCR. (G) Schematic illustration of glutamine metabolism under IDH2 deficiency. (H) Immunoblotting of phosphorylated STAT3 (P-STAT3) and total STAT3 (T-STAT3) in siCON- and siIDH2-treated cells. GAPDH was used as the internal control. Densitometric analysis of P-STAT3 levels is shown below. (I-J) Schematic models illustrating the regulatory pathway by which IDH2 deficiency leads to miR204 induction via reduced glutamine levels and decreased STAT3 phosphorylation. (K) miR204 expression levels were measured using qPCR in siIDH2-treated cells cultured with or without glutamine. (L) Immunoblotting of P-STAT3 and T-STAT3 in cells transfected with β-gal or dominant-negative STAT3 (DN-STAT3). GAPDH was used as the internal control. (M) miR204 expression was measured using qPCR in β-gal and DN-STAT3-transfected cells. All experiments are representative of three independent experiments. These data are presented as means ± SEM. *p \u0026lt; 0.05 vs. siCON or β-gal.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6222927/v1/960d8d76fee53a864204765a.png"},{"id":81145060,"identity":"7a289dc5-966c-4540-bf5d-ab037dfe9d47","added_by":"auto","created_at":"2025-04-22 17:49:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1146344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of miR204 restores mitophagy and apoptosis via enhancing PINK1 expression and parkin recruitment to mitochondria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa cells were treated with siCON-NP or siIDH2-NP for 48 h. or transfected with miR204-I (after 24 h siIDH2 treatment) for 48 h. (A) Expected binding site of pink1 mRNA and miR204 \u003cem\u003ehomo sapiens\u003c/em\u003e and \u003cem\u003eMus musculus\u003c/em\u003e. (B) Overexpression of the miR204 duplex decreased the protein level of WT PINK1. Construction of Myc-tagged WT PINK1-coding region and immunoblotting. (C) Overexpression of the miR204 duplex did no affect the protein level of mutant PINK1. (D) Immunoblotting analysis of PINK1 and Parkin with miR204-I in IDH2-deleted HeLa cells. CCCP was treated as positive control for induction of mitophagy. The protein levels were qualified by densitometric analysis (below panel). (E) Immunoblotting analysis of Parkin translocation in mitochondria fraction HeLa cells. The protein levels were qualified by densitometric analysis (below panel). (F) Immunofluorescence images for Parkin translocation to mitochondria. Scale bar; 20 μm. The stained levels were qualified by densitometric analysis (below panel). (G) Immunoblotting analysis of apoptosis-related proteins with miR204-I in IDH2-deleted HeLa cells. The protein levels were qualified by densitometric analysis (right panel). (H) The apoptotic rare in HeLa cells was measured by TUNEL staining after siIDH2 transfection with miR204-I. GAPDH and TOM20 were used as an internal control. All experiments are representative of three independent experiments. These data are presented as means ± SEM. *p \u0026lt; 0.05 vs. siCON.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6222927/v1/cfa590ef2ff31405e92bb57d.png"},{"id":81144867,"identity":"0d842323-1c79-4bab-aa31-36848a821149","added_by":"auto","created_at":"2025-04-22 17:41:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2122053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR204 suppresses tumor growth in a HeLa Xenograft models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeLa cells were subcutaneously xenografted into nude mice, followed by a 24-day stabilization period. siCON- or siIDH2-loaded nanoparticles were directly injected into the tumor tissues on days 24 and 28. In 24 days after tumor injection, miR204-I and miR204-mimic were injected 100 μl to tail vein using Invivofectamine 3.0 Reagent. (A) Tumor volume was measured over a 9-day period following treatment. (B) Representative images of tumors excised from mice in each treatment group.(C) miR204 expression levels in isolated tumors were quantified by qPCR. RNU6 was used as an internal control. (D) Histological and immunohistochemical analysis of tumor sections. H\u0026amp;E staining was used to assess tissue morphology, and immunostaining for PCNA (proliferation marker), CD31 (neovascularization marker), and MMP9 (metastasis marker) was performed. Quantitative analysis of positively stained cells is shown in the lower panel. Scale bar: 50 μm. (E) Immunoblotting analysis of PINK1 expression in isolated tumors. The protein levels were qualified by densitometric analysis (right panel). GAPDH was used as an internal control. All experiments are representative of three independent experiments. These data are presented as means ± SEM. *p \u0026lt; 0.05 vs. siCON injected HeLa xenograft.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6222927/v1/788eed1e3be89a71911225f2.png"},{"id":85834069,"identity":"afd79d7c-b7af-4213-939f-57320b2b2ee1","added_by":"auto","created_at":"2025-07-02 08:09:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8074994,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6222927/v1/adc7241c-590b-4132-84a7-71bfdfe84b5e.pdf"},{"id":81144858,"identity":"63d933b8-4e21-4e3f-9676-43f75d7b2f6c","added_by":"auto","created_at":"2025-04-22 17:41:33","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":178896,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6222927/v1/f64f4e3d77ad89ebdad4fe60.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"IDH2-siRNA nanoparticles induce apoptosis in HeLa cell by regulating the miR204/mitophagy pathway","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eCervical cancer is the second-leading factor of cancer-related death in women worldwide and it is still increasing in the past decades\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In addition to human papillomavirus (HPV) infection, various genetic and epigenetic factors contribute to the formation and progression of cervical cancer. Treatments of cervical cancer, including radiation therapy, surgery, and systemic therapy such as chemotherapy, targeted therapy, virus-eliminated therapy, immune therapy or any combination of these approaches have been applied in cervical cancer patients\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, identification of new therapeutic agents and biomarkers for cervical cancer remains a significant challenge. Therefore, it is necessary to the development of safe and effective drug delivery systems.\u003c/p\u003e \u003cp\u003eNanoparticles (NPs) provide a platform for the targeted delivery of drugs to treat various diseases\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Poly (lactic-\u003cem\u003eco\u003c/em\u003e-glycolic acid) (PLGA)-based NPs are biodegradable polymeric particles approved by the United States Food and Drug Administration. PLGA particles are suitable for delivering small interfering RNA (siRNA), as they are small enough to penetrate the cell membrane\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. NPs deliver insoluble drugs to both local and distant tumor sites, reducing systemic side effects associated with conventional drug therapies. Furthermore, PLGA NPs are biocompatible, non-immunologic, and non-toxic, offering conventional drug delivery system\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMitochondria have emerged as a critical therapeutic target in cancer treatment, with several anticancer strategies focusing on mitochondrial metabolism\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The proliferation and metastasis of cancer cells can be inhibited by inducing mitochondrial dysfunction through disruption of the Tricarboxylic acid cycle (TCA) and inhibition of the Oxidative phosphorylation (OXPHOS) complex\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. NADP\u003csup\u003e+\u003c/sup\u003e-dependent isocitrate dehydrogenase 2 (IDH2) is a mitochondrial enzyme that converts isocitrate to α-ketoglutarate (α-KG), an essential metabolite for adenosine triphosphate (ATP) production through the TCA cycle. IDH2 deficiency has been reported to impair the function of OXPHOS complex, induces oxidative stress, and mitochondrial dysfunction in endothelial cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Therefore, IDH2 is a potential therapeutic target for cancer treatment.\u003c/p\u003e \u003cp\u003eMitophagy, a selective autophagy process that removes damaged mitochondria, is regulated by key proteins such as PINK1 and Parkin. PINK1 accumulates on the outer mitochondrial membrane during mitochondrial damage, where it recruits Parkin and downstream autophagy-related proteins to initiate mitophagy. Dysregulation of mitophagy can lead to the accumulation of dysfunctional mitochondria, ultimately triggering apoptotic pathways. In IDH2-deficient cells, mitophagy is suppressed due to decreased PINK1 expression and impaired Parkin translocation to the mitochondria.\u003c/p\u003e \u003cp\u003eMicroRNAs (miRNAs) are small non-coding RNAs that inhibit target gene mRNA expression and translation via binding to a target seed sequence in its mRNA. miRNAs are expressed in various tissue and diseases, including many cancers\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. miR204 has been identified as a tumor suppressor in various cancers, including colorectal cancer, glioma, and thyroid carcinoma\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In cervical cancer, however, the role of miR204 in regulating mitophagy and apoptosis remains unclear.\u003c/p\u003e \u003cp\u003eHere, we investigated the role of IDH2 in HeLa cell apoptosis by using PLGA-based NP encapsulating IDH2-siRNA. Our findings suggest that IDH2-siRNA NPs regulate the miR204/mitophagy pathway to induce apoptosis, offering a therapeutic strategy for cervical cancer.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 PLGA (Poly Lactic-\u003cem\u003eco\u003c/em\u003e-Glycolic Acid) nanoparticles preparation\u003c/h2\u003e\n \u003cp\u003eIDH2 siRNA (human siRNA sequence: sense, 5\u0026apos;-CAGUAUGCCAUCCAGAAGA-3\u0026rsquo; and antisense, 5\u0026apos;-UCU UCU GGA UGG CAU ACU G-3\u0026apos;) or negative control siRNA-encapsulating PLGA NPs were purchased from Nanoglia (Daejeon, Korea). PLGA NPs with 1:1 ratio of lactic and glycolic acid carrying IDH2 siRNA or scrambled siRNA were prepared using an emulsification/solvent evaporation method\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e and their physical characteristics were analyzed with the Zetasizer Nano ZS90 (Malvern Instruments, UK). The NP synthesis and characterization are shown in the supplementary Fig.\u0026nbsp;1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Cell culture, transfection, and cell growth\u003c/h2\u003e\n \u003cp\u003eHuman cervical epithelial cell (HCEC), SiHa, C33A and HeLa cells were purchased from Clonetics (San Diego, CA, USA) and cultured in DMEM/HIGH glucose media (HyClone Laboratories Inc., South Logan, UT, USA) including 10% Fetal bovine serum (HyClone Laboratories Inc., South Logan, UT, USA) and 1% antibiotic-antimycotic (gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e according to the manufacturer\u0026rsquo;s instructions. HeLa cells were transfected with siIDH2-NP and siCON-NP treatment, which with miR204 mimic, an inhibitor of miR204 (5\u0026prime;-AGG ATG ACA AAG GGA-3\u0026prime;; miR204-I) (Bioneer, Daejeon, South Korea) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s instructions. These transfected cells were incubated for 2 days at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Cell counting were performed using ADAM MC Automatic Cell Counter (Bulldog Bio, Portsmouth, NH, USA) according to the manufacturer\u0026rsquo;s recommendations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Mouse Xenograft models\u003c/h2\u003e\n \u003cp\u003eAll experiments were approved and conducted at Chungnam National University (CNUH-021-A0029) following the guidelines of the Institutional Animal Care and Use Committee. BALB/c nude Mice (5 weeks, male) were purchased at OrientBio inc (Gyeonggi-do, KOREA). Mice were maintained in a controlled environment (ambient temperature 22\u0026ndash;24\u0026deg;C; humidity 50\u0026ndash;60%; 12-h light/dark cycle). For tumor establishment, mice were anesthetized using Avertin (250 mg/kg), and HeLa cells (1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells/mouse) mixed with Matrigel (Corning, 356230) were injected subcutaneously into the flanks of the mice. For cancer assessment, cancer length and width were measured daily with digital calipers, and the volumes were calculated using the following formula: (length \u0026lowast; width\u003csup\u003e2\u003c/sup\u003e/2). Following the establishment of subcutaneous HeLa cell xenograft in mice 24 and 28 days after tumor cell injection, mice with the same cancer volume (mm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) were divided into two groups (siIDH2 or siCON) and injected 100 \u0026micro;l of PLGA-Nanoparticle (siIDH2, siCON) to tumor directly. Following the establishment of subcutaneous HeLa cell xenograft in mice 24 days after tumor cell injection, an inhibitor of miR204 (5\u0026prime;-AGG ATG ACA AAG GGA-3\u0026prime;; miR204-I) (Bioneer, Daejeon, South Korea) and miR204-mimic were injected 100 \u0026micro;l (100 \u0026micro;M) to tail vein using Invivofectamine 3.0 Reagent (Cat. IVF3001, ThermoFisher, USA), after which the mice were sacrificed by an overdose of Avertin and the tumors were dissected for further experiments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Immunoblotting\u003c/h2\u003e\n \u003cp\u003eCultured HeLa cells and cancer tissues were harvested and lysed in 100 \u0026micro;L RIPA buffer (1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.05 M Tris-HCl pH 8), containing 1\u0026times; Halt protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min on ice. After clearing by centrifugation at 13,000 rpm for 10 min, protein concentration of cell lysate was measured using a bicinchoninic acid (BCA) protein assay kit (iNtRON, cat. 21071). Equal amounts of protein per well were resolved via 10\u0026ndash;15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was then washed with Tris-buffered saline (10 mM Tris, 150 mM NaCl) containing 0.1% Tween 20 (TBST) and blocked in TBST containing 5% bovine serum albumin Fraction V (Roche, Basel, Switzerland). Membranes were then incubated with the following antibodies: anti-IDH2 (ab129180), anti-Parkin (ab77924) and anti-PINK1 (ab23707), (Abcam, Cambridge, UK); anti-P-STAT3 (9145S), anti-STAT3 (4904S), anti-cleaved Cas9 (9509S), anti-cleaved Cas3 (9664S), anti-PARP (9542S) (Cell Signaling Technology, Danvers, MA, USA); anti-TOM20 (sc-17764) and anti-GAPDH (sc-47724) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoblotting of 30 \u0026micro;g of whole-cell lysate or tissue homogenate was performed similarly using appropriate primary and secondary antibodies. The membranes were treated with an appropriate peroxidase-conjugated secondary antibody, and the chemiluminescent signal was developed using Super Signal West Pico or Femto Substrate (Pierce Biotechnology, Rockford, IL, USA). Values were normalized to GAPDH and TOM20 as loading controls.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 RNA extraction and quantitative real-time PCR (RT-qPCR)\u003c/h2\u003e\n \u003cp\u003eTotal RNA was isolated using TRIzol Reagent (Invitrogen) based on the acid guanidinium thiocyanate\u0026ndash;phenol\u0026ndash;chloroform method. Total RNA concentration was determined using a SmartSpec 3000 spectrophotometer (Bio-Rad, Hercules, CA, USA). Complementary DNA was prepared from total RNA using the Maxime RT Premix kit (information). Quantitative real-time PCR was performed using the Prism7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with the Super Script III Platinum SYBR GreenOne-Step qRT-PCR Kit (Invitrogen). The primers used for human PINK1 were sense 5\u0026rsquo;-CAA GAG AGG TCC CAA GCA AC-3\u0026rsquo; and antisense 5\u0026rsquo;-GGC AGC ACA TCA GGG TAG TC-3\u0026rsquo;; human Parkin were sense 5\u0026rsquo;-CGC CGC CAT GAT AGT CTT TGT T-3\u0026rsquo; and antisense 5\u0026rsquo;-TCC TGC AGC GTC AAG TCG TT-3\u0026rsquo;; human GRP78 were sense 5\u0026rsquo;-GAA AGG ATG GTT AAT GAT GCT GAG-3\u0026rsquo; and antisense 5\u0026rsquo;-GTC TTC AAT GTC CGC ATC CTG-3\u0026rsquo;; human eIF2a were sense 5\u0026rsquo;-CAA TGG CAA AAT CTC ACT GC-3\u0026rsquo; and antisense 5\u0026rsquo;-AAC CTC ATC TCT ATT AAA AAC ACC AAA-3\u0026rsquo;; human ATF4 were sense 5\u0026rsquo;-GAG CTT CCT GAA CAG CGA AGT G-3\u0026rsquo; and antisense 5\u0026rsquo;- TGG CCA CCT CCA GAT AGT CAT C-3\u0026rsquo;; human ATF6 were sense 5\u0026rsquo;-TTG ACA TTT TTG GTC TTG TGG-3\u0026rsquo; and antisense 5\u0026rsquo;-GCA GAA GGG GAG ACA CAT TT-3\u0026rsquo;; human XBP1 were sense 5\u0026rsquo;-CAA CCA GGA GTT AAG AAC ACG-3\u0026rsquo; and antisense 5\u0026rsquo;-AGG CAA CAG TGT CAG AGT CC-3\u0026rsquo;; human XBP1 spliced were 5\u0026rsquo;-CTG AGT CCG AAT CAG GTG CAG-3\u0026rsquo; and antisense 5\u0026rsquo;-GTC CAT GGG AAG ATG TTC TGG-3\u0026rsquo;; human CHOP were sense 5\u0026rsquo;-TAT CTC ATC CCC AGG AAA CG-3\u0026rsquo; and antisense 5\u0026rsquo;-GGG CAC TGA CCA CTC TGT TT-3\u0026rsquo;; human ClpP were sense-5\u0026rsquo;- GCC AAG CAC ACC AAA CAG A -3\u0026rsquo; and antisense-5\u0026rsquo;- GGA CCA GAA CCT TGT CTA AG -3\u0026rsquo;; human HSPD1 were sense 5\u0026rsquo;-GAT ATG GCT ATT GCT ACT GGT GGT GC-3\u0026rsquo; and antisense 5\u0026rsquo;-CCT AAG TCA TGA GCT TGA ACA TCT TC-3\u0026rsquo;; human Htra2 were sense 5\u0026rsquo;-CTC CCC GGA GTC AGT ACA ACT-3\u0026rsquo; and antisense 5\u0026rsquo;-AGG ATC TCG ATA TAG ACC ACG G-3\u0026rsquo;; human LonP1 were sense 5\u0026rsquo;-AGC CTT ATG TCG GCG TCT TTC-3\u0026rsquo; and antisense 5\u0026rsquo;-CGT CCC CGT GTG GTA GAT TTC-3\u0026rsquo;. The primers for human glyceraldehyde 3-phosphate dehydrogenase, used as the internal control, were as follows: sense 5\u0026rsquo;-ATGACATCAAGAAGGTGGTG-3\u0026rsquo; and antisense 5\u0026rsquo;-CATACCAGGAAAATGAGCTTG-3\u0026rsquo;. Dissociation curves were monitored to check the aberrant formation of primer-dimers. The fold change in the interest gene expression was calculated by using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 miRNA isolation and qPCR\u003c/h2\u003e\n \u003cp\u003eTotal RNA was isolated using TRIzol Reagent (Invitrogen) based on the acid guanidinium thiocyanate\u0026ndash;phenol\u0026ndash;chloroform method. Total RNA concentration was determined using a SmartSpec 3000 spectrophotometer (Bio-Rad, Hercules, CA, USA). miRNA isolation and cDNA synthesis were synthesized from total RNA used to miScript II RT Kit (Qiagen, cat.218161). Quantitative real-time PCR was performed using the Prism7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with miScript SYBR Green PCR kit (Qiagen, cat.218073). The primers used for miR204-5p were sense-5\u0026rsquo;-CGC TTC CCT TTG TCA TCC TA-3\u0026rsquo; and antisense-universal primer at miScript SYBR Green PCR kit (Qiagen, cat.218073). The primers for U6 snRNA (RNU6), used as the internal control, were as follows: sense-5\u0026rsquo;-GCA AAT TCG TGA AGC GTT CC-3\u0026rsquo; and antisense-universal primer at miScript SYBR Green PCR kit (Qiagen, cat.218073). The fold change in the target gene expression was calculated by using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Histological analysis\u003c/h2\u003e\n \u003cp\u003eAfter washing with phosphate-buffered saline, tumor tissues were fixed with 4% (w/v) paraformaldehyde and then embedded in paraffin. Paraffin sections were deparaffinized and rehydrated according to standard protocols and stained with hematoxylin-eosin (H\u0026amp;E). For immunohistochemistry staining, tumor tissues were stained with primary antibodies anti-MMP9 (diluted in 1:100; Cat.MA5-15886, ThermoFisher), anti-CD31 (diluted in 1:100; Cat.550274, BD science) and anti-PCNA (diluted in 1:100; PC 10; Sigma-Aldrich) overnight at 4\u0026deg;C. HRP-conjugated anti-rabbit and anti-mouse IgG was treated for 60 min at room temperature. Color was developed for 30 sec by incubation with 3,3\u0026rsquo;-diaminobenzidine (DAB). Sections were counterstained with hematoxylin and examined using microscope (Motic, Richmond, BC, Canada) at 100X magnification.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 CCK-8 cell proliferation assay\u003c/h2\u003e\n \u003cp\u003eCells were transfected with siCON or siIDH2 (50 \u0026micro;m) for 48 h. Cell proliferation was then measured using CCK-8 kit (Dojindo, Japan) according to the manufacturer\u0026rsquo;s instructions. Briefly, cells were washed with PBS and resuspended in growth media including CCK-8 reagent added at 1/50 the media volume. These cells were then incubated at 37\u0026deg;C for 1 h in the dark. Cell proliferation was measured using an absorbance detector, with measurements performed at 450 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Cell scratch assay\u003c/h2\u003e\n \u003cp\u003eA wound healing assay was used to assess cell migration. Cells were transfected as described previously with siCON or siIDH2 (50 \u0026micro;m) in 6 well tissue culture plates for 24 h, after which a sterile 200 \u0026micro;L pipette tip was used to detach the cells from the monolayer across the center of the well. Floating cells were flushed out by gently rinsing twice with PBS and replaced with serum-free medium (to rule out cell proliferation as the cause of wound closure) followed by incubation for another 24 h. The total incubation time post transfection was therefore 48 h. Cell movement was monitored using microscopy. Photographs were taken immediately and at 24 h after scratching. The relative wound area was quantitatively evaluated using ImageJ software (NIH, Bethesda, MD, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.10 Transwell assay\u003c/h2\u003e\n \u003cp\u003eTranswell assay was employed to detect cell migration. Cells were transfected with siCON or siIDH2 (50 \u0026micro;m) in 6-well tissue culture plates for 24 h, followed by transfer of 5 X 10\u003csup\u003e5\u003c/sup\u003e/ml cells in the upper transwell chamber (24-well plate; Corning, New York, USA) and culture with FBS free medium. Complete growth medium with 10% FBS was added to the lower chamber and incubated for another 24 h. Then, cells on the upper side (nonmigrating cells) were removed and migrated cells on the lower face were washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI and counted on 5 random high-power fields (200x magnification) under a microscope and averaged.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.11 Measurement of \u0026alpha;-ketoglutarate\u003c/h2\u003e\n \u003cp\u003eCells were transfected siCON or siIDH2 (50 \u0026micro;m) for 48 h. After 2 days transfection, cells were lysed in assay buffer. The \u0026alpha;-KG concentration was determined using an Alpha-Ketoglutarate Colorimetric/Fluorometric Assay Kit (Cat. #K677-100; Biovision, Milpitas, CA, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.12 Measurement of glutamine\u003c/h2\u003e\n \u003cp\u003eCells were transfected siCON or siIDH2 (50 \u0026micro;m) for 48 h. After 2 days transfection, cells were lysed in assay buffer. The glutamine concentration was determined using a Glutamine Colorimetric Assay Kit (Cat. #K556-100; Biovision, Milpitas, CA, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e2.13 TUNEL (detecting DNA fragmentation) assay and Flow cytometer\u003c/h2\u003e\n \u003cp\u003eTUNEL assay is used to detect DNA fragmentation, such as apoptosis. Cells were transfected siCON or siIDH2 (50 \u0026micro;m) for 48 h. After 2 days transfection, cells were washed twice with PBS and detached from plate using trypsin/EDTA and collected 15 ml tube. These cells were fixed in 100% Ethanol for overnight at 4\u0026deg;C. TUNEL was measured using TUNEL Assay Kit - FITC (Abcam, cat.ab66108) according to the manufacturer\u0026rsquo;s instructions. Stained cells were analyzed by flow cytometry for FITC using a NovoCyte flow cytometer, as indicated by the manufacturer (ACEA Biosciences, San Diego, CA, USA). Flow cytometry data were analyzed using NovoExpress software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e2.14 Oncomine data collection\u003c/h2\u003e\n \u003cp\u003eMicroarray datasets were downloaded from public websites or provided by the authors upon request. The web addresses to download particular datasets are listed at ONCOMINE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.oncomine.org\u003c/span\u003e\u003c/span\u003e). All data that were available from the authors were included in processing and analysis. Raw data was uploaded DYRAD. DYRAD address is written in Supporting Information\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e2.15 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analysis was performed using Prism 8 software (GraphPad Software Inc., La Jolla, CA, USA). Data are means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Differences between two groups were evaluated using t-tests. For multiple comparisons, one-way analysis of variance (ANOVA) was performed followed by a Tukey\u0026rsquo;s multiple comparison test. p-Values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered indicative of statistical significance. Data are representative of at least three independent experiments.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 IDH2 is overexpressed in cervical cancer cell lines and tumors\u003c/h2\u003e \u003cp\u003eDeletion of IDH2 in various cell types has been shown to reduce oxidative capacity and triggered mitochondrial dysfunction\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Analysis of the Oncomine Database revealed a significant upregulation of IDH2 mRNA expression in cervical cancer patients compared to normal tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Similarly, IDH2 expression was markedly elevated in cervical cancer cell lines including C33A [HPV negative], SiHa [HPV 16\u003csup\u003e+\u003c/sup\u003e], and HeLa [HPV 18\u003csup\u003e+\u003c/sup\u003e] cells, relative to normal human cervical epithelial cells (HCECs). Notably, HeLa cells exhibited the highest levels of IDH2 mRNA and protein expression among the cervical cell lines examined (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). These findings provided a strong rationale for using HeLa cells as a model system to elucidate the functional consequences of IDH2 deficiency and its role in apoptosis induction via regulation of the miR204/mitophagy pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Downregulation of IDH2 inhibits proliferation and migration by inducing apoptosis in HeLa cells\u003c/h2\u003e \u003cp\u003eTo deliver IDH2 siRNA into HeLa and cancer cells, we used poly-PLGA copolymers, a biodegradable and biocompatible material. The synthesis and characterization of NP are detailed in Supplementary Fig.\u0026nbsp;1A-C. Treatment of siIDH2-encapsulated NPs reduced the expression of IDH2 in HeLa cells within 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Hereafter, siCON-NPs and siIDH2-NPs are referred to as siCON and siIDH2, respectively. Silencing of IDH2 significantly reduced the cell doubling time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and decreased cell proliferation, as confirmed by the CCK-assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In transwell migration assays, siIDH2-treated cells showed a significant reduction in migration, as evident from the decreased number of cells in the lower chamber. Migrated cells were stained with DAPI and quantified under a microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Similarly, a cell scratch-wound healing assay showed a marked reduction in wound closure rates in siIDH2-treated cells compared to siCON-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Collectively, these results indicate that IDH2 knockdown impairs HeLa cell proliferation and migration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the mechanism of the results, we performed immunoblotting for apoptosis-related proteins. IDH2 knockdown led to a significant increase in cleaved caspase-9, which subsequently activated caspase-3 cleavage. The activated caspase-3 induced PARP cleavage, thereby triggering the apoptotic cascade (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). DNA fragmentation, a hallmark of apoptosis, was confirmed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), which demonstrated an increased presence of fragmented DNA in siIDH2-treated cells. Collectively, these findings indicate that IDH2 deficiency promotes apoptosis, contributing to the inhibition of proliferation and migration in HeLa cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 IDH2 knockdown suppresses tumor growth in a HeLa cells xenograft\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of IDH2 knockdown on tumor growth in vivo, HeLa cell xenografts were established in nude mice. After 24 and 28 days of tumor injection, the mice were divided into groups with the same tumor volume (mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e) and the tumors were directly injected with siIDH2 PLGA-NPs or siCON PLGA-NPs. NP injection was well tolerated, with no obvious side-effects such as weight loss or behavioral changes. siIDH2 NPs selectively reduced IDH2 expression in cancer tissues without affecting adjacent or distant tissues, including heart, lung, spleen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOver a 33-day observation period, the volume of tumor (width \u0026times; width \u0026times; length \u0026cedil; 2) in mice treated with siCON PLGA-NPs increased from 600 to 1400 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In contrast, siIDH2 PLGA-NPs treated tumors significantly slower growth, with a mean tumor volume of 800 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). H\u0026amp;E staining of paraffin-embedded tumor sections revealed distinct morphological differences. Tumors from siCON-treated mice displayed a compact epithelial cell structure, while those from siIDH2-treated mice exhibited a looser epithelial cell organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo further investigate the effects of IDH2 knockdown on tumor proliferation, neovascularization, and metastasis, immunohistochemical staining was performed. Tumor sections stained with anti-PCNA antibody, a marker of cell proliferation, showed significantly reduced proliferation in siIDH2-treated mice compared to siCON (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Additionally, siIDH2-treated tumor sections displayed markedly lower neovascularization, as evidenced by reduced CD31 staining, and a diminished metastatic index, as indicated by decreased MMP-9 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These findings demonstrate that IDH2 deficiency effectively suppresses tumor proliferation, neovascularization, and metastatic potential in vivo,\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 IDH2 deficiency disrupts mitochondrial homeostasis via UPR activation and mitophagy suppression\u003c/h2\u003e \u003cp\u003eMitochondrial dysfunction, a critical trigger for apoptosis, often activates the mitochondrial UPRmt and mitophagy\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Reactive oxygen species (ROS) generated by IDH2 deficiency disrupt mitochondrial homeostasis by inhibiting TCA cycle progression\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Damaged mitochondria activate the UPRmt, upregulating stress-response proteins such as CHOP, Hsp60, Htra2, ClpP, and LONP to restore function (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). CHOP, a key transcription factor, binds to the promoter regions of repair genes under mitochondrial stress, promoting mitochondrial recovery\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, IDH2 deficiency impairs mitophagy by reducing PINK1 protein accumulation on the outer mitochondrial membrane, a critical step for mitophagy activation, despite unchanged PINK1 and Parkin mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This disruption prevents Parkin translocation to the mitochondria, further compromising mitophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results suggest that IDH2 deficiency-induced mitochondrial dysfunction activates stress responses, promoting apoptosis in HeLa cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5 IDH2 deficiency induces miR204 expression by reducing the mitochondrial glutamine level\u003c/h2\u003e \u003cp\u003eCancer stem cells in colorectal, brain, breast, and cervical cancers rely on both mitochondrial oxidative metabolism and glycolysis for energy production\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In IDH2-deleted cells, the mitochondrial α-KG concentration was markedly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Glutamine, a key regulator of cancer cell proliferation, migration, and antioxidant defense, is converted into glutamate and transported into mitochondria to replenish α-KG. IDH2 deficiency significantly reduced mitochondrial glutamine levels compared to cytosolic levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eGlutamine is metabolized to α-KG via glutaminolysis, a process involving glutaminase (GLS) and glutamate dehydrogenase 1 (GDH1)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Solute-linked carrier family 1 member A5 (SLCA15) is a transporter of glutamine in rapidly growing epithelial and tumor cells\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In IDH2-deficient cells, GLS and GDH1 mRNA levels were upregulated, while solute carrier family 1 member A5 (SLCA15), a glutamine transporter, remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). This result suggests intracellular glutamine is consumed to compensate for the loss of α-KG, depleting mitochondrial glutamine stores (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eGlutamine also regulates STAT3 phosphorylation, which suppresses miR204 by binding to the TRPM3 promoter region\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. STAT3 phosphorylation was significantly decreased in IDH2 deficient cells miR204 expression was increased in IDH2-deficient HeLa cells without glutamine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Reduced glutamine levels in IDH2-deficient cells led to decreased STAT3 phosphorylation, thereby de-repressing miR204 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, J). Elevated miR204 expression was observed in IDH2-deficient cells under glutamine-deprived conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK). Furthermore, overexpression of dominant-negative STAT3 (DN-STAT3) inhibited miR204 repression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL, M)\u003c/p\u003e \u003cp\u003eThese findings demonstrate that IDH2 deficiency reduces mitochondrial glutamine levels, which decreases STAT3 phosphorylation and upregulates miR204 expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6 miR204 directly inhibits PINK1 expression\u003c/h2\u003e \u003cp\u003emiRNAs regulate target gene expression by binding to target mRNA with the seed region (positions 2\u0026ndash;7 from the miRNA 5\u0026acute;-end) being critical for this interaction. To confirm that miRNA204 directly targets PINK1, we analyzed the PINK1 mRNA coding sequence and identified a conserved miR204-binding site within the coding region (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The binding site includes a complementary sequence to the miR204 seed region, suggesting a post-transcriptional regulatory mechanism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm the interaction, we co-transfected miR204 mimics and plasmids expressing wild-type (WT) or mutant PINK1 fused to a Myc-tag. The mutant PINK1 was designed to disrupt the miR204 seed-binding site. Overexpression of miR204 significantly inhibited Myc-PINK1 expression in cells transfected with the WT construct, while no inhibition was observed in cells transfected with the mutant construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). These results demonstrate that miR204 directly binds to the PINK1 coding region, suppressing the PINK1 expression at the post-transcriptional level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Inhibition of miR204 restores mitophagy and reduces apoptosis\u003c/h2\u003e \u003cp\u003ePINK1 expression rescued IDH2 siRNA and miR204 inhibitor (miR204-I) co-transfected HeLa cells but did not affect Parkin expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Treatment with an miR204 inhibitor (miR204-I) restored PINK1 levels and facilitated Parkin translocation from the cytosol to mitochondria, effectively rescuing mitophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). Immunofluorescence analysis confirmed enhanced Parkin localization to damaged mitochondria in miR204-I-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Levels of cleaved caspase-8, -3, and PARP, key markers of apoptosis, were significantly decreased following miR204 inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Similarly, TUNEL assays showed reduced DNA fragmentation in cells treated with miR204-I, indicating suppression of apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eThese results confirm that miR204 downregulation restores mitochondrial quality control mechanisms and mitigates apoptosis by enhancing PINK1 expression and Parkin recruitment to mitochondria.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.8 miR204 suppressed cancer growth in HeLa cell xenografts\u003c/h2\u003e \u003cp\u003eTo evaluate the role of miR204 in tumor growth, siIDH2 nanoparticles (NPs) were injected directly into tumors on days 24 and 28, with tail-vein injections of either an miR204 mimic or inhibitor (miR204-I) administered on day 24 using Invivofectamine\u0026trade; 3.0. After 33 days, tumor volumes in the control and siIDH2\u0026thinsp;+\u0026thinsp;miR204-I groups increased from 1200 mm\u0026sup3; to 1500 mm\u0026sup3;. In contrast, the tumor volumes in the miR204 mimic group exhibited significantly slower growth, with an average tumor volume of 600 mm\u0026sup3; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Representative images of tumor size confirmed the inhibitory effect of the miR204 mimic compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). miR204-I treatment suppressed miR204 expression in siIDH2-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The siIDH2\u0026thinsp;+\u0026thinsp;miR204-I group showed tumor growth similar to that of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). H\u0026amp;E staining of tumor paraffin sections revealed that tumors from the miR204 mimic group exhibited loose epithelial organization, whereas tumors from the siIDH2\u0026thinsp;+\u0026thinsp;miR204-I and control groups displayed compact epithelial structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Immunohistochemical staining showed significantly reduced levels of PCNA, CD31, and MMP9 in the miR204 mimic group compared to the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The PINK1 expression significantly decreased in the miR204 mimic group, while PINK1 levels was not changed in siIDH2\u0026thinsp;+\u0026thinsp;miR204-I group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These findings suggest that the anticancer effect of IDH2 deficiency is mediated by miR204 upregulation, which suppresses tumor proliferation, neovascularization, and metastasis by targeting PINK1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eCervical cancer remains a leading cause of cancer-related mortality in women, with poor survival rates and frequent relapses, particularly in younger patients\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. It is most commonly associated with high-risk oncogenic HPV types, typically HPV16 and HPV18\u003csup\u003e29\u003c/sup\u003e. In addition, mitochondria participate in carcinogenesis through energy production, macromolecular synthesis, and the regulation of cell survival. Malignant tumors selectively retain the mitochondrial genome and OXPHOS complex function, whereas tumors with pathogenic mitochondrial DNA mutations are benign, indicating the importance of mitochondria for cancer progression. Moreover, mutant TCA cycle enzymes, such as IDH, produce oncometabolites that promote tumorigenesis\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIDH2 mutation is found in several cancer types and has been associated with a poor prognosis\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, the effect of WT IDH2 or IDH2 overexpression in cervical cancer is controversial. Previous studies have shown that IDH2 deficiency induces mitochondrial dysfunction, oxidative stress, inflammation, and endothelium-dependent vasomotor function in endothelial cells\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. To achieve efficient and targeted IDH2 knockdown, we used PLGA-NPs for the delivery of siIDH2. PLGA is a biocompatible and biodegradable polymer approved by the FDA for clinical use, making it a vehicle for siRNA delivery\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In our study, siIDH2-loaded nanoparticles demonstrated IDH2 silencing in HeLa cells and tumor tissues without affecting non-cancerous tissues such as the heart, lung, or spleen. The targeted delivery minimized off-target effects and systemic toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In this study, our findings demonstrate that IDH2 deficiency disrupts mitochondrial homeostasis, resulting in impaired cellular function and increased apoptosis. Specifically, IDH2 knockdown in HPV18\u0026thinsp;+\u0026thinsp;HeLa cells suppressed cancer cell proliferation and migration both in vitro and in vivo. These results suggest that the potential therapeutic implications of targeting IDH2 in cervical cancer.\u003c/p\u003e \u003cp\u003eMitochondrial dysfunction plays a critical role in cancer cell survival and apoptosis. Cancer cells are typically resistant to mitochondrial stress, as mitophagy eliminates dysfunctional organelles and prevents cellular damage \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, apoptosis can be triggered if there is excessive mitochondrial damage\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Our study has shown that IDH2 deficiency induces upregulation of UPRmt markers such as GRP78, ATF4, CHOP, HSPD1, and ClpP and ER stress These markers indicate attempts to restore mitochondrial function under stressful conditions. Despite the activation of UPRmt, IDH2 deficiency inhibits PINK1 accumulation and blocks parkin translocation to mitochondria, thus damaging mitochondrial dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings suggest that IDH2 deficiency disrupts both mitochondrial repair mechanisms and quality control pathways, leading to apoptosis.\u003c/p\u003e \u003cp\u003eIn addition, IDH2 deficiency significantly altered mitochondrial metabolism, reducing α-KG and glutamine levels. Glutamine is a critical substrate for the TCA cycle and supports cancer cell survival by replenishing mitochondrial intermediates through glutaminolysis. Our results demonstrated that IDH2 deficiency depleted mitochondrial glutamine levels while upregulating glutaminase (GLS) and glutamate dehydrogenase 1 (GDH1), key enzymes involved in glutaminolysis. These changes suggest that intracellular glutamine is rapidly consumed to compensate for the loss of α-KG, leading to mitochondrial glutamine depletion and metabolic dysregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn particular, glutamine levels directly regulate STAT3 phosphorylation, which is a key transcription factor involved in cancer progression\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Reduced mitochondrial glutamine levels in IDH2-deficient cells decreased STAT3 phosphorylation, leading to the upregulation of miR204. Elevated miR204 levels were observed under glutamine-deprived conditions, whereas glutamine supplementation had minimal impact on its expression. Furthermore, dominant-negative STAT3 (DN-STAT3) overexpression inhibited STAT3 phosphorylation and its DNA-binding activity, confirming that STAT3 inactivation mediates miR204 upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These findings link mitochondrial glutamine metabolism to transcriptional regulation through the STAT3/miR204 axis.\u003c/p\u003e \u003cp\u003eIDH2 deficiency induces miR204 expression in hepatocytes\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. miR204 has been reported to regulate various physiological and pathological processes, including cardiovascular diseases, tumor suppression, and β-cell proliferation3\u003csup\u003e38\u0026ndash;43\u003c/sup\u003e. Here, we demonstrate that miR204 directly suppresses PINK1 expression by binding to its coding region sequence (CDS). This inhibition of PINK1 translation impairs mitophagy and further promotes apoptosis by preventing Parkin translocation to mitochondria. In particular, inhibition of miR204 restored PINK1 expression, rescued Parkin-mediated mitophagy, and mitigated apoptosis in IDH2-deficient cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results suggest that the critical role of miR204 in mediating the effects of IDH2 deficiency on mitochondrial quality control and apoptosis.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eCollectively, our findings reveal that IDH2 deficiency suppresses cervical cancer progression by disrupting mitochondrial homeostasis through the inhibition of mitophagy. The subsequent upregulation of miR204 contributes to mitophagy suppression and apoptosis by targeting PINK1. These results suggest that the IDH2/miR204/PINK1 axis as a potential therapeutic target in cervical cancer and other malignancies characterized by mitochondrial dysfunction. Future studies should explore the translational potential of targeting this pathway in combination with existing therapies to overcome tumor resistance and improve patient outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003eThe English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/DwAPeK\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003eS.K and C.S.K initiated and designed this study. S.K performed and analysed the majority of experiments. S.K., Y.R.K., and C.S.K wrote the manuscript. S.P, Y.R.K., I.L and H.N performed and analysed individual experiments. I.L, S.P and S.C performed data curation. C.S.K, D.W.K, B.H.J and K.I supervised the study. S.K and Y.R.K contributed to equal this work. C.S.K and K.I were corresponding author in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003eThis work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A6A1029617) and by the research fund of Chungnam National University and by the research fund of Chungnam National University Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003eThe datasets supporting the conclusions of this article are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003eAll experiments were approved and conducted at Chungnam National University (CNUH-021-A0029) following the guidelines of the Institutional Animal Care and Use Committee. Informed consent was obtained from each participate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOlusola, P., Banerjee, H. N., Philley, J. V. \u0026amp; Dasgupta, S. Human Papilloma Virus-Associated Cervical Cancer and Health Disparities. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e (2019). https://doi.org:10.3390/cells8060622\u003c/li\u003e\n\u003cli\u003eBurmeister, C. 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R.\u003cem\u003e et al.\u003c/em\u003e Genetic deletion of miR-204 improves glycemic control despite obesity in db/db mice. \u003cem\u003eBiochemical and biophysical research communications\u003c/em\u003e \u003cstrong\u003e532\u003c/strong\u003e, 167-172 (2020). https://doi.org:10.1016/j.bbrc.2020.08.077\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PLGA nanoparticles, cervical cancer, IDH2, miR204, PINK1, mitophagy","lastPublishedDoi":"10.21203/rs.3.rs-6222927/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6222927/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eMitochondrial dysfunction plays an important role in modulation of cancer and considered as a therapeutic target for anticancer treatment. Mutation of mitochondrial protein isocitrate dehydrogenase 2 (IDH2) has been identified in various cancers. However, the role of wild-type IDH2 in cancer proliferation and migration, particularly in cervical cancer, remains poorly understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn the present study, we examined the antitumor mechanism of IDH2 deficiency in HeLa cervical cancer cells using both \u003cem\u003ein vitro\u003c/em\u003e assay and \u003cem\u003ein vivo\u003c/em\u003e xenograft mouse model. Poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles encapsulating IDH2-specific siRNA (siIDH2-NP) were used IDH2 downregulation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIDH2 siRNA. IDH2 deficiency suppressed cell proliferation, migration, as well as mitochondrial homeostasis in HeLa cells. IDH2 deficiency markedly inhibited cell proliferation and migration while disrupting mitochondrial homeostasis in HeLa cells. The mitochondrial dysfunction led to the upregulation of endoplasmic reticulum (ER) stress and mitochondrial unfolded protein response (UPRmt) markers, accompanied by a significant reduction in PINK1 accumulation and impaired Parkin translocation to mitochondria suppressing mitophagy. Additionally, STAT3 de-phosphorylation in IDH2 deficient cells increased miR204 expression, which inhibited PINK1 expression and suppressed mitophagy. Consistent findings were obtained \u003cem\u003ein vivo\u003c/em\u003e, with significantly reduced tumor volume in treatment of siIDH2-NP.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings suggest that IDH2 deficiency induces apoptotic cell death by disrupting mitochondrial homeostasis and suppressing mitophagy. This study highlights IDH2 as a potential therapeutic target and miR204 as a novel mediator in cervical cancer, providing a promising avenue for the development of anticancer strategies.\u003c/p\u003e","manuscriptTitle":"IDH2-siRNA nanoparticles induce apoptosis in HeLa cell by regulating the miR204/mitophagy pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-22 17:41:29","doi":"10.21203/rs.3.rs-6222927/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e744a1f0-21e5-463b-a995-5d7b1c5f0656","owner":[],"postedDate":"April 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-02T08:09:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-22 17:41:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6222927","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6222927","identity":"rs-6222927","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}