LINC01234 coordinates protein interactions and ceRNA networks to enhance YWHAZ-driven malignancy in triple-negative breast cancer

LINC01234 coordinates protein interactions and ceRNA networks to enhance YWHAZ-driven malignancy in triple-negative breast cancer | 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 LINC01234 coordinates protein interactions and ceRNA networks to enhance YWHAZ-driven malignancy in triple-negative breast cancer Diping Yu, Li Chen, Huimin Li, Shiyao Kang, Fei Hu, Chao Yuan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7330942/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 Triple-negative breast cancer (TNBC) exhibits poor prognosis due to the lack of effective therapeutic targets. This study investigates the molecular mechanism of long non-coding RNA LINC01234 in TNBC progression. Our preliminary work identified significant upregulation of LINC01234 in TNBC cells, and its knockdown suppressed tumor progression. Here, through RNA-pulldown coupled with mass spectrometry, we screened LINC01234-interacting proteins and confirmed its direct binding to the scaffolding protein YWHAZ (14-3-3ζ) via RNA immunoprecipitation (RIP), which promotes YWHAZ phosphorylation. Clinical analysis showed that YWHAZ was highly expressed in TNBC tissues and correlated with poor patient prognosis. Mechanistically, LINC01234 regulated YWHAZ expression via targeting miR-204-5p, thereby influencing tumor progression. Further functional validation demonstrated that either miR-204-5p overexpression or YWHAZ knockdown significantly inhibited TNBC cell proliferation/migration and promoted apoptosis. These findings suggest a dual regulatory mechanism: LINC01234 directly activates YWHAZ's oncogenic function through protein interaction, while indirectly releasing the suppression of YWHAZ expression by sponging miR-204-5p. This study reveals a "protein interaction-ceRNA crosstalk" paradigm by which LINC01234 promotes TNBC progression, providing a theoretical foundation and potential therapeutic strategy for TNBC management. Triple-negative breast cancer LINC01234 YWHAZ progression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Breast cancer represents the most frequently diagnosed malignancy among women worldwide. According to global cancer burden statistics for 2020, approximately 2.26 million new breast cancer cases were reported, surpassing lung cancer (2.20 million cases) to become the leading cause of cancer incidence globally, with an estimated 685,000 associated deaths[ 1 , 2 ]. In China, breast cancer similarly constitutes the predominant female cancer, accounting for 416,000 new cases (18.4% of the global total) and 117,000 deaths (17.1% of global breast cancer mortality) during the same period[ 3 ]. Notably, TNBC - characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expression – constitutes the most aggressive molecular subtype. Patients with TNBC derive no benefit from endocrine therapies or anti-HER2 targeted agents. This subtype frequently presents with high histological grade at diagnosis, elevated rates of postoperative recurrence and distant metastasis, and confers poor prognosis, posing severe threats to patient survival[ 4 , 5 ]. Therefore, developing effective therapeutic strategies to improve the prognosis of TNBC patients remains a critical research priority in the medical community. Advances in transcriptome sequencing technologies have revealed that approximately 70% of the human genome is transcriptionally active, with long non-coding RNAs (lncRNAs) constituting a substantial proportion of these transcripts. Defined as non-protein-coding transcripts exceeding 200 nucleotides in length, lncRNAs function as critical regulatory molecules through multifaceted mechanisms: they modulate gene expression at transcriptional and post-transcriptional levels via interactions with DNA, RNA, or proteins, operating through both cis-regulatory and trans-regulatory modes[ 6 – 9 ]. Emerging evidence implicates dysregulation of lncRNAs in the pathogenesis of diverse malignancies, where they influence oncogenic pathways including proliferation, metastasis, and therapy resistance[ 10 – 13 ]. Consequently, lncRNAs represent promising novel therapeutic targets and prognostic biomarkers for cancer diagnosis and clinical management. Accumulating evidence reveals aberrant expression of numerous lncRNAs in TNBC, with specific lncRNAs functioning as critical regulators of TNBC pathogenesis and progression. For instance, Zhang et al. identified lnc-BTG3-7:1 as a TNBC-specific transcript through RNA sequencing analysis. Their mechanistic investigation demonstrated that upregulation of lnc-BTG3-7:1 enhanced the transcriptional activity of the oncogene C21orf91, thereby activating both PI3K-AKT-GSK3β-β-catenin and MAPK signaling pathways to drive tumor development[ 14 ]. Similarly, Shaath et al. performed single-cell RNA sequencing on 1,758 cells from TNBC patients, identifying metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) as a key lncRNA associated with resistance to neoadjuvant chemotherapy[ 15 ]. Critically, high MALAT1 expression was associated with poorer clinical prognosis in TNBC patients. Although lncRNAs have been demonstrated to regulate multiple pathological processes in TNBC such as proliferation, apoptosis, invasion, and metastasis, only a small fraction of known lncRNAs have been functionally characterized in this aggressive subtype. Critical unresolved questions persist, such as: (i) identification of key lncRNA molecules regulating TNBC progression and metastasis, and (ii) screening of therapeutically targetable lncRNAs for precision intervention. Thus, in-depth research on specific LncRNAs' roles in TNBC or exploring new LncRNAs' functions in TNBC can offer a reliable theoretical basis and new perspectives for TNBC prevention and treatment. LINC01234 is a highly conserved lncRNA located at chromosomal position 12q24.13[ 16 ], whose dysregulation has been implicated in breast cancer pathogenesis[ 17 , 18 ]. Our preliminary studies demonstrated that LINC01234 was highly expressed in breast cancer cell lines, particularly with significantly elevated expression in MDA-MB-231 and MDA-MB-468 cells (TNBC cell lines). Functional validation through both in vitro and in vivo experiments confirmed that LINC01234 knockdown effectively suppressed TNBC tumor progression[ 19 ]. Nevertheless, the precise molecular mechanisms underlying LINC01234's oncogenic functions in TNBC remained unclear. To address this knowledge gap, this current study integrated RNA-pulldown combined with mass spectrometry (MS) and RIP technologies to systematically screen the interaction protein network and regulated signaling pathways of LINC01234, aiming to offer a theoretical basis and strategies for targeted therapy and prognostic evaluation of TNBC. 2. Materials and methods 2.1 Cell culture MDA-MB-231 triple-negative human breast cancer cells and the immortalized, non-tumorigenic breast epithelial line MCF-10A were obtained from the Cell Culture Center, Peking Union Medical College. MDA-MB-231 Cells were maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Cat. C11995500BT) supplemented with 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific, Cat. 10099141). MCF-10A Cells were cultured in DMEM/F-12 (Thermo Fisher Scientific, Cat. 12500-062) containing 10% (v/v) horse serum (HyClone), 1 mg/mL epidermal growth factor (Merck KGaA, Darmstadt, Germany), 1 mg/mL cholera toxin (Merck KGaA), 20 mg/mL insulin (Merck KGaA) and 1 mg/mL hydrocortisone (Merck KGaA). All cultures were incubated at 37℃ in a humidified atmosphere of 95% air / 5% CO₂. Routine mycoplasma testing confirmed the absence of contamination. 2.2 Data acquisition and bioinformatic analysis In this study, we downloaded the gene expression dataset of breast cancer cases with YWHAZ expression from The Cancer Genome Atlas (TCGA) database ( https://portal.gdc.cancer.gov/ ). The subcellular localization of LINC01234 in cells was predicted utilizing the lncLocator website ( http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/ ). Both the nucleic acid sequence of LINC01234 and its known miRNA binding targets were verified via the AnnoLnc platform. Kaplan-Meier survival analysis was applied to analyze the relationship between YWHAZ, hsa-miR-205-5p and the overall survival rate of patients with triple-negative breast cancer (TNBC). Breast Cancer Gene-Expression Miner v5.2 (bc-GenExMiner v5.2) was employed to assess the expression levels of YWHAZ in various subtypes of BC. Additionally, UALCAN ( https://ualcan.path.uab.edu/ ) was employed to assess the expression levels of hsa-miR-204-5p among various breast cancer subtypes. 2.3 Cell transfection For transient transfection assays, TNBC cells were transfected with LINC01234 siRNA, YWHAZ siRNA, miR-204-5p mimic, mimic negative control (mimic-NC), pcDNA3.1-LINC01234 plasmids, and plasmids with different truncated variants of LINC01234 using TransIntro EL transfection reagent (TransGen Biotech, Beijing, China) according to the manufacturer’s protocols. The LINC01234 siRNA, YWHAZ siRNA, miR-204-5p mimic, and corresponding negative controls were procured from RiboBio (Guangzhou, China). The full-length LINC01234 sequence and its various truncated forms were synthesized by General Biosystems (Anhui, China) and subsequently cloned into the pcDNA3.1 vector. A pGreenPuro vector encoding a short hairpin RNA (shRNA) against YWHAZ was constructed by General Biosystems (Anhui, China). For lentivirus production, the lentiviral vector was cotransfected with the packaging vectors psPAX2 and pMD2.G into 293T cells. Viral supernatant was harvested 48 h later, filtered (0.45 µm PVDF) and supplemented with 8 µg mL⁻¹ polybrene (Merck). To establish stable cell lines, MDA-MB-231 cells were infected with lentiviral particles according to the manufactures’s protocol. Twelve hours post-infection, the culture medium containing lentiviral particles was discarded, and 4 mL of fresh medium supplemented with puromycin was added to maintain the culture. After 2 weeks, knockdown efficiency was confirmed by qRT-PCR. All oligonucleotide sequences were provided in Supplementary Table 1. 2.4 RNA extraction and quantitative RT‒PCR Total RNA from cultured cells and tissues was extracted with TRI Reagent® (Merck KGaA, Cat. T9424) following the manufacturer’s instructions. For the quantification of LINC01234 and YWHAZ, mRNA was reverse-transcribed into complementary DNA (cDNA) using the GoScript™ Reverse Transcription Mix with Random Primers (A2801, Promega, Wisconsin, USA). For miR-204 detection, miRNA was reverse-transcribed into cDNA using the GoScript™ Reverse Transcription System (A5001, Promega, Wisconsin, USA). qRT-PCR reactions were performed in triplicate with SYBR Green Master mix (Roche, Basel, Switzerland) on an ABI 7300 system (Applied Biosystems). Dissociation-curve analysis confirmed single-product amplification. Relative expression was calculated using the 2- ΔΔCt method with GAPDH (for LINC01234 and YWHAZ) or U6 (for miR-204) as endogenous controls. All primer sequences were listed in Supplementary Table 1. 2.5 Cell proliferation assays MDA-MB-231 cells were seeded in 24-well plates and transfected 24 h later with 100 nM YWHAZ-targeting siRNA (si-YWHAZ) or non-targeting control siRNA (si-NC) using TransIntro EL. After the indicated incubation periods, 50 µL CCK-8 reagent (RiboBio, Guangzhou, China) was added to each well. The plates were then incubated at 37°C in a 5% CO₂ atmosphere to allow the reaction to proceed. Absorbance was recorded at 450 nm with background subtraction at 630 nm using a microplate reader (BioTek). 2.6 Apoptosis analysis Transfected cells were collected by gentle trypsinization, washed twice with ice-cold PBS, and adjusted to 1 × 10⁶ cells mL⁻¹ in 1 × Annexin V-binding buffer. 100 µL of the suspension (≈ 1 × 10⁵ cells) were transferred to a new tube and stained with 2 µL Annexin V-FITC (Roche, Cat. 11988549001) for 15 min at room temperature in the dark. Propidium iodide (4 µL, 50 µg mL⁻¹) was then added, followed by an additional 5-min incubation under identical conditions. Samples were diluted to 500 µL with binding buffer and immediately analysed on an Accuri C6 flow cytometer (Becton Dickinson, Franklin Lakes, USA)). At least 10 000 events per sample were acquired, and data were processed using FlowJo v10.8 to quantify early (Annexin V⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptotic fractions. 2.7 Western blot analysis Cells were harvested on ice in RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with 1× protease inhibitor cocktail (Roche). Lysates were clarified by centrifugation (12 000 × g, 15 min, 4°C) and total protein determined by the BCA assay (Beyotime). Equal amounts (20 µg per lane) were resolved on SDS-PAGE gels and electro-transferred to 0.22 µm PVDF membranes (Merck KGaA). Membranes were blocked for 1 h at room temperature with 5% (w/v) non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), then incubated overnight at 4°C with primary antibodies diluted in 5% BSA-TBST. After three TBST washes, membranes were probed with HRP-conjugated secondary antibodies (1:10 000, Cell Signaling Technology) for 1 h at room temperature. Immunoreactive bands were detected with Immobilon Western Chemiluminescent HRP Substrate (Merck KGaA) and visualized on a Tanon 5200 imaging system (Tanon 5200, Shanghai, China). The primary antibodies used in this study as follows: p-YWHAZ (abclonal,1:1000), YWHAZ (WanLeiBio, 1:1000), p-PI3K (CST, 1:1000), PI3K (CST, 1:1000), p-AKT (CST, 1:1000), AKT (CST, 1:1000), TSC1 (CST, 1:1000), ZEB1 (ABclonal, 1:1000), E-cadherin (ABclonal, 1:1000), β-catenin (ABclonal, 1:1000), MMP2 (ABclonal, 1:1000), MMP11 (ABclonal, 1:1000), TAGLN2 (ABclonal, 1:1000), CFL1 (ABclonal, 1:1000), snail (CST, 1:1000), Ki67 (CST, 1:1000), vinculin (Bioss, 1:5000), β-actin (ABclonal, 1:5000). 2.8 RNA immunoprecipitation (RIP) RIP assays were performed using a Magna RIP Kit (17‒700, Millipore Corporation, USA) according to the manufacturer’s instructions. Briefly, MDA-MB-231 cells were lysed in ice-cold RIP lysis buffer containing RNase and protease inhibitors (Roche). Protein A/G magnetic beads were pre-conjugated with 5 µg of either anti-YWHAZ rabbit polyclonal antibody (WanLeiBio, WL0361) or control non-immune rabbit IgG (Cell Signaling Technology, 2729) for 30 min at room temperature. After three washes with RIP wash buffer, antibody-bound beads were incubated with 100 µL of clarified lysate overnight at 4°C with gentle rotation. Immune complexes were sequentially washed six times with stringent RIP wash buffer, then resuspended in proteinase K digestion buffer to release RNA. RNA was recovered by Trizol-chloroform RNA extraction method and analyzed by RT-qPCR. Results were expressed as fold-enrichment over input after normalization to the IgG control. 2.9 Transwell assays Transwell analysis was conducted using 24-well Transwell® inserts with 8 µm-pore polycarbonate membranes (Corning, Cat. 3422). Transfected cells (8 × 10⁴ per insert) were resuspended in serum-free medium and seeded into the upper compartment; the lower compartment received 500 µL complete medium. After 24 h incubation (37°C, 5% CO₂), non-migrated cells on the upper chamber were gently removed with a cotton swab. Migrated cells on the lower surface of the membrane were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet. Five random fields per membrane were imaged at 200× magnification using an inverted microscope. Migrated cells were counted by ImageJ and expressed as mean ± SD per field. 2.10 Animal experiments BALB/c nude mice aged 4–6 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and randomly assigned to two groups. Each mouse received a subcutaneous injection of 1×10⁶ MDA-MB-231 cells transfected with lentivirus (sh-NC or sh-YWHAZ). Tumor volume was measured every two days, and mouse body weight was recorded every four days. Tumor volume was calculated using the formula: V = 0.5×length×width². After one month, the mice were euthanized via cervical dislocation, and the tumors were harvested for subsequent analyses. All protocols involving the use of animals were approved by the Animal Ethics Committee of Kunming University of Science and Technology (PZWH(dian)K2024-0001). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines. 2.11 RNA pull-down assays and Mass spectrometry (MS) MDA-MB-231 cells were lysed in RIP buffer, and the supernatant was incubated with biotin-labeled probes targeting LINC01234 (synthesized by Guangzhou Laboratory Biotechnology Co., Ltd) at 4°C overnight. Streptavidin-coated magnetic beads (Thermo Fisher) were subsequently added, and the mixture was incubated at room temperature for 1 hour. Following thorough washing, the bound proteins were eluted using washing buffer and collected from the supernatant. Eluates were separated on 4–12% Bis-Tris SDS-PAGE gels. After brief Coomassie staining, entire lanes were excised, reduced, alkylated, and in-gel digested with trypsin (Promega). Peptides were extracted, and analysed on a Q Exactive™ HF-X mass spectrometer (Thermo Fisher). RAW data were converted to MGF files using Proteome Discoverer 1.4 (v1.4.0.288). These files were then subjected to database searching against the UniProt human protein database with ProteinPilot 4.5 (v1656, AB Sciex). Negative control probes were used to exclude nonspecific interactions. 2.12 Statistical analyses All the data results were shown as mean ± SD through the software GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA). Differences among groups were analyzed with two-way analysis of variance (ANOVA), while the differences among one group were analyzed with unpaired t-test or one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 were considered as significant. All experiments were performed at least thrice with triplicate samples. 3. Results 3.1 LINC01234 binds to YWHAZ and promotes its phosphorylation To investigate the molecular mechanisms of LINC01234, we performed RNA-pull down and mass spectrometry experiments, which preliminarily identified 117 proteins directly interacting with LINC01234 (Fig. 1 A and Supplementary Table 2). By intersecting these results with the predictions from the AnnoLnc database, 8 target proteins were pinpointed. Through literature review, we focused on the YWHAZ gene (Fig. 1 B). To determine whether there is an association between LINC01234 and YWHAZ, we first knocked down or overexpressed LINC01234, and the results showed that the expression levels of YWHAZ and LINC01234 were positively correlated (Fig. 1 C and 1 D). Further RIP experiments confirmed that YWHAZ could directly bind to LINC01234 (Fig. 1 E). LINC01234 has four main structural branches, and to identify the specific binding site with YWHAZ, we truncated LINC01234 (Fig. 1 F). The results revealed that the fragment 1 of LINC01234 bound to YWHAZ (Fig. 1 G- 1 I). Studies have shown that the phosphorylation of YWHAZ is crucial for its function (Fig. 1 J and 1 K). Our experimental results demonstrated that overexpression of LINC01234 significantly promoted the phosphorylation of YWHAZ. 3.2 YWHAZ is highly expressed in TNBC and correlates with poor prognosis Subsequently, we evaluated the expression level of YWHAZ using TCGA, METABRIC, and SCNA-B databases. Compared with the control group, YWHAZ was significantly highly expressed in TNBC tissues (Fig. 2 A- 2 C), and patients with high expression had poorer overall survival (Fig. 2 D). Consistently, cellular validation demonstrated markedly increased YWHAZ expression in the TNBC cell line MDA-MB-231 relative to the normal mammary epithelial cell line MCF-10A (Fig. 2 E and 2 F). These findings suggest that YWHAZ may play an important role in promoting the progression of TNBC. 3.3 MiR-204-5p regulates YWHAZ expression and mediates biological functions To investigate the regulatory mechanism underlying YWHAZ overexpression, we integrated bioinformatic predictions with prior functional evidence. lncLocator analysis localized LINC01234 predominantly in the cytoplasm (Fig. 3 A), suggesting its potential role in post-transcriptional regulation. Subsequent screening via AnnoLnc identified a conserved binding motif for miR-204-5p on LINC01234 (Fig. 3 B). Notably, miR-204-5p was previously reported to directly target YWHAZ in esophageal cancer and osteosarcoma. Based on these findings, we hypothesize that LINC01234 may function as a competitive endogenous RNA (ceRNA) by targeting miR-204-5p to regulate YWHAZ expression. Experimental validation demonstrated that LINC01234 knockdown increased miR-204-5p expression while decreasing YWHAZ levels, whereas LINC01234 overexpression exerted the opposite effects (Fig. 3 C and 3 D). Conversely, miR-204-5p overexpression significantly suppressed YWHAZ expression (Fig. 3 E). Clinically, miR-204-5p was downregulated in TNBC tissues (Fig. 3 F), and its low expression correlated with poor patient prognosis (Fig. 3 G). Functional assays revealed that miR-204-5p overexpression inhibited proliferation, promoted apoptosis, and suppressed PI3K/AKT signaling in MDA-MB-231 cells (Fig. 3 H- 3 K). These findings suggest that LINC01234 can act as a sponge for miR-204-5p, thereby influencing YWHAZ expression and regulating TNBC progression. 3.4 YWHAZ knockdown suppresses proliferation/migration and induces appoptosis in MDA-MB-231 cells To functionally validate the oncogenic role of YWHAZ in TNBC, we transfected si-YWHAZ oligonucleotides into MDA-MB-231 cells and verified the knockdown efficiency using RT-qPCR (Fig. 4 A). Then, we evaluated the effects of YWHAZ on cell proliferation, apoptosis, and migration via CCK-8 assays, flow cytometry, and Transwell assays, respectively (Fig. 4 B- 4 E). The results indicated that YWHAZ knockdown significantly inhibited cell proliferation and migration while promoting apoptosis (Fig. 3 F). Additionally, the activity of the PI3K/AKT signaling pathway was suppressed. These findings confirm that YWHAZ plays a crucial role in TNBC progression. 3.5 YWHAZ knockdown inhibits xenograft tumor growth. Next, we established a xenograft nude mouse model to investigate the effect of YWHAZ on TNBC progression in vivo . Our results showed that, compared to the control group, the YWHAZ knockdown group exhibited significantly reduced tumor volume and weight (Fig. 5 A- 5 D), without any change in the body weight of the mice (Fig. 5 E). Western blot analysis further demonstrated that the activity of the EMT signaling pathway and the levels of Ki67 were markedly inhibited following YWHAZ knockdown (Fig. 5 F). Collectively, these findings indicate that YWHAZ knockdown can significantly suppress the growth of xenograft tumors in mice. 4. Discussion TNBC, the most aggressive and rapidly progressing molecular subtype of breast cancer, presents significant therapeutic challenges due to its unique pathological profile: absence of ER, PR, and HER2 expression.[ 20 ] Clinical data indicate that TNBC patients exhibit higher rates of distant metastasis, advanced histological grade and PFS compared to other subtypes. Despite incremental improvements in surgical resection, adjuvant radio-chemotherapy, and immunotherapy, mortality rates remain substantially elevated[ 5 ]. Targeted therapies, designed to inhibit tumor progression by modulating specific molecular markers, offer theoretical advantages of precision and minimal systemic toxicity[ 21 – 23 ]. However, the complex pathogenesis of TNBC and the lack of well-characterized effective molecular markers have resulted in suboptimal clinical responses to current targeted approaches[ 24 ].Thus, identifying novel tumor markers and elucidating their regulatory mechanisms are critical for improving diagnostic accuracy and therapeutic efficacy in TNBC. lncRNAs are single-stranded RNA molecules exceeding 200 nucleotides in length, which have been functionally implicated in the progression of multiple malignancies including gastric and lung cancers[ 25 , 26 ]. They participate in cellular biological characteristics like umor expansion, infiltration, metastasis, and therapy resistance by modulating their own expression levels or influencing downstream molecular targets[ 27 – 29 ]. In TNBC, numerous lncRNAs exhibit aberrant enrichment or depletion and contribute to disease progression by regulating gene expression at transcriptional/post-transcriptional levels[ 30 , 31 ]. For instance, MALAT1 and HOTAIR can promote TNBC progression by activating EMT and mediating chemoresistance[ 32 , 33 ]. Therefore, in-depth elucidation of the oncogenic mechanisms of TNBC-specific lncRNAs will provide novel perspectives for developing RNA interference (RNAi)-based targeted therapies. Our previous work identified LINC01234, a highly conserved long non-coding RNA, was significantly upregulated in TNBC cells. Overexpression of LINC01234 enhanced proliferation and migration while suppressing apoptosis in MDA-MB-231 cells, whereas its knockdown effectively inhibited xenograft tumor growth in mice. In the current study, RNA-pulldown coupled with MS screened 117 potential LINC01234-interacting proteins. Following cross-verification with AnnoLnc database predictions and literature analysis, we focused on the candidate molecule YWHAZ. Subsequent RIP experiments confirmed direct binding between the 1st truncated fragment of LINC01234 and YWHAZ. YWHAZ, also known as 14-3-3ζ, is located at 8q23.1 and serves as a critical member of the highly conserved 14-3-3 dimeric protein family[ 34 ]. This scaffold protein specifically binds signaling molecules containing phosphorylated serine/threonine motifs, thereby regulating tumor proliferation, migration, and invasion through multiple signaling pathways[ 35 – 37 ]. For example, YWHAZ bound to p-ser83 on the PI3K-P85 regulatory subunit, enhancing PI3K membrane localization and activity to promote tumor cell proliferation[ 38 ]. It also participated in breast cancer invasion, migration, and EMT by modulating the Wnt/β-catenin pathway[ 39 ]. Notably, our study revealed aberrant overexpression of YWHAZ in both TNBC tissues and cell lines, with significant correlation to poor patient prognosis. Functionally, YWHAZ knockdown effectively inhibited proliferation and migration while promoting apoptosis in MDA-MB-231 cells, and significantly suppressed xenograft tumor growth in animal models. To elucidate the upstream regulatory mechanisms underlying YWHAZ overexpression, this study integrated bioinformatic predictions with existing literature evidence. AnnoLnc database analysis predicted miR-204-5p binding sites within the LINC01234 sequence, consistent with Chen et al.’s report of LINC01234 functioning as a ceRNA sponging miR-204 in gastric cancer[ 40 ]; concurrently, independent studies by Shen and Zhao teams confirmed miR-204-5p directly targets YWHAZ mRNA in esophageal cancer and osteosarcoma[ 41 , 42 ]. In our experimental system, we found that LINC01234 overexpression downregulated miR-204-5p and upregulated YWHAZ, while LINC01234 knockdown had the opposite trend; furthermore, miR-204-5p overexpression significantly inhibited MDA-MB-231 cell proliferation, promoted apoptosis, and suppressed PI3K/AKT signaling. These findings suggest LINC01234 regulates YWHAZ through dual mechaniss: acting as an RNA scaffold to directly bind and modulate YWHAZ phosphorylation, while competitively sequestering miR-204-5p via ceRNA activity to release post-transcriptional repression of YWHAZ mRNA, thereby synergistically driving TNBC malignancy progression (Fig. 5 G). Although this study preliminarily revealed a dual regulatory mechanism through which LINC01234 modulated the oncogenic function of YWHAZ, several limitations existed. First, the direct interaction between LINC01234 and miR-204-5p hasn't been verified by dual-luciferase reporter assays. Similarly, miR-204-5p's targeting of YWHAZ's 3'UTR needs confirmation. Second, expanded validation in larger TNBC clinical cohorts is necessary to comprehensively analyze the expression profiles of LINC01234/miR-204-5p/YWHAZ and their correlations with clinicopathological features. Based on these gaps, future research will focus on three aspects: (1) obtaining direct molecular evidence for the ceRNA regulatory network; (2) establishing clinical associations between this molecular axis and pathological characteristics; (3) developing specific inhibitors targeting the LINC01234-YWHAZ interaction interface to provide novel strategies for overcoming TNBC therapeutic resistance. In summary, this study integrates RNA interactomics with functional validation to establish that LINC01234 drives TNBC progression through dual molecular mechanisms: directly binding and activating YWHAZ's oncogenic function as a protein interaction scaffold, while indirectly alleviating YWHAZ suppression via miR-204-5p sequestration. This work not only proposes a lncRNA-mediated "protein binding-ceRNA crosstalk" paradigm in TNBC but also provides foundational insights to overcome the limitations in TNBC targeted therapy. Abbreviations TNBC Triple-negative breast cancer RIP immunoprecipitation HER2 human epidermal growth factor receptor 2 lncRNAs long non-coding RNAs MALAT1 metastasis-associated lung adenocarcinoma transcript 1 MS mass spectrometry TCGA The Cancer Genome Atlas ceRNA competitive endogenous RNA RNAi RNA interference. Declarations Ethics approval and consent to participate All protocols involving the use of animals were approved by the Animal Ethics Committee of Kunming University of Science and Technology (PZWH（dian）K2024-0001). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines. Consent for publication Not applicable. Availability of data and materials All raw and processed data supporting the findings of this study are available within the article and its Supplementary Information files, further inquiries can be directed to the corresponding author. Competing Interests The authors declare no potential conflicts of interest. Funding This work was supported by Yunnan High-level Personnel Training Support Program (YNWR-QNBJ-2020-243) and Kunming University of Science and Technology & Puer People's Hospital Joint Special Project on Medical Research (grant number KUST-PE2022004Y) Authors' contributions Diping Yu, Li Chen, Miaomiao Sheng and Yuan Zhao contributed to the design and conception of the study. Diping Yu, Li Chen, Huimin Li, Shiyao Kang and Fei Hu conducted experiments. Chao Yuan, Hongjun Yuan and Ming Li performed the statistical analysis. Diping Yu, Li Chen, Miaomiao Sheng and Yuan Zhao wrote the first draft of the manuscript. All authors read and approved the submitted the submitted version. Acknowledgements Not applicable. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. 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Tang C, Li C, Chen C, Chen T, Zhu J, Sun M, Wang P, Han C. LINC01234 promoted malignant behaviors of breast cancer cells via hsa-miR-30c-2-3p/CCT4/mTOR signaling pathway. Taiwan J Obstet Gynecol. 2024;63(1):46–56. Guo W, Wang Q, Zhan Y, Chen X, Yu Q, Zhang J, Wang Y, Xu XJ, Zhu L. Transcriptome sequencing uncovers a three-long noncoding RNA signature in predicting breast cancer survival. Sci Rep. 2016;6:27931. Bi M, Zheng L, Chen L, He J, Yuan C, Ma P, Zhao Y, Hu F, Tang W, Sheng M. ln RNA LINC01234 promotes triple-negative breast cancer progression through regulating the miR-429/SYNJ1 axis. Am J Transl Res. 2021;13(10):11399–412. Kim ES. Molecular targets and therapies associated with poor prognosis of triple–negative breast cancer (Review). Int J Oncol 2025, 66(6). Das A, Mandal SK, Arya S, Samanta SK, Kumar A, Panchpuri M, Abdel-Gawad H, Dwibedi V, Das N, Bose S, et al. 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Semin Cancer Biol. 2021;75:38–48. Chen M, Zhao P, Chou J, Zhou L, Feng Z, Hao X, Song H, Yang J. Non-coding RNAs regulating programmed cell death and its implications in cancer chemotherapy resistance. Int J Biol Macromol 2025:144888. Mu D, Han B, Huang H, Zheng Y, Zhang J, Shi Y. Unraveling the advances of non-coding RNAs on the tumor microenvironment: innovative strategies for cancer therapies. J Transl Med. 2025;23(1):614. Bhan A, Soleimani M, Mandal SS. Long Noncoding RNA and Cancer: A New Paradigm. Cancer Res. 2017;77(15):3965–81. Su Y, Bai Q, Zhang W, Xu B, Hu T. The Role of Long Non-Coding RNAs in Modulating the Immune Microenvironment of Triple-Negative Breast Cancer: Mechanistic Insights and Therapeutic Potential. Biomolecules 2025, 15(3). Yang Q, Fu Y, Wang J, Yang H, Zhang X. Roles of lncRNA in the diagnosis and prognosis of triple-negative breast cancer. J Zhejiang Univ Sci B. 2023;24(12):1123–40. Tuluhong D, Dunzhu W, Wang J, Chen T, Li H, Li Q, Wang S. Prognostic Value of Differentially Expressed LncRNAs in Triple-Negative Breast Cancer: A Systematic Review and Meta-Analysis. Crit Rev Eukaryot Gene Expr. 2020;30(5):447–56. Das PK, Siddika A, Rashel KM, Auwal A, Soha K, Rahman MA, Pillai S, Islam F. Roles of long noncoding RNA in triple-negative breast cancer. Cancer Med. 2023;12(20):20365–79. Gan Y, Ye F, He XX. The role of YWHAZ in cancer: A maze of opportunities and challenges. J Cancer. 2020;11(8):2252–64. Yin J, Ma Y, Fu H, Fan Y, Xiang D, Ding L, Huang J. Spartin Promotes Smurf1-Mediated Ubiquitination Modification of YWHAZ to Inhibit Cisplatin Resistance in Ovarian Cancer. FASEB J. 2025;39(10):e70658. Gao X, Feng Q, Zhang Q, Zhang Y, Hu C, Zhang L, Zhang H, Wang G, Hu K, Ma M, et al. Targeting enolase 1 reverses bortezomib resistance in multiple myeloma through YWHAZ/Parkin axis. J Biomed Sci. 2025;32(1):9. Dong N, Gu WW, Yang L, Lian WB, Jiang J, Zhu HJ, Chen CS, Wang BB. MiR-3074-5p suppresses non-small cell lung cancer progression by targeting the YWHAZ/Hsp27 axis. Int Immunopharmacol. 2024;138:112547. Neal CL, Xu J, Li P, Mori S, Yang J, Neal NN, Zhou X, Wyszomierski SL, Yu D. Overexpression of 14-3-3zeta in cancer cells activates PI3K via binding the p85 regulatory subunit. Oncogene. 2012;31(7):897–906. Ren L, Chen H, Song J, Chen X, Lin C, Zhang X, Hou N, Pan J, Zhou Z, Wang L, et al. MiR-454-3p-Mediated Wnt/beta-catenin Signaling Antagonists Suppression Promotes Breast Cancer Metastasis. Theranostics. 2019;9(2):449–65. Chen X, Chen Z, Yu S, Nie F, Yan S, Ma P, Chen Q, Wei C, Fu H, Xu T, et al. Long Noncoding RNA LINC01234 Functions as a Competing Endogenous RNA to Regulate CBFB Expression by Sponging miR-204-5p in Gastric Cancer. Clin Cancer Res. 2018;24(8):2002–14. Shen Z, Chai T, Luo F, Liu Z, Xu H, Zhang P, Kang M, Chen S. Loss of miR-204-5p Promotes Tumor Proliferation, Migration, and Invasion Through Targeting YWHAZ/PI3K/AKT Pathway in Esophageal Squamous Cell Carcinoma. Onco Targets Ther. 2020;13:4679–90. Zhao R, He H, Zhu Y, Wan J, Li Y, Gao S, Zhang C. MiR-204/14-3-3zeta axis regulates osteosarcoma cell proliferation through SATA3 pathway. Pharmazie. 2017;72(10):593–8. Additional Declarations No competing interests reported. Supplementary Files supplementarytable1and2.zip supplementaryFigureWB1.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-7330942","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511697894,"identity":"b16d312f-29c6-4829-b887-ebfc52132ae9","order_by":0,"name":"Diping Yu","email":"","orcid":"","institution":"Puer People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Diping","middleName":"","lastName":"Yu","suffix":""},{"id":511697896,"identity":"c935df28-e7fb-49c7-8cda-87a112230ea7","order_by":1,"name":"Li Chen","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Chen","suffix":""},{"id":511697898,"identity":"9bc2b466-a469-45f1-8f40-8cdfac0fb0a2","order_by":2,"name":"Huimin Li","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Huimin","middleName":"","lastName":"Li","suffix":""},{"id":511697899,"identity":"ed9a9ef3-34d6-4c69-a14a-4bc1ddf56082","order_by":3,"name":"Shiyao Kang","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shiyao","middleName":"","lastName":"Kang","suffix":""},{"id":511697901,"identity":"12ac7617-2392-4af8-b547-4a6d851fb693","order_by":4,"name":"Fei Hu","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Hu","suffix":""},{"id":511697903,"identity":"2895336f-ce62-479b-b4f7-514fe63ff467","order_by":5,"name":"Chao Yuan","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Yuan","suffix":""},{"id":511697905,"identity":"2293d71e-ab31-4af7-a574-7145650ce1f0","order_by":6,"name":"Hongjun Yuan","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Hongjun","middleName":"","lastName":"Yuan","suffix":""},{"id":511697906,"identity":"2295a907-0e09-4cc9-ae32-d655fbd558b7","order_by":7,"name":"Ming Li","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Li","suffix":""},{"id":511697907,"identity":"3b485138-df88-4104-902d-849a5d148ba5","order_by":8,"name":"Miaomiao Sheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYDACCQglB6WZiddiDKQZG0jSkjiDaC3ys5uPPfyasy195oz05w8YKqwTG9jPHsCrxeDOsXRj2W23c2dL5Bg2MJxJT2zgyUvAr0Uix0xaEqhlnkQOYwNj2+HEBgkeA/wOm5H/DaQlXU4i/WED4z8itDDcyGGT/LjtdoK0RIJhA2MDEVoMbqSZSTNuu204s+eN4YwEoMfaeHIIOSz5meTPbbflJY6nP/jwocZatp/9DAGHAQEzD4yVAMRsBNUDAeMPYlSNglEwCkbByAUA6gpGX427mFIAAAAASUVORK5CYII=","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Miaomiao","middleName":"","lastName":"Sheng","suffix":""},{"id":511697908,"identity":"b43e4113-04f2-42a2-89f6-09c978216fa2","order_by":9,"name":"Yuan Zhao","email":"","orcid":"","institution":"Puer People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2025-08-09 03:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7330942/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7330942/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91192488,"identity":"2988c725-13c4-491e-bf62-e148a6aaf1c7","added_by":"auto","created_at":"2025-09-12 14:42:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":245517,"visible":true,"origin":"","legend":"\u003cp\u003eLINC01234 binds to YWHAZ and promotes its phosphorylation.\u003c/p\u003e\n\u003cp\u003eA. Identification of proteins binding to the sense and antisense strands of LINC01234 by mass spectrometry (MS). B. Prediction of LINC01234-binding proteins through MS coupled with AnnoLnc database analysis. C. qRT-PCR analysis of YWHAZ expression following LINC01234 overexpression. D. qRT-PCR analysis of YWHAZ expression upon LINC01234 knockdown. E. Validation of YWHAZ-LINC01234 interaction by RNA immunoprecipitation (RIP). F. Schematic diagram of four truncated fragments of LINC01234. G. qRT-PCR assessment of expression efficiency for four LINC01234 truncation variants. H. qRT-PCR verification of YWHAZ overexpression efficiency. I. RIP assay determining binding affinity between YWHAZ and four LINC01234 truncation fragments. J. qRT-PCR confirmation of LINC01234 overexpression efficiency. K. Western blot detection of YWHAZ phosphorylation status upon LINC01234 overexpression. All experiments were performed at least thrice with triplicate samples. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7330942/v1/214eeb36d7a710e83cbf7185.png"},{"id":91190115,"identity":"a0b65baa-283c-4b8e-aaf6-37bda180531d","added_by":"auto","created_at":"2025-09-12 14:34:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":245864,"visible":true,"origin":"","legend":"\u003cp\u003eYWHAZ is highly expressed in TNBC and correlates with poor prognosis.\u003c/p\u003e\n\u003cp\u003eThe expression levels of YWHAZ in TNBC tissues and normal tissues in the TCGA database. B. The expression levels of YWHAZ in TNBC tissues and non-TNBC tissues in the METABRIC database. C. The expression levels of YWHAZ in TNBC tissues and non-TNBC tissues in the SCNA-B database. D. Kaplan-Meier survival analysis of TNBC patients stratified by YWHAZ expression level. E. qRT-PCR analysis of YWHAZ expression in MCF-10A and MDA-MB-231 cell lines. F. Western blot detection of YWHAZ expression levels in MCF-10A versus MDA-MB-231 cells. All experiments were performed at least thrice with triplicate samples. **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7330942/v1/6b4d68de8d70d1d8492c975d.png"},{"id":91192483,"identity":"24bd0154-f8b9-4f1c-be0e-a6d4c8d82a7b","added_by":"auto","created_at":"2025-09-12 14:42:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":374315,"visible":true,"origin":"","legend":"\u003cp\u003eMiR-204-5p regulates YWHAZ expression and mediates biological functions\u003c/p\u003e\n\u003cp\u003eA. Subcellular localization prediction of LINC01234 using lncLocator database. B. Prediction of miR-204-5p binding sites on LINC01234 sequence via AnnoLnc database. C. qRT-PCR analysis of miR-204-5p and YWHAZ expression levels following LINC01234 knockdown. D. qRT-PCR analysis of miR-204-5p and YWHAZ expression levels after LINC01234 overexpression. E. Western blot analysis of YWHAZ protein expression upon miR-204-5p overexpression. F. TCGA database analysis of miR-204-5p expression levels in TNBC versus normal breast tissues. G. Kaplan-Meier survival analysis of TNBC patients stratified by miR-204-5p expression levels. H. qRT-PCR validation of miR-204-5p overexpression efficiency. I. CCK-8 proliferation assay of MDA-MB-231 cells transfected with miR-204-5p mimic. J. Flow cytometry (FACS) analysis of apoptosis rates in MDA-MB-231 cells after miR-204-5p overexpression. K. Western blot analysis of PI3K/AKT pathway-related proteins in MDA-MB-231 cells following miR-204-5p overexpression. All experiments were performed at least thrice with triplicate samples. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7330942/v1/f33e83bdacf1bb00593ed171.png"},{"id":91194116,"identity":"10b2c949-6203-4afb-96dd-4dfa31315eba","added_by":"auto","created_at":"2025-09-12 14:50:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":534213,"visible":true,"origin":"","legend":"\u003cp\u003eYWHAZ knockdown suppresses proliferation/migration and induces appoptosis in MDA-MB-231 cells.\u003c/p\u003e\n\u003cp\u003eA. qRT-PCR analysis of YWHAZ knockdown efficiency in MDA-MB-231 cells. B. CCK-8 assay evaluating the effect of YWHAZ knockdown on cell proliferation. C. Flow cytometry analysis of apoptosis rates following YWHAZ knockdown. D. Quantitative analysis of the results shown in Panel C. E. Transwell migration assay assessing YWHAZ knockdown effects on MDA-MB-231 cell migration. F. Western blot analysis of PI3K/AKT pathway protein expression after YWHAZ knockdown. All experiments were performed at least thrice with triplicate samples. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7330942/v1/4ed012b70c01fef8f79dbf90.png"},{"id":91190121,"identity":"31760bf4-26c9-4f10-a032-bca6290d42f5","added_by":"auto","created_at":"2025-09-12 14:34:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":443863,"visible":true,"origin":"","legend":"\u003cp\u003eYWHAZ knockdown inhibits xenograft tumor growth.\u003c/p\u003e\n\u003cp\u003eA. Validation of YWHAZ knockdown efficiency. B. Representative images of xenograft tumors from different treatment groups. C. Tumor volume in each group (n = 5). D. Tumor weight in each group (N = 5). E. Body weight changes in mice during treatment periods. F. Western blot analysis of EMT marker expression following YWHAZ knockdown. G. Schematic diagram illustrating the role of LINC01234 in promoting TNBC progression. All experiments were performed at least thrice with triplicate samples. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7330942/v1/c82f5da40239559ab228c539.png"},{"id":97664605,"identity":"2c4505ca-f41a-4caa-a8da-f36f3c2affc5","added_by":"auto","created_at":"2025-12-08 09:11:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2712822,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7330942/v1/3eda8358-4558-4fff-b869-45927a90010e.pdf"},{"id":91190114,"identity":"1a88cc39-af0a-4143-8ee1-50344a367bb9","added_by":"auto","created_at":"2025-09-12 14:34:19","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":40904,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytable1and2.zip","url":"https://assets-eu.researchsquare.com/files/rs-7330942/v1/511bfc566f977f92241d2143.zip"},{"id":91190120,"identity":"22fe4d1d-684a-40d9-a98a-74632ee68609","added_by":"auto","created_at":"2025-09-12 14:34:19","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":439794,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryFigureWB1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7330942/v1/896319761bfb01a82339725b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"LINC01234 coordinates protein interactions and ceRNA networks to enhance YWHAZ-driven malignancy in triple-negative breast cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBreast cancer represents the most frequently diagnosed malignancy among women worldwide. According to global cancer burden statistics for 2020, approximately 2.26\u0026nbsp;million new breast cancer cases were reported, surpassing lung cancer (2.20\u0026nbsp;million cases) to become the leading cause of cancer incidence globally, with an estimated 685,000 associated deaths[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In China, breast cancer similarly constitutes the predominant female cancer, accounting for 416,000 new cases (18.4% of the global total) and 117,000 deaths (17.1% of global breast cancer mortality) during the same period[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, TNBC - characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expression \u0026ndash; constitutes the most aggressive molecular subtype. Patients with TNBC derive no benefit from endocrine therapies or anti-HER2 targeted agents. This subtype frequently presents with high histological grade at diagnosis, elevated rates of postoperative recurrence and distant metastasis, and confers poor prognosis, posing severe threats to patient survival[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, developing effective therapeutic strategies to improve the prognosis of TNBC patients remains a critical research priority in the medical community.\u003c/p\u003e\u003cp\u003eAdvances in transcriptome sequencing technologies have revealed that approximately 70% of the human genome is transcriptionally active, with long non-coding RNAs (lncRNAs) constituting a substantial proportion of these transcripts. Defined as non-protein-coding transcripts exceeding 200 nucleotides in length, lncRNAs function as critical regulatory molecules through multifaceted mechanisms: they modulate gene expression at transcriptional and post-transcriptional levels via interactions with DNA, RNA, or proteins, operating through both cis-regulatory and trans-regulatory modes[\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Emerging evidence implicates dysregulation of lncRNAs in the pathogenesis of diverse malignancies, where they influence oncogenic pathways including proliferation, metastasis, and therapy resistance[\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Consequently, lncRNAs represent promising novel therapeutic targets and prognostic biomarkers for cancer diagnosis and clinical management.\u003c/p\u003e\u003cp\u003eAccumulating evidence reveals aberrant expression of numerous lncRNAs in TNBC, with specific lncRNAs functioning as critical regulators of TNBC pathogenesis and progression. For instance, Zhang et al. identified lnc-BTG3-7:1 as a TNBC-specific transcript through RNA sequencing analysis. Their mechanistic investigation demonstrated that upregulation of lnc-BTG3-7:1 enhanced the transcriptional activity of the oncogene C21orf91, thereby activating both PI3K-AKT-GSK3β-β-catenin and MAPK signaling pathways to drive tumor development[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similarly, Shaath et al. performed single-cell RNA sequencing on 1,758 cells from TNBC patients, identifying metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) as a key lncRNA associated with resistance to neoadjuvant chemotherapy[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Critically, high MALAT1 expression was associated with poorer clinical prognosis in TNBC patients.\u003c/p\u003e\u003cp\u003eAlthough lncRNAs have been demonstrated to regulate multiple pathological processes in TNBC such as proliferation, apoptosis, invasion, and metastasis, only a small fraction of known lncRNAs have been functionally characterized in this aggressive subtype. Critical unresolved questions persist, such as: (i) identification of key lncRNA molecules regulating TNBC progression and metastasis, and (ii) screening of therapeutically targetable lncRNAs for precision intervention. Thus, in-depth research on specific LncRNAs' roles in TNBC or exploring new LncRNAs' functions in TNBC can offer a reliable theoretical basis and new perspectives for TNBC prevention and treatment.\u003c/p\u003e\u003cp\u003eLINC01234 is a highly conserved lncRNA located at chromosomal position 12q24.13[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], whose dysregulation has been implicated in breast cancer pathogenesis[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our preliminary studies demonstrated that LINC01234 was highly expressed in breast cancer cell lines, particularly with significantly elevated expression in MDA-MB-231 and MDA-MB-468 cells (TNBC cell lines). Functional validation through both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments confirmed that LINC01234 knockdown effectively suppressed TNBC tumor progression[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Nevertheless, the precise molecular mechanisms underlying LINC01234's oncogenic functions in TNBC remained unclear. To address this knowledge gap, this current study integrated RNA-pulldown combined with mass spectrometry (MS) and RIP technologies to systematically screen the interaction protein network and regulated signaling pathways of LINC01234, aiming to offer a theoretical basis and strategies for targeted therapy and prognostic evaluation of TNBC.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Cell culture\u003c/h2\u003e\u003cp\u003eMDA-MB-231 triple-negative human breast cancer cells and the immortalized, non-tumorigenic breast epithelial line MCF-10A were obtained from the Cell Culture Center, Peking Union Medical College. MDA-MB-231 Cells were maintained in high-glucose Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Cat. C11995500BT) supplemented with 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific, Cat. 10099141). MCF-10A Cells were cultured in DMEM/F-12 (Thermo Fisher Scientific, Cat. 12500-062) containing 10% (v/v) horse serum (HyClone), 1 mg/mL epidermal growth factor (Merck KGaA, Darmstadt, Germany), 1 mg/mL cholera toxin (Merck KGaA), 20 mg/mL insulin (Merck KGaA) and 1 mg/mL hydrocortisone (Merck KGaA). All cultures were incubated at 37℃ in a humidified atmosphere of 95% air / 5% CO₂. Routine mycoplasma testing confirmed the absence of contamination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Data acquisition and bioinformatic analysis\u003c/h2\u003e\u003cp\u003eIn this study, we downloaded the gene expression dataset of breast cancer cases with YWHAZ expression from The Cancer Genome Atlas (TCGA) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portal.gdc.cancer.gov/\u003c/span\u003e\u003cspan address=\"https://portal.gdc.cancer.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The subcellular localization of LINC01234 in cells was predicted utilizing the lncLocator website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.csbio.sjtu.edu.cn/bioinf/lncLocator/\u003c/span\u003e\u003cspan address=\"http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Both the nucleic acid sequence of LINC01234 and its known miRNA binding targets were verified via the AnnoLnc platform. Kaplan-Meier survival analysis was applied to analyze the relationship between YWHAZ, hsa-miR-205-5p and the overall survival rate of patients with triple-negative breast cancer (TNBC). Breast Cancer Gene-Expression Miner v5.2 (bc-GenExMiner v5.2) was employed to assess the expression levels of YWHAZ in various subtypes of BC. Additionally, UALCAN (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ualcan.path.uab.edu/\u003c/span\u003e\u003cspan address=\"https://ualcan.path.uab.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to assess the expression levels of hsa-miR-204-5p among various breast cancer subtypes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Cell transfection\u003c/h2\u003e\u003cp\u003e For transient transfection assays, TNBC cells were transfected with LINC01234 siRNA, YWHAZ siRNA, miR-204-5p mimic, mimic negative control (mimic-NC), pcDNA3.1-LINC01234 plasmids, and plasmids with different truncated variants of LINC01234 using TransIntro EL transfection reagent (TransGen Biotech, Beijing, China) according to the manufacturer\u0026rsquo;s protocols. The LINC01234 siRNA, YWHAZ siRNA, miR-204-5p mimic, and corresponding negative controls were procured from RiboBio (Guangzhou, China). The full-length LINC01234 sequence and its various truncated forms were synthesized by General Biosystems (Anhui, China) and subsequently cloned into the pcDNA3.1 vector.\u003c/p\u003e\u003cp\u003eA pGreenPuro vector encoding a short hairpin RNA (shRNA) against YWHAZ was constructed by General Biosystems (Anhui, China). For lentivirus production, the lentiviral vector was cotransfected with the packaging vectors psPAX2 and pMD2.G into 293T cells. Viral supernatant was harvested 48 h later, filtered (0.45 \u0026micro;m PVDF) and supplemented with 8 \u0026micro;g mL⁻\u0026sup1; polybrene (Merck). To establish stable cell lines, MDA-MB-231 cells were infected with lentiviral particles according to the manufactures\u0026rsquo;s protocol. Twelve hours post-infection, the culture medium containing lentiviral particles was discarded, and 4 mL of fresh medium supplemented with puromycin was added to maintain the culture. After 2 weeks, knockdown efficiency was confirmed by qRT-PCR. All oligonucleotide sequences were provided in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 RNA extraction and quantitative RT‒PCR\u003c/h2\u003e\u003cp\u003eTotal RNA from cultured cells and tissues was extracted with TRI Reagent\u0026reg; (Merck KGaA, Cat. T9424) following the manufacturer\u0026rsquo;s instructions. For the quantification of LINC01234 and YWHAZ, mRNA was reverse-transcribed into complementary DNA (cDNA) using the GoScript\u0026trade; Reverse Transcription Mix with Random Primers (A2801, Promega, Wisconsin, USA). For miR-204 detection, miRNA was reverse-transcribed into cDNA using the GoScript\u0026trade; Reverse Transcription System (A5001, Promega, Wisconsin, USA).\u003c/p\u003e\u003cp\u003eqRT-PCR reactions were performed in triplicate with SYBR Green Master mix (Roche, Basel, Switzerland) on an ABI 7300 system (Applied Biosystems). Dissociation-curve analysis confirmed single-product amplification. Relative expression was calculated using the 2-\u003csup\u003eΔΔCt\u003c/sup\u003e method with GAPDH (for LINC01234 and YWHAZ) or U6 (for miR-204) as endogenous controls. All primer sequences were listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Cell proliferation assays\u003c/h2\u003e\u003cp\u003eMDA-MB-231 cells were seeded in 24-well plates and transfected 24 h later with 100 nM YWHAZ-targeting siRNA (si-YWHAZ) or non-targeting control siRNA (si-NC) using TransIntro EL. After the indicated incubation periods, 50 \u0026micro;L CCK-8 reagent (RiboBio, Guangzhou, China) was added to each well. The plates were then incubated at 37\u0026deg;C in a 5% CO₂ atmosphere to allow the reaction to proceed. Absorbance was recorded at 450 nm with background subtraction at 630 nm using a microplate reader (BioTek).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Apoptosis analysis\u003c/h2\u003e\u003cp\u003eTransfected cells were collected by gentle trypsinization, washed twice with ice-cold PBS, and adjusted to 1 \u0026times; 10⁶ cells mL⁻\u0026sup1; in 1 \u0026times; Annexin V-binding buffer. 100 \u0026micro;L of the suspension (\u0026asymp;\u0026thinsp;1 \u0026times; 10⁵ cells) were transferred to a new tube and stained with 2 \u0026micro;L Annexin V-FITC (Roche, Cat. 11988549001) for 15 min at room temperature in the dark. Propidium iodide (4 \u0026micro;L, 50 \u0026micro;g mL⁻\u0026sup1;) was then added, followed by an additional 5-min incubation under identical conditions. Samples were diluted to 500 \u0026micro;L with binding buffer and immediately analysed on an Accuri C6 flow cytometer (Becton Dickinson, Franklin Lakes, USA)). At least 10 000 events per sample were acquired, and data were processed using FlowJo v10.8 to quantify early (Annexin V⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptotic fractions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Western blot analysis\u003c/h2\u003e\u003cp\u003eCells were harvested on ice in RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with 1\u0026times; protease inhibitor cocktail (Roche). Lysates were clarified by centrifugation (12 000 \u0026times; g, 15 min, 4\u0026deg;C) and total protein determined by the BCA assay (Beyotime). Equal amounts (20 \u0026micro;g per lane) were resolved on SDS-PAGE gels and electro-transferred to 0.22 \u0026micro;m PVDF membranes (Merck KGaA). Membranes were blocked for 1 h at room temperature with 5% (w/v) non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), then incubated overnight at 4\u0026deg;C with primary antibodies diluted in 5% BSA-TBST. After three TBST washes, membranes were probed with HRP-conjugated secondary antibodies (1:10 000, Cell Signaling Technology) for 1 h at room temperature. Immunoreactive bands were detected with Immobilon Western Chemiluminescent HRP Substrate (Merck KGaA) and visualized on a Tanon 5200 imaging system (Tanon 5200, Shanghai, China). The primary antibodies used in this study as follows: p-YWHAZ (abclonal,1:1000), YWHAZ (WanLeiBio, 1:1000), p-PI3K (CST, 1:1000), PI3K (CST, 1:1000), p-AKT (CST, 1:1000), AKT (CST, 1:1000), TSC1 (CST, 1:1000), ZEB1 (ABclonal, 1:1000), E-cadherin (ABclonal, 1:1000), β-catenin (ABclonal, 1:1000), MMP2 (ABclonal, 1:1000), MMP11 (ABclonal, 1:1000), TAGLN2 (ABclonal, 1:1000), CFL1 (ABclonal, 1:1000), snail (CST, 1:1000), Ki67 (CST, 1:1000), vinculin (Bioss, 1:5000), β-actin (ABclonal, 1:5000).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 RNA immunoprecipitation (RIP)\u003c/h2\u003e\u003cp\u003eRIP assays were performed using a Magna RIP Kit (17‒700, Millipore Corporation, USA) according to the manufacturer\u0026rsquo;s instructions. Briefly, MDA-MB-231 cells were lysed in ice-cold RIP lysis buffer containing RNase and protease inhibitors (Roche). Protein A/G magnetic beads were pre-conjugated with 5 \u0026micro;g of either anti-YWHAZ rabbit polyclonal antibody (WanLeiBio, WL0361) or control non-immune rabbit IgG (Cell Signaling Technology, 2729) for 30 min at room temperature. After three washes with RIP wash buffer, antibody-bound beads were incubated with 100 \u0026micro;L of clarified lysate overnight at 4\u0026deg;C with gentle rotation. Immune complexes were sequentially washed six times with stringent RIP wash buffer, then resuspended in proteinase K digestion buffer to release RNA. RNA was recovered by Trizol-chloroform RNA extraction method and analyzed by RT-qPCR. Results were expressed as fold-enrichment over input after normalization to the IgG control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Transwell assays\u003c/h2\u003e\u003cp\u003eTranswell analysis was conducted using 24-well Transwell\u0026reg; inserts with 8 \u0026micro;m-pore polycarbonate membranes (Corning, Cat. 3422). Transfected cells (8 \u0026times; 10⁴ per insert) were resuspended in serum-free medium and seeded into the upper compartment; the lower compartment received 500 \u0026micro;L complete medium. After 24 h incubation (37\u0026deg;C, 5% CO₂), non-migrated cells on the upper chamber were gently removed with a cotton swab. Migrated cells on the lower surface of the membrane were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet. Five random fields per membrane were imaged at 200\u0026times; magnification using an inverted microscope. Migrated cells were counted by ImageJ and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD per field.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Animal experiments\u003c/h2\u003e\u003cp\u003eBALB/c nude mice aged 4\u0026ndash;6 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and randomly assigned to two groups. Each mouse received a subcutaneous injection of 1\u0026times;10⁶ MDA-MB-231 cells transfected with lentivirus (sh-NC or sh-YWHAZ). Tumor volume was measured every two days, and mouse body weight was recorded every four days. Tumor volume was calculated using the formula: V\u0026thinsp;=\u0026thinsp;0.5\u0026times;length\u0026times;width\u0026sup2;. After one month, the mice were euthanized via cervical dislocation, and the tumors were harvested for subsequent analyses. All protocols involving the use of animals were approved by the Animal Ethics Committee of Kunming University of Science and Technology (PZWH(dian)K2024-0001). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 RNA pull-down assays and Mass spectrometry (MS)\u003c/h2\u003e\u003cp\u003eMDA-MB-231 cells were lysed in RIP buffer, and the supernatant was incubated with biotin-labeled probes targeting LINC01234 (synthesized by Guangzhou Laboratory Biotechnology Co., Ltd) at 4\u0026deg;C overnight. Streptavidin-coated magnetic beads (Thermo Fisher) were subsequently added, and the mixture was incubated at room temperature for 1 hour. Following thorough washing, the bound proteins were eluted using washing buffer and collected from the supernatant.\u003c/p\u003e\u003cp\u003eEluates were separated on 4\u0026ndash;12% Bis-Tris SDS-PAGE gels. After brief Coomassie staining, entire lanes were excised, reduced, alkylated, and in-gel digested with trypsin (Promega). Peptides were extracted, and analysed on a Q Exactive\u0026trade; HF-X mass spectrometer (Thermo Fisher). RAW data were converted to MGF files using Proteome Discoverer 1.4 (v1.4.0.288). These files were then subjected to database searching against the UniProt human protein database with ProteinPilot 4.5 (v1656, AB Sciex). Negative control probes were used to exclude nonspecific interactions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Statistical analyses\u003c/h2\u003e\u003cp\u003eAll the data results were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD through the software GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA). Differences among groups were analyzed with two-way analysis of variance (ANOVA), while the differences among one group were analyzed with unpaired t-test or one-way ANOVA. * \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and **** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 were considered as significant. All experiments were performed at least thrice with triplicate samples.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.1 LINC01234 binds to YWHAZ and promotes its phosphorylation\u003c/h2\u003e\u003cp\u003eTo investigate the molecular mechanisms of LINC01234, we performed RNA-pull down and mass spectrometry experiments, which preliminarily identified 117 proteins directly interacting with LINC01234 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Supplementary Table\u0026nbsp;2). By intersecting these results with the predictions from the AnnoLnc database, 8 target proteins were pinpointed. Through literature review, we focused on the YWHAZ gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo determine whether there is an association between LINC01234 and YWHAZ, we first knocked down or overexpressed LINC01234, and the results showed that the expression levels of YWHAZ and LINC01234 were positively correlated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Further RIP experiments confirmed that YWHAZ could directly bind to LINC01234 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). LINC01234 has four main structural branches, and to identify the specific binding site with YWHAZ, we truncated LINC01234 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The results revealed that the fragment 1 of LINC01234 bound to YWHAZ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). Studies have shown that the phosphorylation of YWHAZ is crucial for its function (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). Our experimental results demonstrated that overexpression of LINC01234 significantly promoted the phosphorylation of YWHAZ.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2 YWHAZ is highly expressed in TNBC and correlates with poor prognosis\u003c/h2\u003e\u003cp\u003eSubsequently, we evaluated the expression level of YWHAZ using TCGA, METABRIC, and SCNA-B databases. Compared with the control group, YWHAZ was significantly highly expressed in TNBC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and patients with high expression had poorer overall survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Consistently, cellular validation demonstrated markedly increased YWHAZ expression in the TNBC cell line MDA-MB-231 relative to the normal mammary epithelial cell line MCF-10A (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These findings suggest that YWHAZ may play an important role in promoting the progression of TNBC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.3 MiR-204-5p regulates YWHAZ expression and mediates biological functions\u003c/h2\u003e\u003cp\u003eTo investigate the regulatory mechanism underlying YWHAZ overexpression, we integrated bioinformatic predictions with prior functional evidence. lncLocator analysis localized LINC01234 predominantly in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), suggesting its potential role in post-transcriptional regulation. Subsequent screening via AnnoLnc identified a conserved binding motif for miR-204-5p on LINC01234 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Notably, miR-204-5p was previously reported to directly target YWHAZ in esophageal cancer and osteosarcoma. Based on these findings, we hypothesize that LINC01234 may function as a competitive endogenous RNA (ceRNA) by targeting miR-204-5p to regulate YWHAZ expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eExperimental validation demonstrated that LINC01234 knockdown increased miR-204-5p expression while decreasing YWHAZ levels, whereas LINC01234 overexpression exerted the opposite effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Conversely, miR-204-5p overexpression significantly suppressed YWHAZ expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Clinically, miR-204-5p was downregulated in TNBC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), and its low expression correlated with poor patient prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Functional assays revealed that miR-204-5p overexpression inhibited proliferation, promoted apoptosis, and suppressed PI3K/AKT signaling in MDA-MB-231 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). These findings suggest that LINC01234 can act as a sponge for miR-204-5p, thereby influencing YWHAZ expression and regulating TNBC progression.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.4 YWHAZ knockdown suppresses proliferation/migration and induces appoptosis in MDA-MB-231 cells\u003c/h2\u003e\u003cp\u003eTo functionally validate the oncogenic role of YWHAZ in TNBC, we transfected si-YWHAZ oligonucleotides into MDA-MB-231 cells and verified the knockdown efficiency using RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Then, we evaluated the effects of YWHAZ on cell proliferation, apoptosis, and migration via CCK-8 assays, flow cytometry, and Transwell assays, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). The results indicated that YWHAZ knockdown significantly inhibited cell proliferation and migration while promoting apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Additionally, the activity of the PI3K/AKT signaling pathway was suppressed. These findings confirm that YWHAZ plays a crucial role in TNBC progression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.5 YWHAZ knockdown inhibits xenograft tumor growth.\u003c/h2\u003e\u003cp\u003eNext, we established a xenograft nude mouse model to investigate the effect of YWHAZ on TNBC progression \u003cem\u003ein vivo\u003c/em\u003e. Our results showed that, compared to the control group, the YWHAZ knockdown group exhibited significantly reduced tumor volume and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), without any change in the body weight of the mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Western blot analysis further demonstrated that the activity of the EMT signaling pathway and the levels of Ki67 were markedly inhibited following YWHAZ knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Collectively, these findings indicate that YWHAZ knockdown can significantly suppress the growth of xenograft tumors in mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eTNBC, the most aggressive and rapidly progressing molecular subtype of breast cancer, presents significant therapeutic challenges due to its unique pathological profile: absence of ER, PR, and HER2 expression.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Clinical data indicate that TNBC patients exhibit higher rates of distant metastasis, advanced histological grade and PFS compared to other subtypes. Despite incremental improvements in surgical resection, adjuvant radio-chemotherapy, and immunotherapy, mortality rates remain substantially elevated[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Targeted therapies, designed to inhibit tumor progression by modulating specific molecular markers, offer theoretical advantages of precision and minimal systemic toxicity[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the complex pathogenesis of TNBC and the lack of well-characterized effective molecular markers have resulted in suboptimal clinical responses to current targeted approaches[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].Thus, identifying novel tumor markers and elucidating their regulatory mechanisms are critical for improving diagnostic accuracy and therapeutic efficacy in TNBC.\u003c/p\u003e\u003cp\u003elncRNAs are single-stranded RNA molecules exceeding 200 nucleotides in length, which have been functionally implicated in the progression of multiple malignancies including gastric and lung cancers[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. They participate in cellular biological characteristics like umor expansion, infiltration, metastasis, and therapy resistance by modulating their own expression levels or influencing downstream molecular targets[\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In TNBC, numerous lncRNAs exhibit aberrant enrichment or depletion and contribute to disease progression by regulating gene expression at transcriptional/post-transcriptional levels[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. For instance, MALAT1 and HOTAIR can promote TNBC progression by activating EMT and mediating chemoresistance[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, in-depth elucidation of the oncogenic mechanisms of TNBC-specific lncRNAs will provide novel perspectives for developing RNA interference (RNAi)-based targeted therapies.\u003c/p\u003e\u003cp\u003eOur previous work identified LINC01234, a highly conserved long non-coding RNA, was significantly upregulated in TNBC cells. Overexpression of LINC01234 enhanced proliferation and migration while suppressing apoptosis in MDA-MB-231 cells, whereas its knockdown effectively inhibited xenograft tumor growth in mice. In the current study, RNA-pulldown coupled with MS screened 117 potential LINC01234-interacting proteins. Following cross-verification with AnnoLnc database predictions and literature analysis, we focused on the candidate molecule YWHAZ. Subsequent RIP experiments confirmed direct binding between the 1st truncated fragment of LINC01234 and YWHAZ.\u003c/p\u003e\u003cp\u003eYWHAZ, also known as 14-3-3ζ, is located at 8q23.1 and serves as a critical member of the highly conserved 14-3-3 dimeric protein family[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This scaffold protein specifically binds signaling molecules containing phosphorylated serine/threonine motifs, thereby regulating tumor proliferation, migration, and invasion through multiple signaling pathways[\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. For example, YWHAZ bound to p-ser83 on the PI3K-P85 regulatory subunit, enhancing PI3K membrane localization and activity to promote tumor cell proliferation[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. It also participated in breast cancer invasion, migration, and EMT by modulating the Wnt/β-catenin pathway[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Notably, our study revealed aberrant overexpression of YWHAZ in both TNBC tissues and cell lines, with significant correlation to poor patient prognosis. Functionally, YWHAZ knockdown effectively inhibited proliferation and migration while promoting apoptosis in MDA-MB-231 cells, and significantly suppressed xenograft tumor growth in animal models.\u003c/p\u003e\u003cp\u003eTo elucidate the upstream regulatory mechanisms underlying YWHAZ overexpression, this study integrated bioinformatic predictions with existing literature evidence. AnnoLnc database analysis predicted miR-204-5p binding sites within the LINC01234 sequence, consistent with Chen et al.\u0026rsquo;s report of LINC01234 functioning as a ceRNA sponging miR-204 in gastric cancer[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]; concurrently, independent studies by Shen and Zhao teams confirmed miR-204-5p directly targets YWHAZ mRNA in esophageal cancer and osteosarcoma[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In our experimental system, we found that LINC01234 overexpression downregulated miR-204-5p and upregulated YWHAZ, while LINC01234 knockdown had the opposite trend; furthermore, miR-204-5p overexpression significantly inhibited MDA-MB-231 cell proliferation, promoted apoptosis, and suppressed PI3K/AKT signaling. These findings suggest LINC01234 regulates YWHAZ through dual mechaniss: acting as an RNA scaffold to directly bind and modulate YWHAZ phosphorylation, while competitively sequestering miR-204-5p via ceRNA activity to release post-transcriptional repression of YWHAZ mRNA, thereby synergistically driving TNBC malignancy progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003eAlthough this study preliminarily revealed a dual regulatory mechanism through which LINC01234 modulated the oncogenic function of YWHAZ, several limitations existed. First, the direct interaction between LINC01234 and miR-204-5p hasn't been verified by dual-luciferase reporter assays. Similarly, miR-204-5p's targeting of YWHAZ's 3'UTR needs confirmation. Second, expanded validation in larger TNBC clinical cohorts is necessary to comprehensively analyze the expression profiles of LINC01234/miR-204-5p/YWHAZ and their correlations with clinicopathological features. Based on these gaps, future research will focus on three aspects: (1) obtaining direct molecular evidence for the ceRNA regulatory network; (2) establishing clinical associations between this molecular axis and pathological characteristics; (3) developing specific inhibitors targeting the LINC01234-YWHAZ interaction interface to provide novel strategies for overcoming TNBC therapeutic resistance.\u003c/p\u003e\u003cp\u003eIn summary, this study integrates RNA interactomics with functional validation to establish that LINC01234 drives TNBC progression through dual molecular mechanisms: directly binding and activating YWHAZ's oncogenic function as a protein interaction scaffold, while indirectly alleviating YWHAZ suppression via miR-204-5p sequestration. This work not only proposes a lncRNA-mediated \"protein binding-ceRNA crosstalk\" paradigm in TNBC but also provides foundational insights to overcome the limitations in TNBC targeted therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTNBC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTriple-negative breast cancer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRIP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eimmunoprecipitation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHER2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehuman epidermal growth factor receptor 2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003elncRNAs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003elong non-coding RNAs\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMALAT1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emetastasis-associated lung adenocarcinoma transcript 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emass spectrometry\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTCGA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eThe Cancer Genome Atlas\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eceRNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecompetitive endogenous RNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRNAi\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRNA interference.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll protocols involving the use of animals were approved by the Animal Ethics Committee of Kunming University of Science and Technology (PZWH（dian）K2024-0001). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll raw and processed data supporting the findings of this study are available within the article and its Supplementary Information files, further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Yunnan High-level Personnel Training Support Program (YNWR-QNBJ-2020-243) and Kunming University of Science and Technology \u0026amp; Puer People\u0026apos;s Hospital Joint Special Project on Medical Research (grant number KUST-PE2022004Y)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDiping Yu, Li Chen, Miaomiao Sheng and Yuan Zhao contributed to the design and conception of the study. Diping Yu, Li Chen, Huimin Li, Shiyao Kang and Fei Hu conducted experiments. Chao Yuan, Hongjun Yuan and Ming Li performed the statistical analysis. Diping Yu, Li Chen, Miaomiao Sheng and Yuan Zhao wrote the first draft of the manuscript. All authors read and approved the submitted the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLei S, Zheng R, Zhang S, Wang S, Chen R, Sun K, Zeng H, Zhou J, Wei W. 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Theranostics. 2019;9(2):449\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen X, Chen Z, Yu S, Nie F, Yan S, Ma P, Chen Q, Wei C, Fu H, Xu T, et al. Long Noncoding RNA LINC01234 Functions as a Competing Endogenous RNA to Regulate CBFB Expression by Sponging miR-204-5p in Gastric Cancer. Clin Cancer Res. 2018;24(8):2002\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShen Z, Chai T, Luo F, Liu Z, Xu H, Zhang P, Kang M, Chen S. Loss of miR-204-5p Promotes Tumor Proliferation, Migration, and Invasion Through Targeting YWHAZ/PI3K/AKT Pathway in Esophageal Squamous Cell Carcinoma. Onco Targets Ther. 2020;13:4679\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao R, He H, Zhu Y, Wan J, Li Y, Gao S, Zhang C. MiR-204/14-3-3zeta axis regulates osteosarcoma cell proliferation through SATA3 pathway. Pharmazie. 2017;72(10):593\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\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":"Triple-negative breast cancer, LINC01234, YWHAZ, progression","lastPublishedDoi":"10.21203/rs.3.rs-7330942/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7330942/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTriple-negative breast cancer (TNBC) exhibits poor prognosis due to the lack of effective therapeutic targets. This study investigates the molecular mechanism of long non-coding RNA LINC01234 in TNBC progression. Our preliminary work identified significant upregulation of LINC01234 in TNBC cells, and its knockdown suppressed tumor progression. Here, through RNA-pulldown coupled with mass spectrometry, we screened LINC01234-interacting proteins and confirmed its direct binding to the scaffolding protein YWHAZ (14-3-3ζ) via RNA immunoprecipitation (RIP), which promotes YWHAZ phosphorylation. Clinical analysis showed that YWHAZ was highly expressed in TNBC tissues and correlated with poor patient prognosis. Mechanistically, LINC01234 regulated YWHAZ expression via targeting miR-204-5p, thereby influencing tumor progression. Further functional validation demonstrated that either miR-204-5p overexpression or YWHAZ knockdown significantly inhibited TNBC cell proliferation/migration and promoted apoptosis. These findings suggest a dual regulatory mechanism: LINC01234 directly activates YWHAZ's oncogenic function through protein interaction, while indirectly releasing the suppression of YWHAZ expression by sponging miR-204-5p. This study reveals a \"protein interaction-ceRNA crosstalk\" paradigm by which LINC01234 promotes TNBC progression, providing a theoretical foundation and potential therapeutic strategy for TNBC management.\u003c/p\u003e","manuscriptTitle":"LINC01234 coordinates protein interactions and ceRNA networks to enhance YWHAZ-driven malignancy in triple-negative breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 14:34:14","doi":"10.21203/rs.3.rs-7330942/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":"25915c69-80cb-4183-911e-b4db9f60ec00","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-03T17:08:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 14:34:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7330942","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7330942","identity":"rs-7330942","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}