Gallbladder cancer (GBC) is a highly malignant and rapidly progressing tumor of the human biliary system, and there is an urgent need to develop new therapeutic targets and modalities. Non-POU domain-containing octamer-binding protein (NONO) is an RNA-binding protein involved in the regulation of transcription, mRNA splicing, and DNA repair. NONO expression is elevated in multiple tumors and can act as an oncogene to promote tumor progression. Here, we found that NONO was highly expressed in GBC and promoted tumor cells growth. The dysregulation of RNA splicing is a molecular feature of almost all tumor types. Accordingly, mRNA-seq and RIP-seq analysis showed that NONO promoted exon6 skipping in DLG1, forming two isomers (DLG1-FL and DLG1-S). Furthermore, lower Percent-Spliced-In (PSI) values of DLG1 were detected in tumor tissue relative to the paraneoplastic tissue, and were associated with poor patient prognosis. Moreover, DLG1-S and DLG1-FL act as tumor promoters and tumor suppressors, respectively, by regulating the YAP1/JUN pathway. N6-methyladenosine (m6A) is the most common and abundant RNA modification involved in alternative splicing processes. We identified an m6A reader, IGF2BP3, which synergizes with NONO to promote exon6 skipping in DLG1 in an m6A-dependent manner. Furthermore, IP/MS results showed that RBM14 was bound to NONO and interfered with NONO-mediated exon6 skipping of DLG1. In addition, IGF2BP3 disrupted the binding of RBM14 to NONO. Overall, our data elucidate the molecular mechanism by which NONO promotes DLG1 exon skipping, providing a basis for new therapeutic targets in GBC treatment.

Background The effect of neoadjuvant chemotherapy (NACT) in gallbladder cancer (GBC) patients remains controversial. The aim of this study was to assess the impact of NACT on overall survival (OS) and cancer specific survival (CSS) in patients with localized or locoregionally advanced GBC, and to explore possible protective predictors for prognosis. Methods Data for patients with localized or locoregionally advanced GBC (i.e., categories cTx-cT4, cN0-2, and cM0) from 2004 to 2020 were collected from the Surveillance, Epidemiology, and End Results (SEER) database. Patients in the NACT and non-NACT groups were propensity score matched (PSM) 1:3, and the Kaplan-Meier method and log-rank test were performed to analyze the impact of NACT on OS and CSS. Univariable and multivariable Cox regression models were applied to identify the possible prognostic factors. Subgroup analysis was conducted to identify patients who would benefit from NACT. Results Of the 2676 cases included, 78 NACT and 234 non-NACT patients remained after PSM. In localized or locoregionally advanced GBC patients, the median OS of the NACT and non-NACT was 31 and 16 months (log-rank P < 0.01), and the median CSS of NACT and non-NACT was 32 and 17 months (log-rank P < 0.01), respectively. Longer median OS (31 vs 17 months, log-rank P < 0.01) and CSS (32 vs 20 months, log-rank P < 0.01) was associated with NACT compared with surgery alone. Multivariable Cox regression analysis showed that NACT, stage, and surgery type were prognostic factors for OS and CSS in GBC patients. Subgroup analysis revealed that the survival hazard ratios (HRs) of NACT vs non-NACT for localized or locoregionally advanced GBC patients were significant in most subgroups. Conclusions NACT may provide therapeutic benefits for localized or locoregionally advanced GBC patients, especially for those with advanced stage, node-positive, poorly differentiated or undifferentiated disease. NACT combined with radical surgery was associated with a survival advantage. Therefore, NACT combined with surgery may provide a better treatment option for resectable GBC patients.

Liver metastasis is common in patients with gallbladder cancer (GBC), imposing a significant challenge in clinical management and serving as a poor prognostic indicator. However, the mechanisms underlying liver metastasis remain largely unknown. Here, we report a crucial role of tyrosine aminotransferase (TAT) in liver metastasis of GBC. TAT is frequently up-regulated in GBC tissues. Increased TAT expression is associated with frequent liver metastasis and poor prognosis of GBC patients. Overexpression of TAT promotes GBC cell migration and invasion in vitro, as well as liver metastasis in vivo. TAT knockdown has the opposite effects. Intriguingly, TAT promotes liver metastasis of GBC by potentiating cardiolipin-dependent mitophagy. Mechanistically, TAT directly binds to cardiolipin and leads to cardiolipin externalization and subsequent mitophagy. Moreover, TRIM21 (Tripartite Motif Containing 21), an E3 ubiquitin ligase, interacts with TAT. The histine residues 336 and 338 at TRIM21 are essential for this binding. TRIM21 preferentially adds the lysine 63 (K63)-linked ubiquitin chains on TAT principally at K136. TRIM21-mediated TAT ubiquitination impairs its dimerization and mitochondrial location, subsequently inhibiting tumor invasion and migration of GBC cells. Therefore, our study identifies TAT as a novel driver of GBC liver metastasis, emphasizing its potential as a therapeutic target.

Gallbladder cancer (GBC) is the most common malignant tumor of the biliary system, with poor response to current treatments. Abnormal alternative splicing has been associated with the development of a variety of tumors. Combining the GEO database and GBC mRNA-seq analysis, it is found high expression of the splicing factor polypyrimidine region- binding protein 3 (PTBP3) in GBC. Multi-omics analysis revealed that PTBP3 promoted exon skipping of interleukin-18 (IL-18), resulting in the expression of ΔIL-18, an isoform specifically expressed in tumors. That ΔIL-18 promotes GBC immune escape by down-regulating FBXO38 transcription levels in CD8+T cells to reduce PD-1 ubiquitin-mediated degradation is revealed. Using a HuPBMC mouse model, the role of PTBP3 and ΔIL-18 in promoting GBC growth is confirmed, and showed that an antisense oligonucleotide that blocked ΔIL-18 production displayed anti-tumor activity. Furthermore, that the H3K36me3 promotes exon skipping of IL-18 by recruiting PTBP3 via MRG15 is demonstrated, thereby coupling the processes of IL-18 transcription and alternative splicing. Interestingly, it is also found that the H3K36 methyltransferase SETD2 binds to hnRNPL, thereby interfering with PTBP3 binding to IL-18 pre-mRNA. Overall, this study provides new insights into how aberrant alternative splicing mechanisms affect immune escape, and provides potential new perspectives for improving GBC immunotherapy.

The prognosis of gallbladder cancer（GBC）is very dismal and surgical resection renders little help for those at advanced stages. Whether neoadjuvant therapy can improve the outcome of GBC has become a hot topic of concern.However, there are still concerns regarding how to achieve effective new adjuvant therapy because of a lack of high-quality evidence-based data and misapprehension in clinical settings. In addition, three core issues need to be clarified,including the criteria of indication of applicable patientss, the specific treatment regimens, and the evaluation of therapeutic effect. The application of novel technological breakthroughs such as artificial intelligence, an organoid and liquid biopsy may shed light on neoadjuvant therapy for gallbladder cancer.

Gallbladder cancer (GBC) is a type of rare but highly aggressive cancer with a dismal prognosis. Runt-related transcription factor 3 (RUNX3), a member of the runt-domain family, and its promoter methylation have been widely observed in a variety of human malignancies. However, the biological function and underlying mechanism of RUNX3 in GBC remain elusive. In this study, bisulfate sequencing PCR (BSP), Western blot, and qPCR were applied to identify the expression level and DNA methylation level of RUNX3 in GBC tissues and cells. The transcriptional relationship between RUNX3 and Inhibitor of growth 1 (ING1) was validated by dual-luciferase reporter assay and ChIP assay. A series of gain-of-function and loss-of-function assays were performed to detect the function and the regulatory relationship of RUNX3 in vitro and in vivo. RUNX3 was aberrantly downregulated in GBC cells and tissues caused by DNA Methyltransferase 1 (DNMT1)-mediated methylation, and downregulation of RUNX3 is associated with poor prognosis of GBC patients. Functional experiments reveal that RUNX3 can induce ferroptosis of GBC cells in vitro and in vivo. Mechanistically, RUNX3 induces ferroptosis by activating ING1 transcription, thereby repressing SLC7A11 in a p53-dependent manner. In conclusion, the downregulation of RUNX3 is mediated by DNA methylation, which promotes the pathogenesis of gallbladder cancer through attenuating SLC7A11-mediated ferroptosis. This study gives novel insights into the role of RUNX3 in the ferroptosis of GBC cells, which may contribute to developing potential treatment targets for GBC.

Objective·To investigate the effects of breast cancer susceptibility gene 1 (BRCA1) R1325K mutation [arginine (R) to lysine (K) mutation at amino acid 1325] on the proliferation and apoptosis of gallbladder cancer cell lines GBC-SD and NOZ.Methods·BRCA1 wild-type overexpression lentivirus, BRCA1 R1325K mutation overexpression lentivirus, and negative control lentivirus were used to construct the stable transgenic strains of gallbladder carcinoma, cell lines GBC-SD and NOZ. The cells were divided into the control group without the target gene, the BRCA1 wild-type group, and the BRCA1 R1325K mutation group. The expression of target protein was verified by Western blotting. The BRCA1 R1325K mutant gallbladder cancer cells were treated with 20 μmol/L Olaparib, a BRCA1 mutation inhibitor. Gallbladder cancer cell lines were divided into the control group, the BRCA1 wild-type group, the BRCA1 R1325K mutation group, and the BRCA1 R1325K mutation+Olaparib group according to the target gene expression and whether or not the inhibitor was added. The effect of BRCA1 R1325K mutation on proliferation and clonogenesis ability of gallbladder cancer cell lines GBC-SD and NOZ was observed by CCK8 assay and clonogenesis assay, respectively. The effect of BRCA1 R1325K mutation on apoptosis of gallbladder cancer cell lines GBC-SD and NOZ was observed by TUNEL assay. The expressions of apoptosis-related proteins, cleaved PARP, Bcl-2 and Bax, were detected by Western blotting. The inhibitor Olaparib was used to treat the BRCA1 R1325K mutant gallbladder cancer cell lines GBC-SD and NOZ. The phenotypic changes (promoting proliferation, enhancing clonogenesis and inhibiting apoptosis) induced by BRCA1 R1325K mutation were tested in the presence of Olaparib to determine whether the changes could be reversed by the inhibitor.Results·The results of CCK8 assay and clonogenesis assay showed that BRCA1 R1325K mutation could promote the proliferation of gallbladder cancer cell lines GBC-SD and NOZ, and improve their clonal formation ability, compared with the control group and the BRCA1 wild-type group. Olaparib inhibited the proliferation of gallbladder cancer cell lines overexpressing BRCA1 R1325K mutation (P<0.05). Through TUNEL and Western blotting, it was found that overexpression of wild-type BRCA1 could induce the apoptosis of gallbladder cancer cell lines GBC-SD and NOZ, compared with the control group. Compared with the control group and the BRCA1 wild-type group, the BRCA1 R1325K mutation group had anti-apoptotic effect, in which the expression of apoptosis-inhibiting protein Bcl-2 increased and the expression of pro-apoptotic protein Bax decreased (P<0.05).Conclusion·BRCA1 R1325K mutation can promote the proliferation of GBC-SD and NOZ cell lines and inhibit their apoptosis.

Abstract Background Three‐dimensional visualization preoperative evaluation (3D‐VPE) and enhanced recovery after surgery (ERAS) have been suggested to improve outcomes of cancer surgery in patients, yet little is known regarding their clinical benefit in patients with gallbladder cancer (GBC). We hypothesized that the combination of 3D‐VPE and ERAS would improve the outcome of patients undergoing surgery for GBC. Objective This study aimed to determine if 3D‐VPE and ERAS can improve the outcomes and overall survival in patients with GBC, establishing a novel patient management strategy for GBC. Methods A total of 227 patients with GBC were recruited and divided into two groups: those who received traditional treatment between January 2000 and December 2010 (n = 86; the control group) and those who underwent 3D‐VPE and ERAS between January 2011 and December 2017 (n = 141). Univariate and multivariate analyses were employed to assess the relationship among disease stages, lymph node invasion, and cell differentiation between the two groups. Cox regression analysis was used to investigate patient survival in these groups. Results Patients who underwent 3D‐VPE and ERAS showed a significantly higher R0 resection rate (67.4% vs. 20.9%, p < 0.001) and dissected lymph node number (26.6 ± 12.6 vs. 16.3 ± 7.6 p < 0.001) compared to the control group. The median survival was 27.4 months, and the 1‐ and 3‐year survival rates were 84.4% and 29.8%, respectively, in patients who received combined management; in the control cohort, the median survival was 12.7 months, and the 1‐ and 3‐year survival rates were 53.5% and 15.1%, respectively. In addition, some postoperative complications and risk factors were diminished relative to the traditionally treated patients. Conclusion The implementation of 3D‐VPE and ERAS can significantly improve the prognosis and outcomes of patients with GBC and should be considered for wide use in clinical practice.

The long non-coding RNAs (lncRNAs) have been implicated in multiple human cancers, which may offer great potential as putative targets for cancer diagnosis and treatment. However, the roles of most lncRNAs in gallbladder cancer (GBC) remain poorly understood. The objective of this research involves investigating the clinical implications and underlying mechanism of lncRNA motor neuron and pancreas homeobo×1 antisense RNA 1 (MNX1-AS1) in GBC. This study shows that MNX1-AS1 expression is elevated in the tissues of GBC patients, and is strongly associated with reduced patient survival. Functionally, MNX1-AS1 significantly stimulates the proliferation and metastasis of GBC cells in vitro and in vivo. Mechanistically, MNX1-AS1 is transcriptionally activated by TEA domain family member 4 (TEAD4), and suppresses insulin-like growing factor 2 mRNA-binding protein 3 (IGF2BP3) degradation by recruiting ubiquitin specific peptidase 16 (USP16). Furthermore, MNX1-AS1/IGF2BP3 axis inhibits the Hippo signaling pathway and subsequently activates TEAD4, thereby forming a positive feedback loop. According to our results, MNX1-AS1 facilitates tumorigenesis, progression and metastasis of GBC through a MNX1-AS1/IGF2BP3/Hippo pathway positive feedback loop, which could be both diagnostically and therapeutically helpful in GBC.

BACKGROUND:The first-line chemotherapy regimen for advanced gallbladder cancer (GBC) is gemcitabine plus platinum (GP), despite its efficacy is limited. The current investigation is a retrospective study to compare the safety and efficacy between the modified FOLFIRINOX (mFOLFIRINOX) and gemcitabine plus oxaliplatin (GEMOX) as the first-line chemotherapy for unresectable locally advanced or metastatic GBC.METHODS:The data of patients with unresectable locally advanced or metastatic GBC, who were treated with mFOLFIRINOX or GEMOX as the first-line therapy between April 2014 and April 2018 at Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, were retrieved. This retrospective study evaluated the clinical characteristics, survival outcomes and adverse events.RESULTS:A total of 44 patients (n=25 in mFOLFIRINOX, n=19 in GEMOX) were included. There were no significant differences between groups in baseline characteristics. The median progression free survival (mPFS) was 5.0 months in the mFOLFIRINOX group and 2.5 months in the GEMOX group [P=0.021; hazard ratio (HR), 0.499; 95% CI, 0.266 to 0.937]. The median overall survival (mOS) was 9.5 months in the mFOLFIRINOX group and 7.0 months in the GEMOX group (P=0.019; HR, 0.471; 95% CI, 0.239 to 0.929). Disease control rate (DCR) was 76.0% in the mFOLFIRINOX group and 47.4% in the GEMOX group (P=0.051). The rate of grade 3-4 adverse events was 48% in the mFOLFIRINOX group and 36.8% in the GEMOX group (P=0.459). The incidence of grade 3-4 neutropenia and diarrhea were more common in the mFOLFIRINOX group, while the incidence of grade 3-4 thrombocytopenia and peripheral neuropathy were more common in the GEMOX group.CONCLUSIONS:mFOLFIRINOX might improve the poor prognosis of unresectable locally advanced or metastatic GBC, and the results need to be further verified by prospective clinical studies.

目的 探讨在胰十二指肠切除术后不留置鼻胃和鼻肠营养管的安全性及可行性.方法 回顾性分析2014年1月至2019年12月上海交通大学医学院附属新华医院普外科收治的238例胰十二指肠切除术病人的临床资料,其中110例不留置鼻胃及鼻肠营养管(未留置组),128例留置鼻胃及鼻肠营养管(留置组).对比分析两组病人术后恢复情况、营养状况及术后并发症.结果 未留置组与留置组病人的手术时间、术中出血量、术后首次肛门排气和排便时间差异均无统计学意义(P＞0.05),但未留置组病人术后住院时间更短,住院费用明显降低,差异有统计学意义(P＜0.01).未留置组病人在消化道不适症状(咽喉疼痛、恶心呕吐)、肺部感染方面明显优于留置组(P＜0.05),且未增加胰瘘、胆瘘、切口感染、胃排空延迟、术后出血、腹腔感染的发生率(P＞0.05);两组在术后30 d死亡及二次手术方面差异无统计学意义(P＞0.05).术后第3、5天未留置组病人血红蛋白(Hb)、白蛋白(ALB)、前白蛋白(PAB)水平明显高于留置组,差异有统计学意义(P＜0.01).结论 胰十二指肠切除术后病人不常规放置鼻胃及鼻肠营养管安全可行,有利于病人的术后康复,减轻痛苦,改善术后营养状况.

AbstractBackgroundLong noncoding RNAs (lncRNA) represent significant factors of the mammalian transcriptome that mediates varied biological and pathological processes. The liver is the most common site for gallbladder cancer (GBC) distant metastasis and contributes to the majority of GBC‐related death. How lncRNA affects GBC metastasis is not completely understood.ResultsA novel lncRNA termed lncGALM (lncRNA in GBC associated with liver metastasis) was discovered to be highly expressed in cancer patients and xenografted tumors with liver metastasis. Elevated lncGALM in GBC patients also correlated to decreased survival. Invasion and migration of GBC cells were enhanced through lncGALM, both in vitro and in vivo. lncGALM functioned as sponges by competitively binding to and inactivating miR‐200 family members, which increase epithelial‐mesenchymal transition‐associated transcription factor ZEB1 and ZEB2, leading to a fibroblastic phenotype and increased expression of N‐cadherin. In addition, lncGALM bound to IL‐1β mRNA and stabilized the IL‐1β gene that mediates liver sinusoidal endothelial cell (LSECs) apoptosis. lncGALM‐expressing LiM2‐NOZ cells acquired a strong ability to migrate and adhere to LSECs, promoting LSECs apoptosis and therefore facilitating tumor cell extravasation and dissemination.ConclusionslncGALM promotes GBC liver metastasis by facilitating GBC cell migration, invasion, liver arrest, and extravasation via the invasion‐metastasis cascade. Targeting lncGALM may be protective against the development of liver metastasis in GBC patients.

NOP2/Sun domain family, member 2 (NSUN2) is a nuclear RNA methyl-transferase catalyzing 5-methylcytosine formation. Evidence shows that NSUN2 is correlated with cell unlimited proliferation. However, its functional role in gallbladder carcinoma (GBC), which is the most common biliary tract malignancy and has a poor prognosis, remains to be determined. Here we found that NSUN2 was highly expressed in GBC tissues as well as cell lines. NSUN2 silencing repressed GBC cell proliferation and tumorigenesis both in vitro and in vivo. Conversely, upregulation of NSUN2 enhanced GBC cell growth and colony formation. We further discovered that RPL6 was a closely interacting partner with NSUN2. Silencing RPL6 resulted in insufficient NSUN2 translational level and accumulative NSUN2 transcriptional level. Exogenous expression of NSUN2 partially rescued the effect of RPL6 in gallbladder cancer progression. Taken together, our data provided novel mechanic insights into the function of NSUN2 in GBC, thus pointing to NSUN2 as a potential and effective therapeutic approach to GBC treatment.

Objectives Patients with gallbladder carcinoma (GBC) lack effective treatment methods largely due to the inadequacy of both molecular characterisation and potential therapeutic targets. We previously uncovered a spectrum of genomic alterations and identified recurrent mutations in the ErbB pathway in GBC. Here, we aimed to study recurrent mutations of genes and pathways in a larger cohort of patients with GBC and investigate the potential mechanisms and clinical significance of these mutations. Design We performed whole-exome sequencing (WES) in 157 patients with GBC. Functional experiments were applied in GBC cell lines to explore the oncogenic roles of ERBB2/ERBB3 hotspot mutations, their correlation with PD-L1 expression and the underlying mechanisms. ERBB inhibitors and a PD-L1 blocker were used to evaluate the anticancer activities in co-culture systems in vitro and in vivo. Results WES identified ERBB2 and ERBB3 mutations at a frequency of 7%–8% in the expanded cohort, and patients with ERBB2/ERBB3 mutations exhibited poorer prognoses. A set of in vitro and in vivo experiments revealed increased proliferation/migration on ERBB2/ERBB3 mutation. Ectopic expression of ERBB2/ERBB3 mutants upregulated PD-L1 expression in GBC cells, effectively suppressed normal T-cell-mediated cytotoxicity in vitro through activation of the PI3K/Akt signalling pathway and contributed to the growth and progression of GBC in vivo. Treatment with an ERBB2/ERBB3 inhibitor or a PD-L1 monoclonal antibody reversed these immunosuppressive effects, and combined therapy revealed promising therapeutic activities. Conclusions ERBB2/ERBB3 mutations may serve as useful biomarkers in identifying patients who are sensitive to ERBB2/ERBB3 inhibitors and PD-L1 monoclonal antibody treatment. Trial registration number NCT02442414;Pre-results.

目的 探讨精准胆道外科理论和技术在手术治疗中的应用及其价值.方法 回顾性分析2000年1月至2015年6月上海交通大学医学院附属新华医院收治的97例胆囊癌病人资料.按照收治的时间将病人分为传统手术组(38例,2000年1月至2010年12月)与精准手术组(59例,2011年1月至2015年6月).比较两组病人的围手术期状况及生存期等指标.结果 两组病人在术前年龄、性别、分化程度、TNM分期、白蛋白、CA19-9、术中出血量、术后住院天数等方面差异均无统计学意义.精准手术组行胆囊癌扩大根治术比例为35.5％(21/59),传统手术组为15.6％(6/38),两组比较差异有统计学意义(x2=4.513,P＜0.05).精准手术组R0切除率为66.1％,淋巴结清扫数目(26.0±12.1)枚;传统手术组RO切除率为18.4％,淋巴结清扫数目(17.0±9.1)枚,两组比较差异有统计学意义(x2=21.075,P＜0.001;t=3.915,P＜0.001).精准手术组病人的中位生存期19.2个月,1、3年存活率分别为62.7％、28.8％;传统手术组病人的中位生存期9.9个月,1、3年存活率分别为42.1％、7.9％,差异有统计学意义(x2=3.960,6.180,P＜0.05).而两组术中出血量、并发症发生率、住院时间等差异均无统计学意义.结论 精准胆道外科理论和技术在胆囊癌病人中的应用,可有效提高根治性手术切除率和术后病人的存活率.

Objective To investigate the surgical anatomical and histological data for the mesopancreas and mesopancreatic excision. Methods The anatomic structures of the peripancreatic space,the important vessels and the retroperitoneal structures of 5 cadaveric specimens between January and December 2016 in Basic Medicine Faculty of Shanghai Jiao Tong University were observed in the study. The structures of the vessels and retroperitoneum were histological studied. Results Posterior pancreatic space was an important anatomical plane of mesopancreatic excision. Celiac trunk and superior mesenteric artery were important anatomic landmarks of mesopancreatic excision. The No.16 lymph node was the extra anatomy structure of mesopancreas. There was a natural surgical space between the adventitia and sheath of the artery. There was a great deal of nerve and lymphatic vessel distribution on and outside the arterial sheath of the peripancreatic arteries. Conclusion Mesopancreas and mesopancreatic excision have the corresponding basis of surgical anatomy. Arterial sheaths dissection strategy for improving the dissection effect has certain anatomical basis.