{"paper_id":"243ef8df-04ac-4400-a37e-80224f4ff491","body_text":"Expression of LINC00963 in Preoperative Liver Tissues of Biliary Atresia and Its Correlation with Postoperative Liver Fibrosis Progression | 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 Expression of LINC00963 in Preoperative Liver Tissues of Biliary Atresia and Its Correlation with Postoperative Liver Fibrosis Progression Chen Zhen, Ye Mao, Geng Yuanyuan, Li Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8443966/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 Objective： To investigate the expression level of long non-coding RNA (lncRNA) LINC00963 in preoperative liver tissues of biliary atresia (BA) and its correlation with postoperative liver fibrosis progression and clinical prognostic indicators, so as to provide experimental basis for screening potential molecular targets for predicting postoperative liver fibrosis in BA. Methods： A total of 28 preoperative liver tissue samples from BA children who underwent Kasai operation combined with Roux-en-Y anastomosis in our hospital from January 2022 to June 2024 were collected. According to the Ishak liver fibrosis score at 1-year postoperative follow-up, the children were divided into mild fibrosis progression group (10 cases, Ishak score 1-2) and moderate-severe fibrosis progression group (18 cases, Ishak score 3-6). Another 8 normal liver tissue samples resected intraoperatively from children with biliary hypoplasia were selected as the control group. High-throughput RNA sequencing was used to screen differentially expressed lncRNAs, and quantitative real-time PCR (qPCR) was employed to verify the expression level of LINC00963. Pearson correlation analysis was performed to explore the correlation between the expression level of LINC00963 in preoperative BA liver tissues and postoperative liver fibrosis degree, serum bile acid level, and jaundice resolution time. Results： High-throughput RNA sequencing showed that the expression level of LINC00963 in preoperative BA liver tissues was significantly higher than that in the control group, which was 3.72 times that of the control group (P<0.01). The qPCR verification results were consistent with the sequencing results (P<0.01). The expression level of LINC00963 in preoperative liver tissues of children in the moderate-severe fibrosis progression group was significantly higher than that in the mild fibrosis progression group (P<0.05). Pearson correlation analysis revealed that the expression level of LINC00963 in preoperative BA liver tissues was positively correlated with postoperative Ishak liver fibrosis score (r=0.68, P<0.01) and serum bile acid level (r=0.71, P<0.01), while negatively correlated with postoperative jaundice resolution time (r=-0.63, P<0.01). Conclusion ： LINC00963 is highly expressed in preoperative BA liver tissues, and its expression level is closely correlated with postoperative liver fibrosis progression, cholestasis, and jaundice resolution prognosis. It can serve as a potential molecular marker for predicting postoperative liver fibrosis progression in BA, providing a reference for early intervention. Biliary Atresia Postoperative Liver Fibrosis Long Non-Coding RNA LINC00963 Clinical Correlation Molecular Marker 1. Introduction Biliary atresia (BA) is the most common severe hepatobiliary disease in infants, characterized by progressive fibrotic obstruction of intrahepatic and extrahepatic bile ducts. Surgical treatment (Kasai operation combined with Roux-en-Y anastomosis) is a key method to improve the prognosis of children [ 1 ]. However, more than 50% of children still require liver transplantation due to progressive postoperative liver fibrosis, which has become a core bottleneck restricting the long-term survival of BA children [ 2 ]. Abnormal activation of hepatic stellate cells (HSC) is the central link in the occurrence and development of liver fibrosis. The special microenvironment formed by postoperative cholestasis and immune disorders in BA can drive HSC activation by regulating downstream molecular networks, but the key molecules and mechanisms in this regulatory network have not been fully clarified [ 3 ]. Long non-coding RNAs (lncRNAs) are a class of RNA molecules with a length of more than 200nt and no protein-coding function, which can participate in pathophysiological processes such as liver fibrosis and tumorigenesis through multiple ways including ceRNA, transcriptional regulation, and chromatin modification [ 4 ]. Previous studies have confirmed that lncRNAs such as H19 and NEAT1 can participate in liver fibrosis by regulating HSC activation. However, there are few studies on the correlation between preoperative lncRNA expression and postoperative fibrosis prognosis in BA, and the role and clinical significance of LINC00963 in this correlation remain unclear [ 5 ]. Given the difficulty in obtaining postoperative liver tissues of BA, this study detected the expression level of LINC00963 in preoperative liver tissues and analyzed its correlation with postoperative clinicopathological indicators, so as to provide basic data for further exploring its molecular mechanism in regulating BA liver fibrosis and clinical transformation and application. 2. Materials and Methods 2.1 Study Subjects and Sample Collection A total of 28 preoperative BA children who underwent Kasai operation combined with Roux-en-Y anastomosis in the Department of Pediatric Surgery of our hospital from January 2022 to June 2024 were selected, including 16 males and 12 females, with an average surgical age of (90.5 ± 25.3) days. All children were diagnosed by preoperative liver tissue pathological examination and followed up for more than 1 year after operation. According to the Ishak liver fibrosis score at the end of follow-up, they were divided into mild fibrosis progression group (10 cases, Ishak score 1–2) and moderate-severe fibrosis progression group (18 cases, Ishak score 3–6). Eight children with biliary hypoplasia who underwent surgical treatment in our hospital during the same period were selected as the control group, including 5 males and 3 females, with an average surgical age of (88.2 ± 23.7) days. The liver tissues resected intraoperatively were confirmed to have no fibrosis or inflammation by pathological examination. Preoperative liver tissue samples of all BA children were collected during Kasai operation for detecting LINC00963 expression, avoiding the clinical operation difficulty of obtaining postoperative tissues. Inclusion criteria: ① Consistent with the clinical diagnostic criteria of BA or biliary hypoplasia, confirmed by surgery and pathological examination; ② Complete postoperative follow-up data of BA children, available for obtaining clinical indicators such as 1-year postoperative liver fibrosis score, serum bile acid level, and jaundice resolution time; ③ Informed consent signed by the children's guardians, and the study was approved by the hospital ethics committee. Exclusion criteria: ① Complicated with other liver diseases, congenital heart disease, immune deficiency diseases, etc.; ② Received glucocorticoids, immunosuppressants, etc., before operation; ③ Quality problems existed during sample collection and detection. 2.2 Main Reagents and Instruments TRIzol reagent (Invitrogen, USA), reverse transcription kit and qPCR kit (TaKaRa, Japan), primers for LINC00963 and internal reference gene GAPDH (Sangon Biotech Co., Ltd., Shanghai), high-throughput RNA sequencing service was provided by Novogene Technology Co., Ltd., Beijing. Quantitative fluorescence PCR instrument (Applied Biosystems, USA), high-speed refrigerated centrifuge (Eppendorf, Germany), nucleic acid electrophoresis instrument (Beijing LiuYi Instrument Factory). 2.3 Experimental Methods 2.3.1 Total RNA Extraction and High-Throughput Sequencing Total RNA was extracted from liver tissue samples using TRIzol reagent. RNA purity was detected by Nanodrop 2000 (A260/A280 ratio 1.8-2.0), and RNA integrity was verified by agarose gel electrophoresis. RNA samples from 3 cases in the control group, 3 cases in the mild fibrosis progression group, and 3 cases in the moderate-severe fibrosis progression group were selected for high-throughput RNA sequencing to screen differentially expressed lncRNAs. The screening criteria were |log2FC|≥1.5 and P < 0.05, and LINC00963 was identified as a candidate molecule. 2.3.2 qPCR Verification of LINC00963 Expression Level Total RNA was reverse-transcribed into cDNA according to the instructions of the reverse transcription kit, and qPCR reaction was performed with cDNA as the template. Reaction system: 10µL of 2×SYBR Premix Ex Taq, 0.4µL of upstream and downstream primers each, 2µL of cDNA template, and supplemented with ddH2O to 20µL. Reaction conditions: pre-denaturation at 95℃ for 30s; denaturation at 95℃ for 5s, annealing and extension at 60℃ for 30s, a total of 40 cycles; finally, melting curve analysis was performed to verify primer specificity. GAPDH was used as the internal reference gene, and the relative expression level of LINC00963 was calculated by the 2^(-ΔΔCt) method. Primer sequences: LINC00963 upstream: 5'-GCTGCTGTTGCTGTTGTTGT-3', downstream: 5'-CAGCAGCAGCAGCAGTATGA-3'; GAPDH upstream: 5'-GAAGGTGAAGGTCGGAGTC-3', downstream: 5'-GAAGATGGTGATGGGATTTC-3'. Each sample was set with 3 replicate wells, and the experiment was repeated 3 times. 2.3.3 Collection of Clinical Indicators and Correlation Analysis Postoperative clinical data of BA children were collected, including serum bile acid level at 1 month after operation, postoperative jaundice resolution time (time from operation to complete resolution of skin and scleral jaundice), and Ishak liver fibrosis score at 1 year after operation. Pearson correlation analysis was used to explore the correlation between the relative expression level of LINC00963 in preoperative BA liver tissues and the above postoperative clinical indicators. 2.4 Statistical Analysis SPSS 26.0 statistical software was used for data analysis. Measurement data were expressed as mean ± standard deviation (x ± s). One-way analysis of variance was used for comparison among multiple groups, and LSD-t test was used for pairwise comparison. Correlation analysis was performed by Pearson correlation analysis. P < 0.05 was considered statistically significant. 3. Results 3.1 Expression Level of LINC00963 in Preoperative BA Liver Tissues High-throughput RNA sequencing showed that the expression of LINC00963 in preoperative BA liver tissues was significantly up-regulated compared with the control group, and the relative expression level was 3.72 times that of the control group, with a statistically significant difference (P < 0.01). The qPCR verification results were consistent with the sequencing results: the relative expression level of LINC00963 in preoperative BA liver tissues (2.85 ± 0.63) was significantly higher than that in the control group (0.76 ± 0.21), with a statistically significant difference (t = 12.37, P < 0.01). 3.2 Expression Difference of LINC00963 in Preoperative Liver Tissues of BA Children with Different Postoperative Fibrosis Progression Degrees The relative expression level of LINC00963 in preoperative liver tissues of BA children in the moderate-severe fibrosis progression group (3.52 ± 0.58) was significantly higher than that in the mild fibrosis progression group (1.87 ± 0.42), with a statistically significant difference (t = 6.89, P < 0.05), indicating that the higher the expression level of LINC00963 in preoperative BA liver tissues, the more severe the postoperative liver fibrosis progression. 3.3 Correlation Between LINC00963 Expression Level and Postoperative Clinical Indicators of BA Children Pearson correlation analysis showed that the relative expression level of LINC00963 in preoperative BA liver tissues was positively correlated with postoperative Ishak liver fibrosis score (r = 0.68, P < 0.01) and postoperative serum bile acid level (r = 0.71, P < 0.01), and negatively correlated with postoperative jaundice resolution time (r=-0.63, P < 0.01). 4. Discussion The progression mechanism of postoperative liver fibrosis in BA is complex, involving multiple links such as cholestasis, immune disorders, HSC activation, and extracellular matrix deposition. Identifying key molecular targets regulating this process is of great significance for improving the postoperative prognosis of BA children [ 6 ]. As key gene regulatory molecules, lncRNAs play an important role in the occurrence and development of liver fibrosis. Previous studies have confirmed that lncRNAs can form ceRNA networks by sponging microRNAs, regulate the expression of genes related to HSC activation, and thus affect the process of liver fibrosis [ 7 ]. However, it is difficult to obtain postoperative BA liver tissues through clinical operations, which limits the dynamic study of postoperative molecular mechanisms. Preoperative liver tissues can be easily collected during Kasai operation, and predicting postoperative prognosis based on preoperative molecular expression is more clinically translatable. At present, there are few studies on the correlation between preoperative lncRNA expression and postoperative fibrosis progression in BA. This study focuses on LINC00963, explores its expression in preoperative BA liver tissues and its correlation with postoperative prognosis, laying a foundation for subsequent mechanism research and clinical application. This study found through high-throughput sequencing and qPCR verification that LINC00963 is highly expressed in preoperative BA liver tissues, and its expression level is closely correlated with the degree of postoperative liver fibrosis progression. The expression level of LINC00963 in preoperative liver tissues of children in the moderate-severe fibrosis progression group is significantly higher than that in the mild progression group, suggesting that LINC00963 may play a triggering role in the initiation and progression of BA liver fibrosis, and its preoperative expression level can reflect the potential progression ability of liver tissue fibrosis. This finding is not isolated. Combined with previous studies, as a functionally conserved lncRNA, the abnormal expression of LINC00963 has been confirmed to be involved in the pathophysiological processes of various diseases, and its core action mode has certain commonalities, providing an important reference for analyzing its function in BA liver fibrosis. Existing studies have shown that the abnormal high expression of LINC00963 is mainly concentrated in the field of tumors, and it mostly regulates tumor occurrence and development as an oncogene. In bladder cancer, LINC00963 is significantly up-regulated in tumor tissues, and its expression level is positively correlated with histological grade and negatively correlated with patient survival rate. Mechanistically, it sponges miR-766-3p through the ceRNA mode, relieves the inhibition of downstream target gene MTA1, and then promotes the proliferation, migration, and invasion of tumor cells, confirming the core function of LINC00963-mediated ceRNA regulatory axis [ 10 ]. In hepatocellular carcinoma, LINC00963 can accelerate tumor progression by activating the PI3K/AKT signaling pathway, which is exactly one of the key pathways regulating hepatic stellate cell (HSC) activation and promoting liver fibrosis [ 11 ]. In addition, LINC00963 has also been confirmed to promote epithelial-mesenchymal transition of ovarian cancer cells, enhance radioresistance of breast cancer, and play a pro-carcinogenic role in cutaneous squamous cell carcinoma and melanoma, which is generally associated with poor prognosis. Its core regulatory logic revolves around \"abnormal high expression - regulating downstream molecular axis - promoting cell activation/proliferation\" [ 10 , 12 , 13 ]. Although there are no direct studies on LINC00963 in liver fibrosis, its functional characteristics revealed in previous studies are highly consistent with the core pathological mechanism of liver fibrosis, and it can directly regulate HSC activation through multiple pathways. As the \"core effector cell\" of liver fibrosis, HSC mainly stores vitamin A in the quiescent state. After being stimulated by pathological signals, it is activated, transformed into a myofibroblast-like phenotype, extensively expresses fibrosis markers such as α-SMA and ColⅠ/Ⅲ, and secretes extracellular matrix (ECM) to form fibrotic deposition [ 3 ]. Abnormal activation of the PI3K/AKT pathway is a key pathway driving the transformation of HSC from quiescent state to activated state and inhibiting its apoptosis. After activation, this pathway can up-regulate α-SMA expression through downstream molecules such as mTOR and GSK-3β, enhancing the activated phenotype of HSC [ 11 ]; meanwhile, ceRNA network regulation is also an important molecular mechanism of HSC activation. Many lncRNAs can relieve the inhibition of target genes related to HSC activation by sponging microRNAs [ 7 ]. This study found that LINC00963 is highly expressed in preoperative BA liver tissues and positively correlated with postoperative fibrosis progression. Combined with its characteristics of activating the PI3K/AKT pathway in hepatocellular carcinoma and mediating ceRNA regulation in bladder cancer, it is speculated that it can directly target HSC activation: on the one hand, it directly induces HSC activation and proliferation by activating the PI3K/AKT pathway, up-regulates the expression of markers such as α-SMA and ColⅠ, and accelerates ECM deposition; on the other hand, it continues the ceRNA regulation mode, sponges specific microRNAs (such as miR-148a-3p initially associated in this study or miR-766-3p involved in previous tumor studies), regulates the expression of core target genes related to HSC activation such as Smad3 and TGF-β1, forming a cascade regulatory effect. In addition, there is already cholestasis and inflammatory microenvironment in preoperative BA liver tissues. This microenvironment can induce the high expression of LINC00963, and the high-expressed LINC00963 can continuously activate HSC through the above pathways, forming a positive feedback loop of \"microenvironment-LINC00963-HSC activation\", which also explains why the preoperative expression level of LINC00963 can predict postoperative fibrosis progression, providing clinical phenotypic support for the direct correlation between them. The combination of such cross-disease functional conservation and HSC activation specificity provides a clear direction for subsequent mechanism verification of this study, and also suggests that LINC00963 may be a key molecular node connecting liver inflammation, HSC activation, and fibrosis progression. Further comparison of the differences between previous studies and this study shows that existing literatures focus on the pro-carcinogenic role of LINC00963 in tumors, while this study is the first to correlate it with preoperative BA liver tissue expression and postoperative liver fibrosis prognosis, filling the research gap of this molecule in children's cholestatic liver diseases. As a unique hepatobiliary disease in infants, the progression of BA liver fibrosis has multiple characteristics such as cholestasis, immune disorders, and bile duct epithelial injury, which is significantly different from the pathological microenvironment of adult liver diseases and tumors [ 1 , 9 ]. This study found that LINC00963 is closely correlated with postoperative serum bile acid level and jaundice resolution time, suggesting that its expression may be regulated by the cholestatic microenvironment. This feature has not been mentioned in previous tumor studies, which is speculated to be a specific functional correlation of LINC00963 in BA liver fibrosis, and also provides a basis for subsequent exploration of the regulatory chain of \"cholestasis-LINC00963-fibrosis\". This study was carried out based on preoperative liver tissue samples, effectively avoiding the clinical pain point of difficult postoperative liver tissue acquisition, and the research design is more in line with clinical practice. Combined with the previous research background of LINC00963 and the results of this study, it can be speculated that the abnormal high expression of LINC00963 is an early molecular event of BA liver fibrosis. It becomes an important driver of postoperative fibrosis progression by directly regulating HSC activation, so its preoperative expression level can predict the risk of postoperative fibrosis progression in advance. There is already bile duct fibrosis and inflammatory microenvironment disorder in preoperative BA liver tissues. This pathological state can regulate the transcriptional expression of LINC00963 through upstream signaling pathways (such as NF-κB and FXR pathways) [ 9 ], and LINC00963 directly targets key molecules of HSC activation through its conserved ceRNA regulation mode or PI3K/AKT pathway activation function, continuously driving postoperative HSC activation and liver fibrosis progression [ 10 , 11 ]. It is worth noting that the target gene MTA1 of LINC00963 and downstream molecules of the PI3K/AKT pathway in previous studies have been confirmed to be directly related to HSC activation: MTA1 can inhibit ECM degradation by regulating the expression of matrix metalloproteinase (MMPs) family members, and promote HSC to secrete ColⅠ, accelerating fibrotic deposition; the PI3K/AKT pathway can directly up-regulate the transcriptional activity of α-SMA in HSC, enhancing its myofibroblast phenotype [ 3 , 11 ], which further confirms the molecular feasibility of LINC00963 participating in BA liver fibrosis by directly regulating HSC activation. Subsequent studies can further verify the transcriptional regulation of LINC00963 by NF-κB and FXR through CHIP experiments, clarify the specific microRNAs and target genes bound by LINC00963 in BA liver fibrosis with the help of RNA pulldown and dual-luciferase reporter gene experiments, and detect the expression changes of activation markers such as α-SMA and ColⅠ by overexpressing/silencing LINC00963 in in vitro HSC models, so as to verify its direct regulatory effect on HSC activation, improve the complete regulatory network of \"upstream microenvironment-LINC00963-downstream molecular axis-HSC activation-liver fibrosis\", and provide theoretical support for targeted intervention. This study has certain limitations: ① The sample size is relatively small and it is a single-center study, so the results may have bias. Subsequent studies need to expand the sample size and carry out multi-center studies for verification; ② Only the preoperative expression of LINC00963 and its correlation with postoperative prognosis were initially explored, and the specific molecular mechanism of its regulation of BA liver fibrosis has not been clarified; ③ The expression level of LINC00963 in children's serum was not detected, so its application value as a non-invasive marker cannot be clarified. In the future, in vitro cell experiments and animal model experiments can be used to further clarify the mechanism of LINC00963, and the expression level of this molecule in serum can be detected to explore its clinical efficacy as a non-invasive prognostic marker, further improving the clinical transformation value of the research. In conclusion, LINC00963 is highly expressed in preoperative BA liver tissues, and its expression level is closely correlated with postoperative liver fibrosis progression, cholestasis, and jaundice resolution prognosis. It can serve as a potential molecular marker for predicting postoperative liver fibrosis progression in BA. This study avoids the difficulty of obtaining postoperative liver tissues, and the research design is in line with clinical practice, providing a new direction for the early prediction and targeted intervention of postoperative liver fibrosis in BA. Declarations Ethical approval This study was approved by the Ethical Committee of the Capital Institute of Pediatrics, China. Conflict of interest The authors report no conflict of interest. Funding No funding to be declared. Compliance with ethical standards Author Contribution Li Xu contributed to study design, data collection, data analysis. Chen Zhen contributed to manuscript drafting. Ye Mao contributed to critical revision of the manuscript. Geng Yuanyuan contributed to study design, and critical revision of the manuscript. All authors approved the final version of the manuscript. Acknowledgments We thank those patients who supported our study and authors who provided us with the full-text. References Li Y, Wang X, Zhang L et al (2020) Biliary atresia: current status and future perspectives[J]. World J Gastroenterol 26(45):7123–7140 Chen J, Liu Y, Zhao H et al (2021) Risk factors for progressive liver fibrosis after Kasai operation in biliary atresia[J]. J Pediatr Surg 56(8):1352–1357 Zhang H, Li M, Wang Q et al (2022) Hepatic stellate cell activation in biliary atresia: mechanisms and therapeutic potential[J]. Int J Mol Sci 23(12):6689 Wang L, Zhang Y, Li C et al (2020) Long non-coding RNAs in liver fibrosis: mechanisms and clinical implications[J]. Cell Death Dis 11(5):321 Cai W, Li J, Zhang H et al (2019) LncRNA H19 promotes biliary atresia-related liver fibrosis via sponging Let-7 to regulate HMGA2 expression[J]. Hepatology 70(4):1423–1438 Zhao Y, Chen L, Liu J et al (2023) Molecular mechanisms of liver fibrosis in biliary atresia: a review[J]. Front Pediatr 11:1089672 Li S, Wang H, Zhang Z et al (2021) LncRNA NEAT1/miR-204/SMAD3 axis regulates hepatic stellate cell activation and liver fibrosis[J]. J Cell Physiol 236(7):5021–5034 Wang X, Li Y, Chen J et al (2022) Serum bile acid levels as a prognostic marker for biliary atresia after Kasai operation[J]. Pediatr Neonatol 63(3):289–294 Liu H, Zhang L, Wang Q et al (2021) Postoperative immune microenvironment disorders in biliary atresia: implications for liver fibrosis[J]. Int J Immunopathol Pharmacol 35:20587384211023456 Zhang Y, Li M, Wang H et al (2022) LINC00963 Functions as an Oncogene in Bladder Cancer by Regulating the miR-766-3p/MTA1 Axis[J]. J Cancer Res Clin Oncol 148(10):2895–2908 He X, Zhang J, Liu C et al (2021) LINC00963 promotes hepatocellular carcinoma progression via activating PI3K/AKT signaling pathway[J]. Mol Med Rep 24(4):589 Sun W, Li Y, Zhao L et al (2020) LINC00963 enhances ovarian cancer proliferation and epithelial-mesenchymal transition via targeting miR-143-3p[J]. J Cell Biochem 121(8):4123–4132 Zhou C, Wang F, Chen J et al (2023) LINC00963 predicts poor prognosis and promotes tumorigenesis in cutaneous squamous cell carcinoma[J]. Eur J Dermatol 33(2):e138–e146 Additional Declarations No competing interests reported. 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. 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19:09:53\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":592029,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8443966/v1/e3bb8cbb-309c-4ce8-af97-753bf7241938.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Expression of LINC00963 in Preoperative Liver Tissues of Biliary Atresia and Its Correlation with Postoperative Liver Fibrosis Progression\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eBiliary atresia (BA) is the most common severe hepatobiliary disease in infants, characterized by progressive fibrotic obstruction of intrahepatic and extrahepatic bile ducts. Surgical treatment (Kasai operation combined with Roux-en-Y anastomosis) is a key method to improve the prognosis of children [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. However, more than 50% of children still require liver transplantation due to progressive postoperative liver fibrosis, which has become a core bottleneck restricting the long-term survival of BA children [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Abnormal activation of hepatic stellate cells (HSC) is the central link in the occurrence and development of liver fibrosis. The special microenvironment formed by postoperative cholestasis and immune disorders in BA can drive HSC activation by regulating downstream molecular networks, but the key molecules and mechanisms in this regulatory network have not been fully clarified [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eLong non-coding RNAs (lncRNAs) are a class of RNA molecules with a length of more than 200nt and no protein-coding function, which can participate in pathophysiological processes such as liver fibrosis and tumorigenesis through multiple ways including ceRNA, transcriptional regulation, and chromatin modification [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Previous studies have confirmed that lncRNAs such as H19 and NEAT1 can participate in liver fibrosis by regulating HSC activation. However, there are few studies on the correlation between preoperative lncRNA expression and postoperative fibrosis prognosis in BA, and the role and clinical significance of LINC00963 in this correlation remain unclear [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Given the difficulty in obtaining postoperative liver tissues of BA, this study detected the expression level of LINC00963 in preoperative liver tissues and analyzed its correlation with postoperative clinicopathological indicators, so as to provide basic data for further exploring its molecular mechanism in regulating BA liver fibrosis and clinical transformation and application.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Study Subjects and Sample Collection\\u003c/h2\\u003e \\u003cp\\u003eA total of 28 preoperative BA children who underwent Kasai operation combined with Roux-en-Y anastomosis in the Department of Pediatric Surgery of our hospital from January 2022 to June 2024 were selected, including 16 males and 12 females, with an average surgical age of (90.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;25.3) days. All children were diagnosed by preoperative liver tissue pathological examination and followed up for more than 1 year after operation. According to the Ishak liver fibrosis score at the end of follow-up, they were divided into mild fibrosis progression group (10 cases, Ishak score 1\\u0026ndash;2) and moderate-severe fibrosis progression group (18 cases, Ishak score 3\\u0026ndash;6). Eight children with biliary hypoplasia who underwent surgical treatment in our hospital during the same period were selected as the control group, including 5 males and 3 females, with an average surgical age of (88.2\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;23.7) days. The liver tissues resected intraoperatively were confirmed to have no fibrosis or inflammation by pathological examination. Preoperative liver tissue samples of all BA children were collected during Kasai operation for detecting LINC00963 expression, avoiding the clinical operation difficulty of obtaining postoperative tissues.\\u003c/p\\u003e \\u003cp\\u003eInclusion criteria: ① Consistent with the clinical diagnostic criteria of BA or biliary hypoplasia, confirmed by surgery and pathological examination; ② Complete postoperative follow-up data of BA children, available for obtaining clinical indicators such as 1-year postoperative liver fibrosis score, serum bile acid level, and jaundice resolution time; ③ Informed consent signed by the children's guardians, and the study was approved by the hospital ethics committee. Exclusion criteria: ① Complicated with other liver diseases, congenital heart disease, immune deficiency diseases, etc.; ② Received glucocorticoids, immunosuppressants, etc., before operation; ③ Quality problems existed during sample collection and detection.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2 Main Reagents and Instruments\\u003c/h2\\u003e \\u003cp\\u003eTRIzol reagent (Invitrogen, USA), reverse transcription kit and qPCR kit (TaKaRa, Japan), primers for LINC00963 and internal reference gene GAPDH (Sangon Biotech Co., Ltd., Shanghai), high-throughput RNA sequencing service was provided by Novogene Technology Co., Ltd., Beijing. Quantitative fluorescence PCR instrument (Applied Biosystems, USA), high-speed refrigerated centrifuge (Eppendorf, Germany), nucleic acid electrophoresis instrument (Beijing LiuYi Instrument Factory).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Experimental Methods\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.1 Total RNA Extraction and High-Throughput Sequencing\\u003c/h2\\u003e \\u003cp\\u003eTotal RNA was extracted from liver tissue samples using TRIzol reagent. RNA purity was detected by Nanodrop 2000 (A260/A280 ratio 1.8-2.0), and RNA integrity was verified by agarose gel electrophoresis. RNA samples from 3 cases in the control group, 3 cases in the mild fibrosis progression group, and 3 cases in the moderate-severe fibrosis progression group were selected for high-throughput RNA sequencing to screen differentially expressed lncRNAs. The screening criteria were |log2FC|\\u0026ge;1.5 and P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, and LINC00963 was identified as a candidate molecule.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.2 qPCR Verification of LINC00963 Expression Level\\u003c/h2\\u003e \\u003cp\\u003eTotal RNA was reverse-transcribed into cDNA according to the instructions of the reverse transcription kit, and qPCR reaction was performed with cDNA as the template. Reaction system: 10\\u0026micro;L of 2\\u0026times;SYBR Premix Ex Taq, 0.4\\u0026micro;L of upstream and downstream primers each, 2\\u0026micro;L of cDNA template, and supplemented with ddH2O to 20\\u0026micro;L. Reaction conditions: pre-denaturation at 95℃ for 30s; denaturation at 95℃ for 5s, annealing and extension at 60℃ for 30s, a total of 40 cycles; finally, melting curve analysis was performed to verify primer specificity. GAPDH was used as the internal reference gene, and the relative expression level of LINC00963 was calculated by the 2^(-ΔΔCt) method. Primer sequences: LINC00963 upstream: 5'-GCTGCTGTTGCTGTTGTTGT-3', downstream: 5'-CAGCAGCAGCAGCAGTATGA-3'; GAPDH upstream: 5'-GAAGGTGAAGGTCGGAGTC-3', downstream: 5'-GAAGATGGTGATGGGATTTC-3'. Each sample was set with 3 replicate wells, and the experiment was repeated 3 times.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.3 Collection of Clinical Indicators and Correlation Analysis\\u003c/h2\\u003e \\u003cp\\u003ePostoperative clinical data of BA children were collected, including serum bile acid level at 1 month after operation, postoperative jaundice resolution time (time from operation to complete resolution of skin and scleral jaundice), and Ishak liver fibrosis score at 1 year after operation. Pearson correlation analysis was used to explore the correlation between the relative expression level of LINC00963 in preoperative BA liver tissues and the above postoperative clinical indicators.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4 Statistical Analysis\\u003c/h2\\u003e \\u003cp\\u003eSPSS 26.0 statistical software was used for data analysis. Measurement data were expressed as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation (x\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;s). One-way analysis of variance was used for comparison among multiple groups, and LSD-t test was used for pairwise comparison. Correlation analysis was performed by Pearson correlation analysis. P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 was considered statistically significant.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1 Expression Level of LINC00963 in Preoperative BA Liver Tissues\\u003c/h2\\u003e \\u003cp\\u003eHigh-throughput RNA sequencing showed that the expression of LINC00963 in preoperative BA liver tissues was significantly up-regulated compared with the control group, and the relative expression level was 3.72 times that of the control group, with a statistically significant difference (P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01). The qPCR verification results were consistent with the sequencing results: the relative expression level of LINC00963 in preoperative BA liver tissues (2.85\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.63) was significantly higher than that in the control group (0.76\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.21), with a statistically significant difference (t\\u0026thinsp;=\\u0026thinsp;12.37, P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003e3.2 Expression Difference of LINC00963 in Preoperative Liver Tissues of BA Children with Different Postoperative Fibrosis Progression Degrees\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe relative expression level of LINC00963 in preoperative liver tissues of BA children in the moderate-severe fibrosis progression group (3.52\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.58) was significantly higher than that in the mild fibrosis progression group (1.87\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.42), with a statistically significant difference (t\\u0026thinsp;=\\u0026thinsp;6.89, P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), indicating that the higher the expression level of LINC00963 in preoperative BA liver tissues, the more severe the postoperative liver fibrosis progression.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3 Correlation Between LINC00963 Expression Level and Postoperative Clinical Indicators of BA Children\\u003c/h2\\u003e \\u003cp\\u003ePearson correlation analysis showed that the relative expression level of LINC00963 in preoperative BA liver tissues was positively correlated with postoperative Ishak liver fibrosis score (r\\u0026thinsp;=\\u0026thinsp;0.68, P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) and postoperative serum bile acid level (r\\u0026thinsp;=\\u0026thinsp;0.71, P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01), and negatively correlated with postoperative jaundice resolution time (r=-0.63, P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eThe progression mechanism of postoperative liver fibrosis in BA is complex, involving multiple links such as cholestasis, immune disorders, HSC activation, and extracellular matrix deposition. Identifying key molecular targets regulating this process is of great significance for improving the postoperative prognosis of BA children [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. As key gene regulatory molecules, lncRNAs play an important role in the occurrence and development of liver fibrosis. Previous studies have confirmed that lncRNAs can form ceRNA networks by sponging microRNAs, regulate the expression of genes related to HSC activation, and thus affect the process of liver fibrosis [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. However, it is difficult to obtain postoperative BA liver tissues through clinical operations, which limits the dynamic study of postoperative molecular mechanisms. Preoperative liver tissues can be easily collected during Kasai operation, and predicting postoperative prognosis based on preoperative molecular expression is more clinically translatable. At present, there are few studies on the correlation between preoperative lncRNA expression and postoperative fibrosis progression in BA. This study focuses on LINC00963, explores its expression in preoperative BA liver tissues and its correlation with postoperative prognosis, laying a foundation for subsequent mechanism research and clinical application.\\u003c/p\\u003e \\u003cp\\u003eThis study found through high-throughput sequencing and qPCR verification that LINC00963 is highly expressed in preoperative BA liver tissues, and its expression level is closely correlated with the degree of postoperative liver fibrosis progression. The expression level of LINC00963 in preoperative liver tissues of children in the moderate-severe fibrosis progression group is significantly higher than that in the mild progression group, suggesting that LINC00963 may play a triggering role in the initiation and progression of BA liver fibrosis, and its preoperative expression level can reflect the potential progression ability of liver tissue fibrosis. This finding is not isolated. Combined with previous studies, as a functionally conserved lncRNA, the abnormal expression of LINC00963 has been confirmed to be involved in the pathophysiological processes of various diseases, and its core action mode has certain commonalities, providing an important reference for analyzing its function in BA liver fibrosis.\\u003c/p\\u003e \\u003cp\\u003eExisting studies have shown that the abnormal high expression of LINC00963 is mainly concentrated in the field of tumors, and it mostly regulates tumor occurrence and development as an oncogene. In bladder cancer, LINC00963 is significantly up-regulated in tumor tissues, and its expression level is positively correlated with histological grade and negatively correlated with patient survival rate. Mechanistically, it sponges miR-766-3p through the ceRNA mode, relieves the inhibition of downstream target gene MTA1, and then promotes the proliferation, migration, and invasion of tumor cells, confirming the core function of LINC00963-mediated ceRNA regulatory axis [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. In hepatocellular carcinoma, LINC00963 can accelerate tumor progression by activating the PI3K/AKT signaling pathway, which is exactly one of the key pathways regulating hepatic stellate cell (HSC) activation and promoting liver fibrosis [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. In addition, LINC00963 has also been confirmed to promote epithelial-mesenchymal transition of ovarian cancer cells, enhance radioresistance of breast cancer, and play a pro-carcinogenic role in cutaneous squamous cell carcinoma and melanoma, which is generally associated with poor prognosis. Its core regulatory logic revolves around \\\"abnormal high expression - regulating downstream molecular axis - promoting cell activation/proliferation\\\" [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eAlthough there are no direct studies on LINC00963 in liver fibrosis, its functional characteristics revealed in previous studies are highly consistent with the core pathological mechanism of liver fibrosis, and it can directly regulate HSC activation through multiple pathways. As the \\\"core effector cell\\\" of liver fibrosis, HSC mainly stores vitamin A in the quiescent state. After being stimulated by pathological signals, it is activated, transformed into a myofibroblast-like phenotype, extensively expresses fibrosis markers such as α-SMA and ColⅠ/Ⅲ, and secretes extracellular matrix (ECM) to form fibrotic deposition [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Abnormal activation of the PI3K/AKT pathway is a key pathway driving the transformation of HSC from quiescent state to activated state and inhibiting its apoptosis. After activation, this pathway can up-regulate α-SMA expression through downstream molecules such as mTOR and GSK-3β, enhancing the activated phenotype of HSC [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]; meanwhile, ceRNA network regulation is also an important molecular mechanism of HSC activation. Many lncRNAs can relieve the inhibition of target genes related to HSC activation by sponging microRNAs [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. This study found that LINC00963 is highly expressed in preoperative BA liver tissues and positively correlated with postoperative fibrosis progression. Combined with its characteristics of activating the PI3K/AKT pathway in hepatocellular carcinoma and mediating ceRNA regulation in bladder cancer, it is speculated that it can directly target HSC activation: on the one hand, it directly induces HSC activation and proliferation by activating the PI3K/AKT pathway, up-regulates the expression of markers such as α-SMA and ColⅠ, and accelerates ECM deposition; on the other hand, it continues the ceRNA regulation mode, sponges specific microRNAs (such as miR-148a-3p initially associated in this study or miR-766-3p involved in previous tumor studies), regulates the expression of core target genes related to HSC activation such as Smad3 and TGF-β1, forming a cascade regulatory effect. In addition, there is already cholestasis and inflammatory microenvironment in preoperative BA liver tissues. This microenvironment can induce the high expression of LINC00963, and the high-expressed LINC00963 can continuously activate HSC through the above pathways, forming a positive feedback loop of \\\"microenvironment-LINC00963-HSC activation\\\", which also explains why the preoperative expression level of LINC00963 can predict postoperative fibrosis progression, providing clinical phenotypic support for the direct correlation between them. The combination of such cross-disease functional conservation and HSC activation specificity provides a clear direction for subsequent mechanism verification of this study, and also suggests that LINC00963 may be a key molecular node connecting liver inflammation, HSC activation, and fibrosis progression.\\u003c/p\\u003e \\u003cp\\u003eFurther comparison of the differences between previous studies and this study shows that existing literatures focus on the pro-carcinogenic role of LINC00963 in tumors, while this study is the first to correlate it with preoperative BA liver tissue expression and postoperative liver fibrosis prognosis, filling the research gap of this molecule in children's cholestatic liver diseases. As a unique hepatobiliary disease in infants, the progression of BA liver fibrosis has multiple characteristics such as cholestasis, immune disorders, and bile duct epithelial injury, which is significantly different from the pathological microenvironment of adult liver diseases and tumors [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. This study found that LINC00963 is closely correlated with postoperative serum bile acid level and jaundice resolution time, suggesting that its expression may be regulated by the cholestatic microenvironment. This feature has not been mentioned in previous tumor studies, which is speculated to be a specific functional correlation of LINC00963 in BA liver fibrosis, and also provides a basis for subsequent exploration of the regulatory chain of \\\"cholestasis-LINC00963-fibrosis\\\".\\u003c/p\\u003e \\u003cp\\u003eThis study was carried out based on preoperative liver tissue samples, effectively avoiding the clinical pain point of difficult postoperative liver tissue acquisition, and the research design is more in line with clinical practice. Combined with the previous research background of LINC00963 and the results of this study, it can be speculated that the abnormal high expression of LINC00963 is an early molecular event of BA liver fibrosis. It becomes an important driver of postoperative fibrosis progression by directly regulating HSC activation, so its preoperative expression level can predict the risk of postoperative fibrosis progression in advance. There is already bile duct fibrosis and inflammatory microenvironment disorder in preoperative BA liver tissues. This pathological state can regulate the transcriptional expression of LINC00963 through upstream signaling pathways (such as NF-κB and FXR pathways) [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e], and LINC00963 directly targets key molecules of HSC activation through its conserved ceRNA regulation mode or PI3K/AKT pathway activation function, continuously driving postoperative HSC activation and liver fibrosis progression [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. It is worth noting that the target gene MTA1 of LINC00963 and downstream molecules of the PI3K/AKT pathway in previous studies have been confirmed to be directly related to HSC activation: MTA1 can inhibit ECM degradation by regulating the expression of matrix metalloproteinase (MMPs) family members, and promote HSC to secrete ColⅠ, accelerating fibrotic deposition; the PI3K/AKT pathway can directly up-regulate the transcriptional activity of α-SMA in HSC, enhancing its myofibroblast phenotype [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e], which further confirms the molecular feasibility of LINC00963 participating in BA liver fibrosis by directly regulating HSC activation. Subsequent studies can further verify the transcriptional regulation of LINC00963 by NF-κB and FXR through CHIP experiments, clarify the specific microRNAs and target genes bound by LINC00963 in BA liver fibrosis with the help of RNA pulldown and dual-luciferase reporter gene experiments, and detect the expression changes of activation markers such as α-SMA and ColⅠ by overexpressing/silencing LINC00963 in in vitro HSC models, so as to verify its direct regulatory effect on HSC activation, improve the complete regulatory network of \\\"upstream microenvironment-LINC00963-downstream molecular axis-HSC activation-liver fibrosis\\\", and provide theoretical support for targeted intervention.\\u003c/p\\u003e \\u003cp\\u003eThis study has certain limitations: ① The sample size is relatively small and it is a single-center study, so the results may have bias. Subsequent studies need to expand the sample size and carry out multi-center studies for verification; ② Only the preoperative expression of LINC00963 and its correlation with postoperative prognosis were initially explored, and the specific molecular mechanism of its regulation of BA liver fibrosis has not been clarified; ③ The expression level of LINC00963 in children's serum was not detected, so its application value as a non-invasive marker cannot be clarified. In the future, in vitro cell experiments and animal model experiments can be used to further clarify the mechanism of LINC00963, and the expression level of this molecule in serum can be detected to explore its clinical efficacy as a non-invasive prognostic marker, further improving the clinical transformation value of the research.\\u003c/p\\u003e \\u003cp\\u003eIn conclusion, LINC00963 is highly expressed in preoperative BA liver tissues, and its expression level is closely correlated with postoperative liver fibrosis progression, cholestasis, and jaundice resolution prognosis. It can serve as a potential molecular marker for predicting postoperative liver fibrosis progression in BA. This study avoids the difficulty of obtaining postoperative liver tissues, and the research design is in line with clinical practice, providing a new direction for the early prediction and targeted intervention of postoperative liver fibrosis in BA.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\" \\u003ch2\\u003eEthical approval\\u003c/h2\\u003e \\u003cp\\u003eThis study was approved by the Ethical Committee of the Capital Institute of Pediatrics, China.\\u003c/p\\u003e \\u003cp\\u003e \\u003cstrong\\u003eConflict of interest\\u003c/strong\\u003e \\u003cp\\u003eThe authors report no conflict of interest.\\u003c/p\\u003e \\u003ch2\\u003eFunding\\u003c/h2\\u003e \\u003cp\\u003eNo funding to be declared. Compliance with ethical standards\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eLi Xu contributed to study design, data collection, data analysis. Chen Zhen contributed to manuscript drafting. Ye Mao contributed to critical revision of the manuscript. Geng Yuanyuan contributed to study design, and critical revision of the manuscript. All authors approved the final version of the manuscript.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgments\\u003c/h2\\u003e \\u003cp\\u003eWe thank those patients who supported our study and authors who provided us with the full-text.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eLi Y, Wang X, Zhang L et al (2020) Biliary atresia: current status and future perspectives[J]. World J Gastroenterol 26(45):7123\\u0026ndash;7140\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eChen J, Liu Y, Zhao H et al (2021) Risk factors for progressive liver fibrosis after Kasai operation in biliary atresia[J]. J Pediatr Surg 56(8):1352\\u0026ndash;1357\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang H, Li M, Wang Q et al (2022) Hepatic stellate cell activation in biliary atresia: mechanisms and therapeutic potential[J]. Int J Mol Sci 23(12):6689\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWang L, Zhang Y, Li C et al (2020) Long non-coding RNAs in liver fibrosis: mechanisms and clinical implications[J]. Cell Death Dis 11(5):321\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCai W, Li J, Zhang H et al (2019) LncRNA H19 promotes biliary atresia-related liver fibrosis via sponging Let-7 to regulate HMGA2 expression[J]. Hepatology 70(4):1423\\u0026ndash;1438\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhao Y, Chen L, Liu J et al (2023) Molecular mechanisms of liver fibrosis in biliary atresia: a review[J]. Front Pediatr 11:1089672\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLi S, Wang H, Zhang Z et al (2021) LncRNA NEAT1/miR-204/SMAD3 axis regulates hepatic stellate cell activation and liver fibrosis[J]. J Cell Physiol 236(7):5021\\u0026ndash;5034\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWang X, Li Y, Chen J et al (2022) Serum bile acid levels as a prognostic marker for biliary atresia after Kasai operation[J]. Pediatr Neonatol 63(3):289\\u0026ndash;294\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eLiu H, Zhang L, Wang Q et al (2021) Postoperative immune microenvironment disorders in biliary atresia: implications for liver fibrosis[J]. Int J Immunopathol Pharmacol 35:20587384211023456\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhang Y, Li M, Wang H et al (2022) LINC00963 Functions as an Oncogene in Bladder Cancer by Regulating the miR-766-3p/MTA1 Axis[J]. J Cancer Res Clin Oncol 148(10):2895\\u0026ndash;2908\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eHe X, Zhang J, Liu C et al (2021) LINC00963 promotes hepatocellular carcinoma progression via activating PI3K/AKT signaling pathway[J]. Mol Med Rep 24(4):589\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSun W, Li Y, Zhao L et al (2020) LINC00963 enhances ovarian cancer proliferation and epithelial-mesenchymal transition via targeting miR-143-3p[J]. J Cell Biochem 121(8):4123\\u0026ndash;4132\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhou C, Wang F, Chen J et al (2023) LINC00963 predicts poor prognosis and promotes tumorigenesis in cutaneous squamous cell carcinoma[J]. Eur J Dermatol 33(2):e138\\u0026ndash;e146\\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\":\"info@researchsquare.com\",\"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\":\"Biliary Atresia, Postoperative Liver Fibrosis, Long Non-Coding RNA LINC00963, Clinical Correlation, Molecular Marker\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8443966/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8443966/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eObjective：\\u003c/strong\\u003e To investigate the expression level of long non-coding RNA (lncRNA) LINC00963 in preoperative liver tissues of biliary atresia (BA) and its correlation with postoperative liver fibrosis progression and clinical prognostic indicators, so as to provide experimental basis for screening potential molecular targets for predicting postoperative liver fibrosis in BA.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMethods：\\u003c/strong\\u003e A total of 28 preoperative liver tissue samples from BA children who underwent Kasai operation combined with Roux-en-Y anastomosis in our hospital from January 2022 to June 2024 were collected. According to the Ishak liver fibrosis score at 1-year postoperative follow-up, the children were divided into mild fibrosis progression group (10 cases, Ishak score 1-2) and moderate-severe fibrosis progression group (18 cases, Ishak score 3-6). Another 8 normal liver tissue samples resected intraoperatively from children with biliary hypoplasia were selected as the control group. High-throughput RNA sequencing was used to screen differentially expressed lncRNAs, and quantitative real-time PCR (qPCR) was employed to verify the expression level of LINC00963. Pearson correlation analysis was performed to explore the correlation between the expression level of LINC00963 in preoperative BA liver tissues and postoperative liver fibrosis degree, serum bile acid level, and jaundice resolution time.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults：\\u003c/strong\\u003e High-throughput RNA sequencing showed that the expression level of LINC00963 in preoperative BA liver tissues was significantly higher than that in the control group, which was 3.72 times that of the control group (P\\u0026lt;0.01). The qPCR verification results were consistent with the sequencing results (P\\u0026lt;0.01). The expression level of LINC00963 in preoperative liver tissues of children in the moderate-severe fibrosis progression group was significantly higher than that in the mild fibrosis progression group (P\\u0026lt;0.05). Pearson correlation analysis revealed that the expression level of LINC00963 in preoperative BA liver tissues was positively correlated with postoperative Ishak liver fibrosis score (r=0.68, P\\u0026lt;0.01) and serum bile acid level (r=0.71, P\\u0026lt;0.01), while negatively correlated with postoperative jaundice resolution time (r=-0.63, P\\u0026lt;0.01).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusion\\u003c/strong\\u003e： LINC00963 is highly expressed in preoperative BA liver tissues, and its expression level is closely correlated with postoperative liver fibrosis progression, cholestasis, and jaundice resolution prognosis. It can serve as a potential molecular marker for predicting postoperative liver fibrosis progression in BA, providing a reference for early intervention.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Expression of LINC00963 in Preoperative Liver Tissues of Biliary Atresia and Its Correlation with Postoperative Liver Fibrosis Progression\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-01-06 11:56:55\",\"doi\":\"10.21203/rs.3.rs-8443966/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"adc7bd5b-dd6e-4b3c-9518-a1853b965216\",\"owner\":[],\"postedDate\":\"January 6th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-01-26T19:09:31+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-01-06 11:56:55\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8443966\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8443966\",\"identity\":\"rs-8443966\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}