The rearrangement of PRKD3 caused by long noncoding RNA mediated H3K27me3 demethylation led to tumorigenesis

The rearrangement of PRKD3 caused by long noncoding RNA mediated H3K27me3 demethylation led to tumorigenesis | 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 The rearrangement of PRKD3 caused by long noncoding RNA mediated H3K27me3 demethylation led to tumorigenesis Yitang Liu, Jie Yang, Jiayan Fan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9294400/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Gastrointestinal cancer, one of the most prevalent and lethal malignancies worldwide, poses a significant global health burden. Protein kinase D3 (PRKD3) is a member of protein kinase which was demonstrated to be critical in growth regulation. However, the potential role of PRKD3 in gastrointestinal tumors remains unexplored. Here, we found that PRKD3 was abnormally expressed in gastric cancer and colorectal cancer cells. Silencing of PRKD3 markedly inhibits tumor cell proliferation and metastasis, underscoring its functional importance. Notably, this rearrangement of PRKD3 in gastrointestinal cancer was caused by long noncoding RNA ROR (ROR lncRNA) mediated H3K27me3 demethylation. Bring down the expression of ROR lncRNA can restore the trimethylation of H3K27 at PRKD3 promoter region and lead to the suppression of PRKD3 . Our findings reveal a novel mechanism by which the ROR lncRNA mediated H3K27me3 demethylation may rearrange the transcription of PRKD3 and lead to tumorigenesis in gastrointestinal tumor. Collectively, we represent a novel kinase target PRKD3 , offering new avenues for the development of targeted therapies against gastrointestinal cancers. Long non-coding RNAs PRKD3 H3K27me3 demethylation tumorigenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Gastrointestinal malignancies, including gastric cancer and colorectal cancer (CRC), are among the most prevalent cancers worldwide and remain leading contributors to global cancer incidence and mortality profiles [ 1 ]. In recent years, the overall burden of gastrointestinal cancers has remained high, with marked geographic heterogeneity [ 2 , 3 ]. According to the World Health Organization estimates for 2022, CRC accounted for approximately 1.9 million new cases and more than 0.9 million deaths, ranking as the second leading cause of cancer-related mortality globally. In China, gastric cancer and CRC likewise constitute major public health challenges. Owing to the large population base, accelerated ageing, and widespread exposure to modifiable risk factors, the national burden of gastric cancer remains higher than the global average [ 4 ]. Meanwhile, the incidence of CRC continues to rise in China, and both its incidence and mortality have consistently ranked among the top five cancers in the population [ 5 ]. Although treatment strategies for gastrointestinal cancers have evolved substantially—supported by advances in systemic therapy, optimization of therapeutic decision-making, and expanded options for targeted therapy, immunotherapy, and locoregional surgical interventions—clinical outcomes remain suboptimal for a considerable proportion of patients [ 6 , 7 ]. Moreover, multimodal regimens frequently lead to treatment-related complications, which may compromise tolerability, quality of life, and overall effectiveness. Therefore, the development of more effective and safer therapeutic agents for gastrointestinal cancers remains a critical research priority. In this context, elucidating the molecular and pathogenic mechanisms underlying gastrointestinal tumorigenesis is essential for identifying actionable targets and developing novel therapeutic approaches for clinical translation. Tumorigenesis is a complicated process and caused by multiple factors. Although somatic mutations and clonal expansions are widespread even in histologically normal tissues, only a small fraction ultimately progresses to cancer. This observation suggests that additional driving events are required to propel these clones toward an irreversible, highly heterogeneous, and invasive state. Historically, our ability to detect such events was limited; however, with the advent of innovative technologies, the defining features of transforming cells and their interactions with the surrounding microenvironment are being progressively elucidated [ 8 ]. In recent years, increasing emphasis has been placed on environmental cancer risk factors and epigenetic alterations, which can profoundly influence early clonal expansion and malignant evolution without directly inducing new mutations [ 9 ]. Canonical oncogenic signaling pathways—including Wnt/β-catenin, JAK/STAT3, RAS–RAF–MEK–ERK (MAPK), and PI3K/AKT/mTOR—have been extensively shown to play central roles in gastrointestinal tumor biology. These pathways regulate key processes such as tumor cell proliferation, apoptosis evasion, epithelial–mesenchymal transition (EMT), invasion and metastasis, and resistance to therapy, thereby driving tumor initiation and progression [ 10 , 11 ]. In addition, beyond well-established tumor-suppressive microRNAs (miRNAs) such as the miR-34 and let-7 families [ 12 , 13 ], accumulating evidence indicates that multiple miRNAs (e.g., miR-21) contribute to gastrointestinal carcinogenesis by modulating cell-cycle control, apoptosis, and migration-related networks and by reshaping the tumor microenvironment [ 14 ]. Notably, exosome-associated miRNAs have attracted substantial attention for their roles in promoting tumor progression and for their potential utility as noninvasive diagnostic and prognostic biomarkers [ 15 ]. However, most of these target genes are hardly to be used in clinic for the lacking of targeted drugs. For this reason, researchers now prefer to focus on the targets which can easily design and produce small molecule compound[ 16 ]. And kinase is one of such an ideal research target [ 17 , 18 ]. Protein kinase D3 (PRKD3) is a serine/threonine kinase of the protein kinase D (PKD) family and acts as a diacylglycerol-responsive effector downstream of PKC signaling. Beyond its established roles in epithelial barrier regulation and intracellular vesicle trafficking [ 19 ], PRKD3 integrates multiple oncogenic cues to regulate transcriptional programs, metabolism, and cell fate decisions. Accumulating evidence over the past decade supports an oncogenic role of PRKD3 across solid tumors, where it promotes proliferation, survival, migration, and invasion and has been proposed as a druggable target [ 20 , 21 ]. Importantly, emerging studies have begun to implicate PRKD3 in gastrointestinal malignancies. In gastric cancer, PRKD3 overexpression has been linked to enhanced tumor cell growth and metabolic reprogramming through the PRKD3–NF-κB/p65–PFKFB3 axis[ 22 ], and recent work further suggests that PRKD3 may influence cell-cycle progression and malignant phenotypes[ 23 ]. Therefore, we focused on this kinase to further elucidate its potential roles in gastrointestinal malignancies. In this study, we have attempted to identify the function of PRKD3 in gastrointestinal tumors and find out its transcriptional regulatory mechanism from long noncoding RNA level. Here, we show that the demethylation of PRKD3 promoter mediated by ROR determine the transcriptional activation of PRKD3 and affect tumorigenesis. 2 Materials and methods 2.1 Cell culture The AGS cell line, derived from human gastric adenocarcinoma, and the HT29 cell line, derived from human colorectal adenocarcinoma, were obtained from the National Collection of Authenticated Cell Cultures, China. All cell lines were utilized in the cytotoxicity assessment of the MVs derived from L. buchneri. The cell lines were developed in a liquid medium consisting of RPMI1640 and DMEM (Gibco, Carlsbad, USA), which was further enriched with 10% Fetal Bovine Serum (Gibco, Carlsbad, USA), 100 µ/mL penicillin (Gibco, Carlsbad, USA), and 100 µg/mL streptomycin (Gibco, Carlsbad, USA). The cells were cultivated at 37°C in a humid environment containing 5% CO2. 2.2 RNA extraction, reverse transcription-PCR analysis and Real-time RT-PCR Total RNA was extracted using TRI-REAGENT (Invitrogen, USA), according to the manufacturer’s instructions, and cDNA was synthesized using the PrimeScript RT reagent kit (Takara, Japan). Real-time RT-PCR reactions were performed with SYBR Premix Ex Taq (TaKaRa) in a Bio-Rad CFX96 Real-Time PCR System (Bio-rad, Hercules, CA). GAPDH and β-actin was used as an internal control. The primer sequences are listed in Supplementary Table S1 . 2.3 Western blot analysis Cells were harvested at the indicated time and washed twice with cold PBS. Cell extracts were prepared with lysis buffer and centrifuged at 13,000 g for 30 min at 4°C. Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 7.5% (wt/vol) polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Membranes were used to immunoblot with anti-PRKD3 (Proteintech, USA). Membranes were incubated with a secondary antibody conjugated to a fluorescent tag. The bands were visualized using the Odyssey infrared Imaging System (LI-COR, Lincoln, USA). 2.4 shRNA Retrovirus and Selection ROR shRNA-mir constructs and verified nonsilencing shRNA were purchased from Open Biosystems (Lafayette, CO) ( Supplemental Table S2 ). Selection was performed by incubating with 4µg/ml puromycin for 2 weeks; the clones were screened for GFP expression. 2.5 Small Interfering RNA PRKD3 knockdown was achieved using small interfering RNA (siRNA). ( Supplemental Table S3 ). Cells were seeded at 200,000 cells per well in six-well plates and transfected using Lipofectamine 2000 (Invitrogen) in Opti-MEM I Reduced Serum Medium with 50 nM siRNA (Invitrogen). Forty-eight hours posttransfection, cells were harvested in TRIzol for RNA isolation (Invitrogen) or lysed in radioimmunoprecipitation assay (RIPA) lysis buffer for western blot. 2.6 MTT Assay The MTT assay was performed as previously described [ 24 ]. In short, cells were seeded at 5,000 cells per well in flat-bottomed 96-well plates. At the end of the incubation time, 20 µl of 5 mg/ml 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma, St Louis, MO) in PBS was added to each well. After 4 hours, the media was discarded, and the cells were lysed with 100 µl of dimethylsulfoxide. The cells were incubated for a further 30 minutes at 37°C with gentle shaking. The optical density was determined with a microplate reader at 490 nm. 2.7 Transwell assay The migratory ability of the cells was evaluated using a 24-well transwell system (Corning, USA) equipped with 8-µm pore size polycarbonate filters according to the manufacturer's instructions. The upper compartment contained 10,000 cells suspended in appropriate medium supplemented with 5% fetal bovine serum, and the lower compartment contained 10% fetal bovine serum. After the appropriate days of incubation at 37°C, the lower compartment was fixed with 100% methanol and stained with 0.1% crystal violet before photographing. The crystal violet was washed down from the migrated cells using 100 µl of 33% acetic acid. The absorbance of the washed down liquid was determined with a microplate reader at 490 nm. 2.8 Soft agar tumor formation assay A soft agar colony formation assay was performed in 6-well plates. One milliliter of the bottom layer comprised of 0.6% agar in complete medium was spread in each 6-well plates. A total of 20,000 cells were suspended in 1.0 milliliter of complete medium containing 0.3% agar and was seeded into each well. The cultures were fed every 3 to 4 days with 300 µl of complete medium for 3–4 weeks. For quantification, the colonies grown in soft agar were stained with 0.005% crystal violet. The size of the colonies was determined using Adobe Photoshop. 2.9 Chromatin immunoprecipitation (ChIP) ChIP assays were performed as previously described. One hundred million cells were fixed with 1% formaldehyde and sonicated for 8 min (10 s on and 15 s off) on ice with a 2-mm microtip at 40% output control and 90% duty cycle settings. The sonicated chromatin (1 ml) was clarified by centrifugation and snap-frozen in liquid nitrogen. To perform ChIP, sonicated chromatin (150 µl) was diluted 10-fold and added protein G-agarose (60 µl) (Millipore, USA) with shaking at 4°C for 2 h. Then briefly centrifuged at 1000 rpm for 5 min at 4°C and the supernatant was collected into a new tube. Antibodies to SMC1 and MED12 were obtained from Abcam Inc (Abcam, USA) nd added to the supernatant overnight at 4°C. PureProteome™ Protein A and Protein G Magnetic Beads (60 µl) (Millipore, USA) were used to pull down the protein at 4°C for 6 h. The DNA that was released from the bound chromatin after cross-linking reversal and proteinase K treatment was precipitated and diluted in 100 µl of 0.2 M glycine. The PCRs (3 µl under liquid wax) contained 2 µl ChIP (or input) DNA, 0.5 mM appropriate primer pairs, 50 µM deoxynucleotide triphosphate and 0.2 U Klen-Taq I (Ab Peptides, USA). The PCR conditions were 95°C for 5 min, followed by 34 cycles of 95°C for 30 s, 30 s of optimal annealing temperature, and 72°C for 30s of extension ( Supplemental Table S4 ). The PCR products were separated on 8% polyacrylamide–urea gel. 3 Results 3.1 PRKD3 is significantly over-expressed in gastrointestinal tumors To identify the function of PRKD3 in gastrointestinal tumors, we firstly tried to exam its expression level in several gastrointestinal tumor cell lines (AGS, SW620, 1299 and HT29). According to the results of western blot, we found that PRKD3 was remarkably over-expressed in gastric cancer and colorectal cancer cell lines (Fig. 1 A). And this result was quantified according to the gray value (Fig. 1 B). In order to carry out further experiments, we then chose two representative gastrointestinal tumor cell lines (AGS and HT29) and used siRNA to knockdown PRKD3 . Realtime PCR results showed that the knockdown efficiency is remarkable (Fig. 1 C and 1 D). And the outcome was confirmed by western blot (Fig. 1 E and 1 F). The expression level of PRKD3 can be significantly knocked down by siRNA. 3.2 PRKD3 kinase contributes to tumor cell proliferation and metastasis We were then interested in whether the up-regulated PRKD3 is critical to the tumorigenesis of the gastrointestinal tumors. An in vitro , cell proliferation assay was then carried out by CCK-8. The result showed that the proliferation of both AGS and HT29 was significantly reduced after PRKD3 knockdown (Fig. 2 A and 2 B). Then we used transwell assay to verify whether PRKD3 can affect tumor metastasis or not. The result suggested that knock down PRKD3 can dramatically suppress tumor metastasis (Fig. 2 C and 2 D). Furthermore, we used soft agar assay to analyze the clone formation ability of AGS and HT29 in vitro . As we supposed, once the PRKD3 was knocked down, the clones were much smaller (Fig. 2 E). And colony count statistics demonstrated that significant reduction ultimately occurred in the PRKD3 -disturbed cells (Fig. 2 F). These results suggest that knockdown PRKD3 can significantly reduce the proliferation and clone formation ability. 3.3 lncRNA ROR is highly expressed in gastrointestinal cancer cells and its knockdown suppresses malignant phenotypes During the previous study, we have demonstrated that long noncoding RNA ROR is a crucial tumorigenic factor in gastrointestinal tumors[ 25 ]. Next, we examined the expression pattern of the long noncoding RNA ROR (lncRNA ROR ) in gastrointestinal cancer cell models. Semi-quantitative RT–PCR showed that ROR was readily detectable in multiple gastrointestinal cancer cell lines, including AGS and HT29, whereas its expression was relatively low in fibroblasts (Fig. 3 A). To investigate its functional significance, ROR was silenced in AGS and HT29 cells using shRNA, and efficient knockdown was confirmed by RT–PCR and qRT–PCR (Fig. 3 B and 3 C). Functionally, ROR depletion significantly inhibited cell proliferation in both AGS and HT29 cells, as assessed by CCK-8 assays (Fig. 3 D). In addition, ROR knockdown markedly suppressed tumor metastasis in transwell assay (Fig. 3 E and 3 F). Consistently, anchorage-independent growth in soft agar was substantially impaired after ROR silencing (Fig. 3 G). Collectively, these results indicate that ROR is aberrantly expressed in gastrointestinal cancer cells and promotes proliferative and transformation-associated phenotypes in vitro . 3.4 The abnormally expressed PRKD3 is caused by long noncoding RNA ROR Given the pro-tumorigenic effects of ROR , we next sought to identify downstream effectors that may mediate ROR -driven phenotypes. Because PRKD3 is upregulated and contributes to gastrointestinal cancer cell growth, we wonder whether there is a relationship between ROR and PRKD3 . Then, we examined whether PRKD3 expression is regulated by ROR . Notably, ROR knockdown resulted in a significant decrease in PRKD3 mRNA levels in both AGS and HT29 cells (Fig. 4 A and 4 B). This reduction was further validated at the protein level by western blot, which showed markedly diminished PRKD3 abundance upon ROR silencing (Fig. 4 C and 4 D). These findings support a regulatory axis in which aberrant ROR expression sustains PRKD3 upregulation in gastrointestinal cancer cells, providing a rationale for subsequent mechanistic investigations into how ROR controls PRKD3 . 3.5 ROR abolishes the trimethylation of H3K27 at PRKD3 promoter In order to find out the reason why PRKD3 could be affected by ROR , we carried out a chromatin immunoprecipitation assay (ChIP). We chose three analyzing sites upstream the PRKD3 transcription start site (TSS). Site C is in the PRKD3 promoter region, site B is nearby the promoter region and site A is far from the PRKD3 promoter which is a negative control (Fig. 5 A). After testing the H3K27 methylation status of these three sites, we found that ROR decreases H3K27me3 levels at the PRKD3 promoter (Fig. 5 B). In AGS and HT29, the ROR was highly expressed which leads to the demethylation of H3K27me3 of the PRKD3 promoter site. Once the ROR was knockdown, the methylation level of H3K27me3 at PRKD3 promoter site became high and this can lead to the repression of PRKD3 (Fig. 5 C). However, these phenomena cannot be observed at sit A and site B (Fig. 5 B). These results demonstrate that ROR can abolish histone H3K27 methylation of the PRKD3 gene which lead to the abnormal expression of PRKD3 . Discussion During the past several decades, extensive efforts have been devoted to elucidating the molecular mechanisms underlying tumorigenesis and to identifying effective therapeutic targets [ 1 , 26 ]. Gastrointestinal cancers remain among the most prevalent malignancies worldwide and have therefore been intensively investigated at the genetic, epigenetic, and signaling levels [ 6 , 27 ]. Despite the rapid progress in cancer biology, only a limited proportion of candidate targets have successfully translated into clinically applicable therapies. One major challenge is that many potential targets lack “druggability,” making them difficult to modulate effectively in clinical settings. Consequently, increasing attention has been directed toward molecular targets whose pharmacological inhibitors can be readily developed or optimized [ 28 ]. Protein kinases represent one of the most attractive classes of such targets. Because of their well-defined catalytic domains and regulatory mechanisms, kinase inhibitors have become an important category of targeted anti-cancer drugs in precision oncology [ 29 ]. Moreover, kinases frequently function as central nodes in signaling networks controlling cell proliferation, survival, and metastasis, and their dysregulation—including overexpression, mutation, or genomic rearrangement—can play critical roles in tumorigenesis [ 30 – 32 ]. Therefore, identifying kinase-related oncogenic mechanisms remains highly relevant for therapeutic development. PRKD3 represents one such protein kinase. It belongs to the protein kinase D family which function as serine/threonine kinases [ 33 ]. During previous studies, researchers found that PRKD3 has been involved in a variety of functions. For example, PRKD3 was reported to negatively regulate human airway epithelial barrier formation and integrity through down-regulation of claudin-1 [ 19 ]. Besides, Ryvkin V et al. found that loss of PRKD3 resulted in a progressive proliferation defect, loss of clonogenicity and diminished tissue regenerative ability in human keratinocytes [ 34 ]. These phenomena suggest that the disorder of PRKD3 may cause the uncontrollability of cell growth and metastasis which can be remarkably observed in tumors. Then researchers found that PRKD3 contributes to the proliferation and malignant growth of prostate cancer and breast cancer cells [ 35 – 37 ]. In this study, we found that PRKD3 is abnormally expressed in gastric cancer and colorectal cancer. And knock down the expression of PRKD3 to eliminate this change in gastric cancer and colorectal cancer can significantly suppress their proliferation and metastasis. These findings suggest that PRKD3 is a potential target for curing gastrointestinal cancer. According to the previous studies, we fund that many researches have studied the targets and downstream signaling pathways of PRKD3 [ 38 ]. However, there are only a few studies tried to investigate the mechanism of the abnormal expression of PRKD3 in tumors. He J et al. found that snail-activated long non-coding RNA PCA3 can up-regulate PRKD3 expression by miR-1261 sponging in prostate cancer cells[ 37 ]. According to their study, we found that long noncoding RNAs may affect the rearrangement of PRKD3 . Interestingly, in our previous study, we demonstrated that ROR lncRNA which acts as a decoy oncoRNA plays an important regulatory role in tumorigenesis of several tumors including gastrointestinal cancers [ 25 ]. Recent studies have increasingly highlighted the critical regulatory roles of long noncoding RNAs (lncRNAs) in human diseases, particularly in cancer initiation and progression [ 39 – 41 ]. Aberrant expression of lncRNAs can profoundly influence cellular processes such as proliferation, apoptosis, migration, and metastasis by modulating transcriptional networks and epigenetic landscapes [ 42 ]. In the present study, we found that the lncRNA ROR positively regulates PRKD3 expression in gastrointestinal cancer cells, including gastric and colorectal cancer models. Mechanistically, upregulation of ROR was associated with a significant reduction of the repressive histone modification H3K27me3 at the PRKD3 promoter region, leading to transcriptional activation of PRKD3 . Conversely, silencing ROR restored H3K27 trimethylation at the PRKD3 promoter and consequently suppressed PRKD3 expression. Histone methylation represents one of the most important epigenetic regulatory mechanisms controlling gene transcription [ 43 ]. Distinct histone methylation marks exert different regulatory effects on chromatin activity. For example, trimethylation of H3K4 and H3K36 is generally associated with transcriptional activation, whereas H3K9me3 and H3K27me3 are classical repressive marks that contribute to chromatin condensation and gene silencing [ 44 ]. Notably, H3K27me3 is catalyzed by the polycomb repressive complex 2 (PRC2) and can be dynamically removed by histone demethylases such as KDM6A and KDM6B, allowing precise epigenetic control of gene expression[ 45 ]. Our findings suggest that aberrant ROR expression may disrupt the regulatory balance of H3K27 methylation machinery at the PRKD3 promoter, thereby facilitating H3K27me3 demethylation and promoting PRKD3 activation. Importantly, restoring ROR expression levels reversed this epigenetic alteration and suppressed PRKD3 transcription, ultimately inhibiting tumor cell growth and transformation. Together, these observations support a model in which lncRNA-mediated epigenetic remodeling contributes to PRKD3 dysregulation and gastrointestinal tumorigenesis. In summary, our results reveal a novel target PRKD3 for curing gastrointestinal tumors. And we found a novel mechanism in which the trimethylation of H3K27 affected by ROR lncRNA can cause the expression level of PRKD3 in tumor. Most importantly, since PRKD3 is a kinase which allow us to find targeted drugs easily, it is possible for researchers to design a new targeted drug to cure patients with gastrointestinal cancer in the future. Declarations Acknowledgments None Funding This work was supported by the Science and Technology Commission of Shanghai, China (20DZ2270800). Author information Authors and Affiliations Department of Ophthalmology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200001, China. Yitang Liu, Jie Yang & Jiayan Fan State Key Laboratory of Eye Health, Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai 200001, China. Yitang Liu, Jie Yang & Jiayan Fan Contributions In this article, J.Y.F., Y.T.L. and J.Y. designed and performed the experiments and drafted the manuscript; J.Y.F. discussed and revised the manuscript. J.Y.F., J.Y. and Y.T.L. were the originators of the concept of this article and wrote and approved the manuscript. All authors approved this manuscript. Corresponding authors: Correspondence to Jie Yang and Jiayan Fan. Ethics declarations Ethics approval and consent to participate: Not applicable. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper can be obtained from the corresponding author upon reasonable request. References Sung, H., et al., Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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Supplementary Files Supplementarymaterials.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 20 Apr, 2026 Reviewers agreed at journal 19 Apr, 2026 Reviewers invited by journal 19 Apr, 2026 Editor assigned by journal 10 Apr, 2026 Submission checks completed at journal 08 Apr, 2026 First submitted to journal 01 Apr, 2026 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-9294400","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626648002,"identity":"52a3863a-d5e0-4175-a07f-fc4b44f8b588","order_by":0,"name":"Yitang Liu","email":"","orcid":"","institution":"Shanghai Ninth People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yitang","middleName":"","lastName":"Liu","suffix":""},{"id":626648003,"identity":"c40f6249-2f7b-4d64-ad3d-6ab7d54af307","order_by":1,"name":"Jie Yang","email":"","orcid":"","institution":"Shanghai Ninth People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Yang","suffix":""},{"id":626648004,"identity":"03ac6925-9778-427a-ab07-abf3986d4417","order_by":2,"name":"Jiayan Fan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDACZijNz8CQAKIZG4jWItnAkNhAnBYYMDgAUU1YC99x3sOveWru2G0+f+D5Yx4GG9kNB5ifPcCnRfIwX5o1z7FnydtuJCQ28zCkGW84wGZugNc9h3nMjHnYDieb3WAAaTmcuOEAD5sEYS3/Dicb9x8AaflPlBbjx7xth+0MGMAOO0BYiyTQFsa5fYcTJIB+mTnHINl45mE2M7xa+M6fMf7w5tthe/7+Mwkf3lTYyfYdb36GVwvDAQY2KR4GUDTyJADdyYCIXDxamD/+YGCwZ2BgP0BI7SgYBaNgFIxQAACue03kCIIy8QAAAABJRU5ErkJggg==","orcid":"","institution":"Shanghai Ninth People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Jiayan","middleName":"","lastName":"Fan","suffix":""}],"badges":[],"createdAt":"2026-04-01 16:24:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9294400/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9294400/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108003532,"identity":"587cf81c-b182-41bc-ae75-7e25c6829245","added_by":"auto","created_at":"2026-04-28 12:25:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":183503,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression in cancer cells and knockdown of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e PRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in AGS and HT29\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. Western blot analysis showing PRKD3 protein expression in normal fibroblasts and cancer cell lines, including gastric cancer (AGS) and colorectal cancer (SW620, 1299, and HT29). β-Actin was used as the loading control.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. Quantification of PRKD3 protein expression levels normalized to β-Actin based on the gray value analysis of the western blot bands shown in panel A. PRKD3 expression in cancer cell lines was significantly higher than that in fibroblasts. Data are presented as mean ± SD. **P \u0026lt; 0.01 versus fibroblast.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC and D. qRT–PCR analysis of relative \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mRNA expression in AGS and HT29 cells transfected with si\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e or negative control siRNA (NC). *P \u0026lt; 0.05 versus Control.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE and F. Western blot analysis of PRKD3 protein levels in AGS and HT29 cells after si\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transfection; β-actin was used as the loading control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9294400/v1/c0c1b3e0c63e02925f36f95b.png"},{"id":108003738,"identity":"d432ef46-b8b3-4e3b-a0d8-7f0d67668e51","added_by":"auto","created_at":"2026-04-28 12:25:39","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":537806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e knockdown suppresses proliferation, metastasis and clonogenic growth in AGS and HT29 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA and B.\u003c/strong\u003e \u003cstrong\u003eCCK-8 assay showing growth curves (growth index) of AGS and HT29 cells in the Control, negative control (NC), and si\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e groups at the indicated time points.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e \u003cstrong\u003eRepresentative images of transwell migration assays in AGS and HT29 cells following \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e knockdown. Cells that migrated to the lower surface of the membrane were fixed and stained with crystal violet.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. Quantitative analysis of the transwell assay in AGS and HT29 cells. Knockdown of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e significantly reduced the metastatic ability of both AGS and HT29 cells compared with the control and NC groups. Data are presented as relative OD value. **P \u0026lt; 0.01 versus control.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. Representative images of soft-agar colony formation in AGS and HT29 cells in the indicated groups.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. Quantification of colony formation in the soft agar assay. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e knockdown markedly decreased the number of colonies formed by AGS and HT29 cells compared with the control and NC groups. Data are presented as mean ± SD. **P \u0026lt; 0.01 versus control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9294400/v1/8b80218b303cfc6363ffa5b1.jpeg"},{"id":108003531,"identity":"18842b94-b0cf-49ef-9520-018e1c8343ec","added_by":"auto","created_at":"2026-04-28 12:25:05","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":625191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eROR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression and functional impact of its knockdown in cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e \u003cstrong\u003eSemi-quantitative RT–PCR analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eROR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression in fibroblast, iPSC, ASPC, AGS, HT29, and HRT18 cells; β-actin served as an internal control.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. RT–PCR verification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eROR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eknockdown in AGS and HT29 cells (WT, shROR, and Mock groups); GAPDH served as an internal control.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. qRT–PCR quantification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eROR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eexpression and knockdown efficiency in the indicated groups. Data are presented as mean ± SD from at least three independent experiments. *P \u0026lt; 0.05 versus WT.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. CCK-8 assays showing growth curves (growth index) of AGS and HT29 cells in WT, sh\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eROR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and Mock groups at the indicated time points.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. Representative images of transwell assays in AGS and HT29 cells in the WT, sh\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eROR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and Mock groups. Cells that traversed the membrane were fixed and stained with crystal violet.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. Quantification of migrated cells in transwell assays. Cells were counted in multiple randomly selected fields per insert. Data are presented as mean ± SD from at least three independent experiments. *P \u0026lt; 0.05 versus WT.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG. Representative images of soft-agar colony formation in AGS and HT29 cells.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9294400/v1/5893f619a01207a0016cfd83.jpeg"},{"id":108003755,"identity":"505c47a9-dd7e-4345-906f-eebaba732e5a","added_by":"auto","created_at":"2026-04-28 12:25:43","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":204797,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression is downregulated after \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eROR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eknockdown in gastrointestinal cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA and B. qRT–PCR analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mRNA levels in AGS (A) and HT29 (B) cells following \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eROR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e knockdown (WT, sh\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eROR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and Mock), with fibroblast included as a reference where indicated; expression was normalized to an internal control gene (as described in Methods). Data are presented as mean ± SD from at least three independent experiments. *P \u0026lt; 0.05 versus WT.\u003cbr\u003e\nC and D. Western blot analysis of PRKD3 protein levels in AGS (C) and HT29 (D) cells after \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eROR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e knockdown; β-actin was used as the loading control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9294400/v1/72ddbb21cadc151756242246.jpeg"},{"id":108003586,"identity":"8d995c7f-ec11-4c9f-b846-b924828c55a0","added_by":"auto","created_at":"2026-04-28 12:25:20","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":365052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eROR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e knockdown restores H3K27me3 enrichment at the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoter.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. Schematic illustration of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKD3 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epromoter region and the locations of ChIP-PCR/qPCR amplicons. Three sites upstream of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePRKD3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e TSS were analyzed: site A (distal negative-control region), site B (proximal region), and site C (promoter region). Primer pairs (P1–P6) and expected product sizes are indicated.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. Representative ChIP-PCR results showing H3K27me3 occupancy at sites A, B, and C in AGS and HT29 cells (WT, sh\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eROR\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and Mock), with fibroblasts included as a reference. IgG served as a negative immunoprecipitation control and input DNA was used as loading control.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. ChIP-qPCR quantification of H3K27me3 enrichment (normalized to input) at the indicated sites in AGS and HT29 cells. Data are presented as mean ± SD from at least three independent experiments. **P \u0026lt; 0.01 versus WT.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9294400/v1/882a2ec39f51e4c0c1694773.jpeg"},{"id":108007458,"identity":"3436e7c4-199e-4896-be3c-f105f575f9b5","added_by":"auto","created_at":"2026-04-28 13:00:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2248096,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9294400/v1/3a8cf69b-3174-4e34-9a61-a89b7368064e.pdf"},{"id":108003506,"identity":"707f0cec-3baa-4599-8ba1-f2d7aec05996","added_by":"auto","created_at":"2026-04-28 12:25:02","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19195,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9294400/v1/380b3f41266b376bc5d5f94c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The rearrangement of PRKD3 caused by long noncoding RNA mediated H3K27me3 demethylation led to tumorigenesis","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eGastrointestinal malignancies, including gastric cancer and colorectal cancer (CRC), are among the most prevalent cancers worldwide and remain leading contributors to global cancer incidence and mortality profiles [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In recent years, the overall burden of gastrointestinal cancers has remained high, with marked geographic heterogeneity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. According to the World Health Organization estimates for 2022, CRC accounted for approximately 1.9\u0026nbsp;million new cases and more than 0.9\u0026nbsp;million deaths, ranking as the second leading cause of cancer-related mortality globally. In China, gastric cancer and CRC likewise constitute major public health challenges. Owing to the large population base, accelerated ageing, and widespread exposure to modifiable risk factors, the national burden of gastric cancer remains higher than the global average [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Meanwhile, the incidence of CRC continues to rise in China, and both its incidence and mortality have consistently ranked among the top five cancers in the population [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough treatment strategies for gastrointestinal cancers have evolved substantially\u0026mdash;supported by advances in systemic therapy, optimization of therapeutic decision-making, and expanded options for targeted therapy, immunotherapy, and locoregional surgical interventions\u0026mdash;clinical outcomes remain suboptimal for a considerable proportion of patients [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, multimodal regimens frequently lead to treatment-related complications, which may compromise tolerability, quality of life, and overall effectiveness. Therefore, the development of more effective and safer therapeutic agents for gastrointestinal cancers remains a critical research priority. In this context, elucidating the molecular and pathogenic mechanisms underlying gastrointestinal tumorigenesis is essential for identifying actionable targets and developing novel therapeutic approaches for clinical translation.\u003c/p\u003e \u003cp\u003eTumorigenesis is a complicated process and caused by multiple factors. Although somatic mutations and clonal expansions are widespread even in histologically normal tissues, only a small fraction ultimately progresses to cancer. This observation suggests that additional driving events are required to propel these clones toward an irreversible, highly heterogeneous, and invasive state. Historically, our ability to detect such events was limited; however, with the advent of innovative technologies, the defining features of transforming cells and their interactions with the surrounding microenvironment are being progressively elucidated [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In recent years, increasing emphasis has been placed on environmental cancer risk factors and epigenetic alterations, which can profoundly influence early clonal expansion and malignant evolution without directly inducing new mutations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCanonical oncogenic signaling pathways\u0026mdash;including Wnt/β-catenin, JAK/STAT3, RAS\u0026ndash;RAF\u0026ndash;MEK\u0026ndash;ERK (MAPK), and PI3K/AKT/mTOR\u0026mdash;have been extensively shown to play central roles in gastrointestinal tumor biology. These pathways regulate key processes such as tumor cell proliferation, apoptosis evasion, epithelial\u0026ndash;mesenchymal transition (EMT), invasion and metastasis, and resistance to therapy, thereby driving tumor initiation and progression [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, beyond well-established tumor-suppressive microRNAs (miRNAs) such as the miR-34 and let-7 families [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], accumulating evidence indicates that multiple miRNAs (e.g., miR-21) contribute to gastrointestinal carcinogenesis by modulating cell-cycle control, apoptosis, and migration-related networks and by reshaping the tumor microenvironment [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Notably, exosome-associated miRNAs have attracted substantial attention for their roles in promoting tumor progression and for their potential utility as noninvasive diagnostic and prognostic biomarkers [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, most of these target genes are hardly to be used in clinic for the lacking of targeted drugs. For this reason, researchers now prefer to focus on the targets which can easily design and produce small molecule compound[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. And kinase is one of such an ideal research target [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProtein kinase D3 (PRKD3) is a serine/threonine kinase of the protein kinase D (PKD) family and acts as a diacylglycerol-responsive effector downstream of PKC signaling. Beyond its established roles in epithelial barrier regulation and intracellular vesicle trafficking [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], \u003cem\u003ePRKD3\u003c/em\u003e integrates multiple oncogenic cues to regulate transcriptional programs, metabolism, and cell fate decisions. Accumulating evidence over the past decade supports an oncogenic role of \u003cem\u003ePRKD3\u003c/em\u003e across solid tumors, where it promotes proliferation, survival, migration, and invasion and has been proposed as a druggable target [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Importantly, emerging studies have begun to implicate \u003cem\u003ePRKD3\u003c/em\u003e in gastrointestinal malignancies. In gastric cancer, \u003cem\u003ePRKD3\u003c/em\u003e overexpression has been linked to enhanced tumor cell growth and metabolic reprogramming through the PRKD3\u0026ndash;NF-κB/p65\u0026ndash;PFKFB3 axis[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and recent work further suggests that \u003cem\u003ePRKD3\u003c/em\u003e may influence cell-cycle progression and malignant phenotypes[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, we focused on this kinase to further elucidate its potential roles in gastrointestinal malignancies.\u003c/p\u003e \u003cp\u003eIn this study, we have attempted to identify the function of \u003cem\u003ePRKD3\u003c/em\u003e in gastrointestinal tumors and find out its transcriptional regulatory mechanism from long noncoding RNA level. Here, we show that the demethylation of \u003cem\u003ePRKD3\u003c/em\u003e promoter mediated by \u003cem\u003eROR\u003c/em\u003e determine the transcriptional activation of \u003cem\u003ePRKD3\u003c/em\u003e and affect tumorigenesis.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell culture\u003c/h2\u003e \u003cp\u003eThe AGS cell line, derived from human gastric adenocarcinoma, and the HT29 cell line, derived from human colorectal adenocarcinoma, were obtained from the National Collection of Authenticated Cell Cultures, China. All cell lines were utilized in the cytotoxicity assessment of the MVs derived from L. buchneri. The cell lines were developed in a liquid medium consisting of RPMI1640 and DMEM (Gibco, Carlsbad, USA), which was further enriched with 10% Fetal Bovine Serum (Gibco, Carlsbad, USA), 100 \u0026micro;/mL penicillin (Gibco, Carlsbad, USA), and 100 \u0026micro;g/mL streptomycin (Gibco, Carlsbad, USA). The cells were cultivated at 37\u0026deg;C in a humid environment containing 5% CO2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 RNA extraction, reverse transcription-PCR analysis and Real-time RT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRI-REAGENT (Invitrogen, USA), according to the manufacturer\u0026rsquo;s instructions, and cDNA was synthesized using the PrimeScript RT reagent kit (Takara, Japan). Real-time RT-PCR reactions were performed with SYBR Premix Ex Taq (TaKaRa) in a Bio-Rad CFX96 Real-Time PCR System (Bio-rad, Hercules, CA). GAPDH and β-actin was used as an internal control. The primer sequences are listed in \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Western blot analysis\u003c/h2\u003e \u003cp\u003eCells were harvested at the indicated time and washed twice with cold PBS. Cell extracts were prepared with lysis buffer and centrifuged at 13,000 g for 30 min at 4\u0026deg;C. Protein samples were separated by sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis in 7.5% (wt/vol) polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Membranes were used to immunoblot with anti-PRKD3 (Proteintech, USA). Membranes were incubated with a secondary antibody conjugated to a fluorescent tag. The bands were visualized using the Odyssey infrared Imaging System (LI-COR, Lincoln, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 shRNA Retrovirus and Selection\u003c/h2\u003e \u003cp\u003e \u003cem\u003eROR\u003c/em\u003e shRNA-mir constructs and verified nonsilencing shRNA were purchased from Open Biosystems (Lafayette, CO) (\u003cb\u003eSupplemental Table S2\u003c/b\u003e). Selection was performed by incubating with 4\u0026micro;g/ml puromycin for 2 weeks; the clones were screened for GFP expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Small Interfering RNA\u003c/h2\u003e \u003cp\u003e \u003cem\u003ePRKD3\u003c/em\u003e knockdown was achieved using small interfering RNA (siRNA). (\u003cb\u003eSupplemental Table S3\u003c/b\u003e). Cells were seeded at 200,000 cells per well in six-well plates and transfected using Lipofectamine 2000 (Invitrogen) in Opti-MEM I Reduced Serum Medium with 50 nM siRNA (Invitrogen). Forty-eight hours posttransfection, cells were harvested in TRIzol for RNA isolation (Invitrogen) or lysed in radioimmunoprecipitation assay (RIPA) lysis buffer for western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 MTT Assay\u003c/h2\u003e \u003cp\u003eThe MTT assay was performed as previously described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In short, cells were seeded at 5,000 cells per well in flat-bottomed 96-well plates. At the end of the incubation time, 20 \u0026micro;l of 5 mg/ml 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma, St Louis, MO) in PBS was added to each well. After 4 hours, the media was discarded, and the cells were lysed with 100 \u0026micro;l of dimethylsulfoxide. The cells were incubated for a further 30 minutes at 37\u0026deg;C with gentle shaking. The optical density was determined with a microplate reader at 490 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Transwell assay\u003c/h2\u003e \u003cp\u003eThe migratory ability of the cells was evaluated using a 24-well transwell system (Corning, USA) equipped with 8-\u0026micro;m pore size polycarbonate filters according to the manufacturer's instructions. The upper compartment contained 10,000 cells suspended in appropriate medium supplemented with 5% fetal bovine serum, and the lower compartment contained 10% fetal bovine serum. After the appropriate days of incubation at 37\u0026deg;C, the lower compartment was fixed with 100% methanol and stained with 0.1% crystal violet before photographing. The crystal violet was washed down from the migrated cells using 100 \u0026micro;l of 33% acetic acid. The absorbance of the washed down liquid was determined with a microplate reader at 490 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Soft agar tumor formation assay\u003c/h2\u003e \u003cp\u003eA soft agar colony formation assay was performed in 6-well plates. One milliliter of the bottom layer comprised of 0.6% agar in complete medium was spread in each 6-well plates. A total of 20,000 cells were suspended in 1.0 milliliter of complete medium containing 0.3% agar and was seeded into each well. The cultures were fed every 3 to 4 days with 300 \u0026micro;l of complete medium for 3\u0026ndash;4 weeks. For quantification, the colonies grown in soft agar were stained with 0.005% crystal violet. The size of the colonies was determined using Adobe Photoshop.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Chromatin immunoprecipitation (ChIP)\u003c/h2\u003e \u003cp\u003eChIP assays were performed as previously described. One hundred million cells were fixed with 1% formaldehyde and sonicated for 8 min (10 s on and 15 s off) on ice with a 2-mm microtip at 40% output control and 90% duty cycle settings. The sonicated chromatin (1 ml) was clarified by centrifugation and snap-frozen in liquid nitrogen. To perform ChIP, sonicated chromatin (150 \u0026micro;l) was diluted 10-fold and added protein G-agarose (60 \u0026micro;l) (Millipore, USA) with shaking at 4\u0026deg;C for 2 h. Then briefly centrifuged at 1000 rpm for 5 min at 4\u0026deg;C and the supernatant was collected into a new tube. Antibodies to SMC1 and MED12 were obtained from Abcam Inc (Abcam, USA) nd added to the supernatant overnight at 4\u0026deg;C. PureProteome\u0026trade; Protein A and Protein G Magnetic Beads (60 \u0026micro;l) (Millipore, USA) were used to pull down the protein at 4\u0026deg;C for 6 h. The DNA that was released from the bound chromatin after cross-linking reversal and proteinase K treatment was precipitated and diluted in 100 \u0026micro;l of 0.2 M glycine.\u003c/p\u003e \u003cp\u003eThe PCRs (3 \u0026micro;l under liquid wax) contained 2 \u0026micro;l ChIP (or input) DNA, 0.5 mM appropriate primer pairs, 50 \u0026micro;M deoxynucleotide triphosphate and 0.2 U Klen-Taq I (Ab Peptides, USA). The PCR conditions were 95\u0026deg;C for 5 min, followed by 34 cycles of 95\u0026deg;C for 30 s, 30 s of optimal annealing temperature, and 72\u0026deg;C for 30s of extension (\u003cb\u003eSupplemental Table S4\u003c/b\u003e). The PCR products were separated on 8% polyacrylamide\u0026ndash;urea gel.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 \u003cem\u003ePRKD3\u003c/em\u003e is significantly over-expressed in gastrointestinal tumors\u003c/h2\u003e\n \u003cp\u003eTo identify the function of \u003cem\u003ePRKD3\u003c/em\u003e in gastrointestinal tumors, we firstly tried to exam its expression level in several gastrointestinal tumor cell lines (AGS, SW620, 1299 and HT29). According to the results of western blot, we found that \u003cem\u003ePRKD3\u003c/em\u003e was remarkably over-expressed in gastric cancer and colorectal cancer cell lines (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). And this result was quantified according to the gray value (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In order to carry out further experiments, we then chose two representative gastrointestinal tumor cell lines (AGS and HT29) and used siRNA to knockdown \u003cem\u003ePRKD3\u003c/em\u003e. Realtime PCR results showed that the knockdown efficiency is remarkable (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). And the outcome was confirmed by western blot (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The expression level of \u003cem\u003ePRKD3\u003c/em\u003e can be significantly knocked down by siRNA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 \u003cem\u003ePRKD3\u003c/em\u003e kinase contributes to tumor cell proliferation and metastasis\u003c/h2\u003e\n \u003cp\u003eWe were then interested in whether the up-regulated \u003cem\u003ePRKD3\u003c/em\u003e is critical to the tumorigenesis of the gastrointestinal tumors. An in \u003cem\u003evitro\u003c/em\u003e, cell proliferation assay was then carried out by CCK-8. The result showed that the proliferation of both AGS and HT29 was significantly reduced after \u003cem\u003ePRKD3\u003c/em\u003e knockdown (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Then we used transwell assay to verify whether \u003cem\u003ePRKD3\u003c/em\u003e can affect tumor metastasis or not. The result suggested that knock down \u003cem\u003ePRKD3\u003c/em\u003e can dramatically suppress tumor metastasis (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Furthermore, we used soft agar assay to analyze the clone formation ability of AGS and HT29 in \u003cem\u003evitro\u003c/em\u003e. As we supposed, once the \u003cem\u003ePRKD3\u003c/em\u003e was knocked down, the clones were much smaller (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). And colony count statistics demonstrated that significant reduction ultimately occurred in the \u003cem\u003ePRKD3\u003c/em\u003e-disturbed cells (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results suggest that knockdown \u003cem\u003ePRKD3\u003c/em\u003e can significantly reduce the proliferation and clone formation ability.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 lncRNA \u003cem\u003eROR\u003c/em\u003e is highly expressed in gastrointestinal cancer cells and its knockdown suppresses malignant phenotypes\u003c/h2\u003e\n \u003cp\u003eDuring the previous study, we have demonstrated that long noncoding RNA \u003cem\u003eROR\u003c/em\u003e is a crucial tumorigenic factor in gastrointestinal tumors[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Next, we examined the expression pattern of the long noncoding RNA \u003cem\u003eROR\u003c/em\u003e (lncRNA \u003cem\u003eROR\u003c/em\u003e) in gastrointestinal cancer cell models. Semi-quantitative RT\u0026ndash;PCR showed that \u003cem\u003eROR\u003c/em\u003e was readily detectable in multiple gastrointestinal cancer cell lines, including AGS and HT29, whereas its expression was relatively low in fibroblasts (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To investigate its functional significance, \u003cem\u003eROR\u003c/em\u003e was silenced in AGS and HT29 cells using shRNA, and efficient knockdown was confirmed by RT\u0026ndash;PCR and qRT\u0026ndash;PCR (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Functionally, \u003cem\u003eROR\u003c/em\u003e depletion significantly inhibited cell proliferation in both AGS and HT29 cells, as assessed by CCK-8 assays (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In addition, \u003cem\u003eROR\u003c/em\u003e knockdown markedly suppressed tumor metastasis in transwell assay (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Consistently, anchorage-independent growth in soft agar was substantially impaired after \u003cem\u003eROR\u003c/em\u003e silencing (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Collectively, these results indicate that \u003cem\u003eROR\u003c/em\u003e is aberrantly expressed in gastrointestinal cancer cells and promotes proliferative and transformation-associated phenotypes in \u003cem\u003evitro\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 The abnormally expressed \u003cem\u003ePRKD3\u003c/em\u003e is caused by long noncoding RNA \u003cem\u003eROR\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eGiven the pro-tumorigenic effects of \u003cem\u003eROR\u003c/em\u003e, we next sought to identify downstream effectors that may mediate \u003cem\u003eROR\u003c/em\u003e-driven phenotypes. Because \u003cem\u003ePRKD3\u003c/em\u003e is upregulated and contributes to gastrointestinal cancer cell growth, we wonder whether there is a relationship between \u003cem\u003eROR\u003c/em\u003e and \u003cem\u003ePRKD3\u003c/em\u003e. Then, we examined whether \u003cem\u003ePRKD3\u003c/em\u003e expression is regulated by \u003cem\u003eROR\u003c/em\u003e. Notably, \u003cem\u003eROR\u003c/em\u003e knockdown resulted in a significant decrease in \u003cem\u003ePRKD3\u003c/em\u003e mRNA levels in both AGS and HT29 cells (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This reduction was further validated at the protein level by western blot, which showed markedly diminished PRKD3 abundance upon \u003cem\u003eROR\u003c/em\u003e silencing (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These findings support a regulatory axis in which aberrant \u003cem\u003eROR\u003c/em\u003e expression sustains \u003cem\u003ePRKD3\u003c/em\u003e upregulation in gastrointestinal cancer cells, providing a rationale for subsequent mechanistic investigations into how \u003cem\u003eROR\u003c/em\u003e controls \u003cem\u003ePRKD3\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 \u003cem\u003eROR\u003c/em\u003e abolishes the trimethylation of H3K27 at \u003cem\u003ePRKD3\u003c/em\u003e promoter\u003c/h2\u003e\n \u003cp\u003eIn order to find out the reason why \u003cem\u003ePRKD3\u003c/em\u003e could be affected by \u003cem\u003eROR\u003c/em\u003e, we carried out a chromatin immunoprecipitation assay (ChIP). We chose three analyzing sites upstream the \u003cem\u003ePRKD3\u003c/em\u003e transcription start site (TSS). Site C is in the \u003cem\u003ePRKD3\u003c/em\u003e promoter region, site B is nearby the promoter region and site A is far from the \u003cem\u003ePRKD3\u003c/em\u003e promoter which is a negative control (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). After testing the H3K27 methylation status of these three sites, we found that \u003cem\u003eROR\u003c/em\u003e decreases H3K27me3 levels at the \u003cem\u003ePRKD3\u003c/em\u003e promoter (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In AGS and HT29, the \u003cem\u003eROR\u003c/em\u003e was highly expressed which leads to the demethylation of H3K27me3 of the \u003cem\u003ePRKD3\u003c/em\u003e promoter site. Once the \u003cem\u003eROR\u003c/em\u003e was knockdown, the methylation level of H3K27me3 at \u003cem\u003ePRKD3\u003c/em\u003e promoter site became high and this can lead to the repression of \u003cem\u003ePRKD3\u003c/em\u003e (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). However, these phenomena cannot be observed at sit A and site B (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These results demonstrate that \u003cem\u003eROR\u003c/em\u003e can abolish histone H3K27 methylation of the \u003cem\u003ePRKD3\u003c/em\u003e gene which lead to the abnormal expression of \u003cem\u003ePRKD3\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDuring the past several decades, extensive efforts have been devoted to elucidating the molecular mechanisms underlying tumorigenesis and to identifying effective therapeutic targets [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Gastrointestinal cancers remain among the most prevalent malignancies worldwide and have therefore been intensively investigated at the genetic, epigenetic, and signaling levels [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Despite the rapid progress in cancer biology, only a limited proportion of candidate targets have successfully translated into clinically applicable therapies. One major challenge is that many potential targets lack \u0026ldquo;druggability,\u0026rdquo; making them difficult to modulate effectively in clinical settings. Consequently, increasing attention has been directed toward molecular targets whose pharmacological inhibitors can be readily developed or optimized [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Protein kinases represent one of the most attractive classes of such targets. Because of their well-defined catalytic domains and regulatory mechanisms, kinase inhibitors have become an important category of targeted anti-cancer drugs in precision oncology [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Moreover, kinases frequently function as central nodes in signaling networks controlling cell proliferation, survival, and metastasis, and their dysregulation\u0026mdash;including overexpression, mutation, or genomic rearrangement\u0026mdash;can play critical roles in tumorigenesis [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, identifying kinase-related oncogenic mechanisms remains highly relevant for therapeutic development.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePRKD3\u003c/em\u003e represents one such protein kinase. It belongs to the protein kinase D family which function as serine/threonine kinases [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. During previous studies, researchers found that \u003cem\u003ePRKD3\u003c/em\u003e has been involved in a variety of functions. For example, \u003cem\u003ePRKD3\u003c/em\u003e was reported to negatively regulate human airway epithelial barrier formation and integrity through down-regulation of claudin-1 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Besides, Ryvkin V et al. found that loss of \u003cem\u003ePRKD3\u003c/em\u003e resulted in a progressive proliferation defect, loss of clonogenicity and diminished tissue regenerative ability in human keratinocytes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These phenomena suggest that the disorder of \u003cem\u003ePRKD3\u003c/em\u003e may cause the uncontrollability of cell growth and metastasis which can be remarkably observed in tumors. Then researchers found that \u003cem\u003ePRKD3\u003c/em\u003e contributes to the proliferation and malignant growth of prostate cancer and breast cancer cells [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, we found that \u003cem\u003ePRKD3\u003c/em\u003e is abnormally expressed in gastric cancer and colorectal cancer. And knock down the expression of \u003cem\u003ePRKD3\u003c/em\u003e to eliminate this change in gastric cancer and colorectal cancer can significantly suppress their proliferation and metastasis. These findings suggest that \u003cem\u003ePRKD3\u003c/em\u003e is a potential target for curing gastrointestinal cancer.\u003c/p\u003e \u003cp\u003eAccording to the previous studies, we fund that many researches have studied the targets and downstream signaling pathways of \u003cem\u003ePRKD3\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, there are only a few studies tried to investigate the mechanism of the abnormal expression of \u003cem\u003ePRKD3\u003c/em\u003e in tumors. He J et al. found that snail-activated long non-coding RNA PCA3 can up-regulate \u003cem\u003ePRKD3\u003c/em\u003e expression by miR-1261 sponging in prostate cancer cells[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. According to their study, we found that long noncoding RNAs may affect the rearrangement of \u003cem\u003ePRKD3\u003c/em\u003e. Interestingly, in our previous study, we demonstrated that \u003cem\u003eROR\u003c/em\u003e lncRNA which acts as a decoy oncoRNA plays an important regulatory role in tumorigenesis of several tumors including gastrointestinal cancers [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have increasingly highlighted the critical regulatory roles of long noncoding RNAs (lncRNAs) in human diseases, particularly in cancer initiation and progression [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Aberrant expression of lncRNAs can profoundly influence cellular processes such as proliferation, apoptosis, migration, and metastasis by modulating transcriptional networks and epigenetic landscapes [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In the present study, we found that the lncRNA \u003cem\u003eROR\u003c/em\u003e positively regulates \u003cem\u003ePRKD3\u003c/em\u003e expression in gastrointestinal cancer cells, including gastric and colorectal cancer models. Mechanistically, upregulation of \u003cem\u003eROR\u003c/em\u003e was associated with a significant reduction of the repressive histone modification H3K27me3 at the \u003cem\u003ePRKD3\u003c/em\u003e promoter region, leading to transcriptional activation of \u003cem\u003ePRKD3\u003c/em\u003e. Conversely, silencing \u003cem\u003eROR\u003c/em\u003e restored H3K27 trimethylation at the \u003cem\u003ePRKD3\u003c/em\u003e promoter and consequently suppressed \u003cem\u003ePRKD3\u003c/em\u003e expression.\u003c/p\u003e \u003cp\u003eHistone methylation represents one of the most important epigenetic regulatory mechanisms controlling gene transcription [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Distinct histone methylation marks exert different regulatory effects on chromatin activity. For example, trimethylation of H3K4 and H3K36 is generally associated with transcriptional activation, whereas H3K9me3 and H3K27me3 are classical repressive marks that contribute to chromatin condensation and gene silencing [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Notably, H3K27me3 is catalyzed by the polycomb repressive complex 2 (PRC2) and can be dynamically removed by histone demethylases such as KDM6A and KDM6B, allowing precise epigenetic control of gene expression[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our findings suggest that aberrant \u003cem\u003eROR\u003c/em\u003e expression may disrupt the regulatory balance of H3K27 methylation machinery at the \u003cem\u003ePRKD3\u003c/em\u003e promoter, thereby facilitating H3K27me3 demethylation and promoting \u003cem\u003ePRKD3\u003c/em\u003e activation. Importantly, restoring \u003cem\u003eROR\u003c/em\u003e expression levels reversed this epigenetic alteration and suppressed \u003cem\u003ePRKD3\u003c/em\u003e transcription, ultimately inhibiting tumor cell growth and transformation. Together, these observations support a model in which lncRNA-mediated epigenetic remodeling contributes to \u003cem\u003ePRKD3\u003c/em\u003e dysregulation and gastrointestinal tumorigenesis.\u003c/p\u003e \u003cp\u003eIn summary, our results reveal a novel target \u003cem\u003ePRKD3\u003c/em\u003e for curing gastrointestinal tumors. And we found a novel mechanism in which the trimethylation of H3K27 affected by \u003cem\u003eROR\u003c/em\u003e lncRNA can cause the expression level of \u003cem\u003ePRKD3\u003c/em\u003e in tumor. Most importantly, since \u003cem\u003ePRKD3\u003c/em\u003e is a kinase which allow us to find targeted drugs easily, it is possible for researchers to design a new targeted drug to cure patients with gastrointestinal cancer in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Science and Technology Commission of Shanghai, China (20DZ2270800).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Ophthalmology, Ninth People\u0026apos;s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200001, China.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYitang Liu, Jie Yang \u0026amp; Jiayan Fan\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eState Key Laboratory of Eye Health, Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai 200001, China.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYitang Liu, Jie Yang \u0026amp; Jiayan Fan\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this article,\u0026nbsp;J.Y.F., Y.T.L. and J.Y. designed and performed the experiments and drafted the manuscript; J.Y.F. discussed and revised the manuscript. J.Y.F., J.Y. and Y.T.L. were the originators of the concept of this article and wrote and approved the manuscript. All authors approved this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Jie Yang and Jiayan Fan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions in the paper can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung, H., et al., Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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Nature, 2011. 469(7330): p. 343\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1038/nature09784\u003c/span\u003e\u003cspan address=\"10.1038/nature09784\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"holistic-integrative-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Holistic Integrative Oncology](https://link.springer.com/journal/44178)","snPcode":"44178","submissionUrl":"https://submission.springernature.com/new-submission/44178/3","title":"Holistic Integrative Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Long non-coding RNAs, PRKD3, H3K27me3 demethylation, tumorigenesis","lastPublishedDoi":"10.21203/rs.3.rs-9294400/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9294400/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGastrointestinal cancer, one of the most prevalent and lethal malignancies worldwide, poses a significant global health burden. Protein kinase D3 (PRKD3) is a member of protein kinase which was demonstrated to be critical in growth regulation. However, the potential role of \u003cem\u003ePRKD3\u003c/em\u003e in gastrointestinal tumors remains unexplored. Here, we found that \u003cem\u003ePRKD3\u003c/em\u003e was abnormally expressed in gastric cancer and colorectal cancer cells. Silencing of \u003cem\u003ePRKD3\u003c/em\u003e markedly inhibits tumor cell proliferation and metastasis, underscoring its functional importance. Notably, this rearrangement of \u003cem\u003ePRKD3\u003c/em\u003e in gastrointestinal cancer was caused by long noncoding RNA ROR (ROR lncRNA) mediated H3K27me3 demethylation. Bring down the expression of \u003cem\u003eROR\u003c/em\u003e lncRNA can restore the trimethylation of H3K27 at \u003cem\u003ePRKD3\u003c/em\u003e promoter region and lead to the suppression of \u003cem\u003ePRKD3\u003c/em\u003e. Our findings reveal a novel mechanism by which the \u003cem\u003eROR\u003c/em\u003e lncRNA mediated H3K27me3 demethylation may rearrange the transcription of \u003cem\u003ePRKD3\u003c/em\u003e and lead to tumorigenesis in gastrointestinal tumor. Collectively, we represent a novel kinase target \u003cem\u003ePRKD3\u003c/em\u003e, offering new avenues for the development of targeted therapies against gastrointestinal cancers.\u003c/p\u003e","manuscriptTitle":"The rearrangement of PRKD3 caused by long noncoding RNA mediated H3K27me3 demethylation led to tumorigenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-28 12:24:04","doi":"10.21203/rs.3.rs-9294400/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-21T01:12:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156837728982255662874325835742798595996","date":"2026-04-20T03:08:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-20T02:42:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-10T09:05:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T14:07:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Holistic Integrative Oncology","date":"2026-04-01T16:10:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"holistic-integrative-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Holistic Integrative Oncology](https://link.springer.com/journal/44178)","snPcode":"44178","submissionUrl":"https://submission.springernature.com/new-submission/44178/3","title":"Holistic Integrative Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"516b9894-5198-496a-81ba-721f21cc3314","owner":[],"postedDate":"April 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T12:24:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-28 12:24:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9294400","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9294400","identity":"rs-9294400","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}