Positive Correlation between PMS2 Deficiency and PD-L1 Expression in Pancreatic Cancer

Background: Pancreatic cancer is one of the most lethal types of cancer, and immunotherapy has become a promising remedy with advancements in tumor immunology. However, predicting the clinical response to immunotherapy in pancreatic cancer remains a dilemma for clinicians. Methods: GEPIA database was used to analyze the differential expression of MMR and PD-L1 genes in 33 common cancer types including pancreatic cancer. The expression levels of MMR and PD-L1 genes were downloaded from the GEPIA and GEO databases to analyze the correlation between MMR genes and PD-L1, and the clinicopathological and survival information were downloaded from the TCGA databases to analyze the relationship between the expression of MMR, PD-L1 and clinicopathological characteristics, prognosis. Meanwhile, the tumor tissue samples of 41 patients with pancreatic cancer were collected, and the protein expression levels of MMR and PD-L1 were detected by immunohistochemical assay. Furthermore, we analyzed the correlation between MMR and PD-L1, and the correlation between the expression of MMR, PD-L1 and clinicopathological characteristics, prognosis of pancreatic cancer patients. Results: Bioinformatics analysis showed that MLH1, MLH3, MSH2, MSH3, and PMS2 were highly expressed in most cancer types including pancreatic cancer (P P = 0.012), clinical stage (I vs II: P = 0.016), MSH2 expression was related to clinical stage (P < 0.05), T stage (T3 vs T4: P = 0.039), and MSH3 expression was related to T stage (P < 0.05). Besides, both MSH2 expression (P P = 0.044) were significantly associated with prognosis. GEPIA data also showed that MSH2 expression was related to prognosis (P = 0.008). The correlation analysis revealed that the expressions MSH2, MLH1, PMS2 had strong correlations with PD-L1 both in GEPIA and GEO databases. Real-world data indicated that of the 41 pancreatic cancer patients, 5 cases had MLH1 deletion, 5 cases had MSH2 deletion, 4 cases had PMS2 deletion, and 12 cases had PD-L1 positive expression. Notably, PMS2 deletion was associated with PD-L1 positive expression (P = 0.035). In addition, MLH1 was related to clinical stage (P = 0.033), age (P = 0.048), and MSH2 was related to clinical stage (P = 0.033). However, MLH1 (P = 0.697), MSH2 (P = 0.956), PMS2 (P = 0.341), and PD-L1 (P = 0.734) appeared to have no impact on overall survival among patients with pancreatic cancer. Conclusion: Both bioinformatics and real-world data showed that there were correlation between PMS2 deletion and PD-L1 expression, and correlation between MLH1, MSH2 and clinical stage.

高糖通过调控miR-429/ZEB1轴对胰腺癌细胞免疫逃逸的影响

目的探究高糖干预对胰腺癌细胞免疫逃逸的影响及作用分子机制。方法采用不同浓度葡萄糖(0、7.5、15、30 mmol/L)处理PANC-1细胞24 h构建高糖干预的PANC-1细胞。将miR-429 mimics及其阴性对照(mimics NC)转染至PANC-1细胞,分为对照组、HG组、HG+mimics NC组、HG+mimics组、HG+mimics+oe-NC组和HG+mimics+oe-ZEB1组。流式细胞术检测细胞表面分子细胞程序性死亡-配体1(PD-L1)表达水平;qRT-PCR检测细胞miR-429和锌指E-盒结合同源盒蛋白1(ZEB1)mRNA表达水平;Western blot检测细胞ZEB1蛋白表达水平。将以上各组PANC-1细胞与CD8^(+)T细胞建立共培养体系,CCK-8检测细胞增殖活性;流式细胞术检测细胞凋亡水平;乳酸脱氢酶(LDH)释放法检测CD8^(+)T细胞对PANC-1细胞的杀伤作用;采用双荧光素酶报告系统验证miR-429和ZEB1的靶向调控关系。结果HG可促进PANC-1细胞表面分子PD-L1及ZEB1表达(P<0.05),抑制miR-429表达,且呈浓度依赖性。miR-429过表达可显著抑制HG诱导的PANC-1细胞表面分子PD-L1表达,而过表达ZEB1可逆转miR-429过表达对HG诱导PANC-1细胞表面分子PD-L1表达的抑制作用。建立与CD8^(+)T细胞共培养体系后,与对照组比较,HG组PANC-1细胞增殖活性明显增加,细胞凋亡率和杀伤活性明显降低(P<0.05);与HG+mimics NC组比较,HG+mimics组PANC-1细胞增殖活性明显降低,细胞凋亡水平和杀伤活性明显升高(P<0.05)。与HG+mimics+oe-NC组比较,HG+mimics+oe-ZEB1组PANC-1细胞增殖活性明显增加,细胞凋亡率和杀伤活性明显降低(P<0.05)。双荧光素酶报告基因实验证实,miR-429靶向负调控ZEB1。结论高糖通过下调miR-429表达水平,靶向负调控ZEB1 mRNA的表达,提高PANC-1细胞表面分子PD-L1表达水平,进而促进PANC-1细胞免疫逃逸。

Insulin-like growth factor 2 mRNA-binding protein 1 promotes cell proliferation via activation of AKT and is directly targeted by microRNA-494 in pancreatic cancer

BACKGROUND Studies have shown that insulin-like growth factor 2 mRNA-binding protein 1(IGF2BP1)plays critical roles in the genesis and development of human cancers.AIM To investigate the clinical significance and role of IGF2BP1 in pancreatic cancer.METHODS Expression levels of IGF2BP1 and microRNA-494(miR-494)were mined based on Gene Expression Omnibus datasets and validated in both clinical samples and cell lines by quantitative real-time polymerase chain reaction and Western blot.The relationship between IGF2BP1 expression and clinicopathological factors of pancreatic cancer patients was analyzed.The effect and mechanism of IGF2BP1 on pancreatic cancer cell proliferation were investigated in vitro and in vivo.Analyses were performed to explore underlying mechanisms of IGF2BP1 upregulation in pancreatic cancer and assays were carried out to verify the posttranscriptional regulation of IGF2BP1 by miR-494.RESULTS We found that IGF2BP1 was upregulated and associated with a poor prognosis in pancreatic cancer patients.We showed that downregulation of IGF2BP1 inhibited pancreatic cancer cell growth in vitro and in vivo via the AKT signaling pathway.Mechanistically,we showed that the frequent upregulation of IGF2BP1 was attributed to the downregulation of miR-494 expression in pancreatic cancer.Furthermore,we discovered that reexpression of miR-494 could partially abrogate the oncogenic role of IGF2BP1.CONCLUSION Our results revealed that upregulated IGF2BP1 promotes the proliferation of pancreatic cancer cells via the AKT signaling pathway and confirmed that the activation of IGF2BP1 is partly due to the silencing of miR-494.

Analysis of the autophagy gene expression profile of pancreatic cancer based on autophagy-related protein microtubule-associated protein 1A/1B-light chain 3

BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.

Combining protein arginine methyltransferase inhibitor and antiprogrammed death-ligand-1 inhibits pancreatic cancer progression

BACKGROUND Immunotherapy targeting programmed death-1(PD-1)or programmed deathligand-1(PD-L1)has been shown to be effective in a variety of malignancies but has poor efficacy in pancreatic ductal adenocarcinoma(PDAC).Studies have shown that PD-L1 expression in tumors is an important indicator of the efficacy of immunotherapy.Tumor cells usually evade chemotherapy and host immune surveillance by epigenetic changes.Protein arginine methylation is a common posttranslational modification.Protein arginine methyltransferase(PRMT)1 is deregulated in a wide variety of cancer types,whose biological role in tumor immunity is undefined.AIM To investigate the combined effects and underlying mechanisms of anti-PD-L1 and type I PRMT inhibitor in pancreatic cancer in vivo.METHODS PT1001B is a novel type I PRMT inhibitor with strong activity and good selectivity.A mouse model of subcutaneous Panc02-derived tumors was used to evaluate drug efficacy,toxic and side effects,and tumor growth in vivo.By flow cytometry,we determined the expression of key immune checkpoint proteins,detected the apoptosis in tumor tissues,and analyzed the immune cells.Immunohistochemistry staining for cellular proliferation-associated nuclear protein Ki67,TUNEL assay,and PRMT1/PD-L1 immunofluorescence were used to elucidate the underlying molecular mechanism of the antitumor effect.RESULTS Cultured Panc02 cells did not express PD-L1 in vitro,but tumor cells derived from Panc02 transplanted tumors expressed PD-L1.The therapeutic efficacy of anti-PD-L1 mAb was significantly enhanced by the addition of PT1001B as measured by tumor volume(1054.00±61.37 mm3 vs 555.80±74.42 mm3,P<0.01)and tumor weight(0.83±0.06 g vs 0.38±0.02 g,P<0.05).PT1001B improved antitumor immunity by inhibiting PD-L1 expression on tumor cells(32.74%±5.89%vs 17.95%±1.92%,P<0.05).The combination therapy upregulated tumorinfiltrating CD8+T lymphocytes(23.75%±3.20%vs 73.34%±4.35%,P<0.01)and decreased PD-1+leukocytes(35.77%±3.30%vs 6.48%±1.08%,P<0.001)in tumor tissue compared to the control.In addition,PT1001B amplified the inhibitory effect of anti-PD-L1 on tumor cell proliferation and enhanced the induction of tumor cell apoptosis.PRMT1 downregulation was correlated with PD-L1 downregulation.CONCLUSION PT1001B enhances antitumor immunity and combining it with anti-PD-L1 checkpoint inhibitors provides a potential strategy to overcome anti-PD-L1 resistance in PDAC.

Expression of c-erbB-2 oncogene protein, epidermal growth factor receptor, and TGF-β1 in human pancreatic ductal adenocarcinoma

Objective: To detect the relations of c-erbB-2 onco-gene protein, epidermal growth factor receptor (EG-FR) and transforming growth factor-β1 (TGF-β1)to the progression or metastasis of pancreatic carci-noma.Methods: Using streptavidinbiotin complex (SABC)method, c-erbB-2 oncongene protein, we examinedimmunohistochemically EGFR and TGF-β1 expres-sions in wax-tissue sections from 10 individuals withnormal pancreas (NP), 13 patients with chronic pan-creatitis (CP) and 36 patients with pancreatic ductaladenocarcinoma (PC).Results: The positive expression rates of c-cerbB-2oncogene protein, EGFR and TGF-β1 in the NP, CPand PC groups were 0, 0, 10%; 7.7%, 7.7%,7.7%; and 41.7%, 50.0%, 44.4%, respectively.The positive expression rates of the three specific pro-teins increased more significantly in the PC groupthan in the NP and CP groups (P【0.05). The indi-vidual expression of c-erbB-2, EGFR and TGF-β1was not related to the age and sex of the patients aswell as the site, size and histopathological grade oftumors (P】0.05), but to the clinical stage of tumors(P【0.01). The coexpression rate of the three pro-teins was 27.8 % (10/36). This coexpression in thePC group was correlated with the histopathologicalgrades and clinical stages of tumors (P【0.01).Conclusion: Detection of c-erbB-2 oncogene protein,EGFR, and TGF-β1 expressions in pancreatic tissueis helpful to judge the malignancy, progression, andmetastasis of PC.

Transcription factors specificity protein and nuclear receptor 4A1 in pancreatic cancer

Specificity protein(Sp)transcription factors(TFs)Sp1,Sp3 and Sp4,and the orphan nuclear receptor 4A1(NR4A1)are highly expressed in pancreatic tumors and Sp1 is a negative prognostic factor for pancreatic cancer patient survival.Results of knockdown and overexpression of Sp1,Sp3 and Sp4 in pancreatic and other cancer lines show that these TFs are individually pro-oncogenic factors and loss of one Sp TF is not compensated by other members.NR4A1 is also a prooncogenic factor and both NR4A1 and Sp TFs exhibit similar functions in pancreatic cancer cells and regulate cell growth,survival,migration and invasion.There is also evidence that Sp TFs and NR4A1 regulate some of the same genes including survivin,epidermal growth factor receptor,PAX3-FOXO1,α5-andα6-integrins,β1-,β3-andβ4-integrins;this is due to NR4A1 acting as a cofactor and mediating NR4A1/Sp1/4-regulated gene expression through GC-rich gene promoter sites.Several studies show that drugs targeting Sp downregulation or NR4A1 antagonists are highly effective inhibitors of Sp/NR4A1-regulated pathways and genes in pancreatic and other cancer cells,and the triterpenoid celastrol is a novel dual-acting agent that targets both Sp TFs and NR4A1.

Potential involvement of heat shock proteins in pancreaticduodenal homeobox-1-mediated effects on the genesis of gastric cancer: A 2D gel-based proteomic study

AIM To identify functional proteins involved in pancreaticduodenal homeobox-1(PDX1)-mediated effects on gastric carcinogenesis.METHODS A PDX1-overexpressed model was established by transfecting gastric cancer cell line SGC7901 with pcDNA3.1(+)-PDX1 vector(SGC-PDX1). Transfection with empty pcDNA3.1 vector(SGC-pcDNA) served as control. Comparative protein profiles of the two groups were analyzed by two-dimensional electrophoresis basedproteomics(2 DE gel-based proteomics). The differential proteins identified by 2 DE were further validated by qRTPCR and immunoblotting. Finally, co-immunoprecipitation was used to determine any direct interactions between PDX1 and the differential proteins.RESULTS2 DE gel proteomics identified seven differential proteins in SGC-PDX1 when compared with those in SGC-pcDNA. These included four heat shock proteins(HSPs; HSP70 p1 B, HSP70 p8, HSP60, HSP27) and three other proteins(ER60, laminin receptor 1, similar to epsilon isoform of 14-3-3 protein). Immunoblotting validated the expression of the HSPs(HSP70, HSP60, HSP27). Furthermore, their expressions were lowered to 80%, 20% and 24%, respectively, in SGC-PDX1, while PDX1 exhibited a 9-fold increase, compared to SGC-pcDNA. However, qRT-PCR analysis revealed that mRNA levels of the HSP s were increased in SGC-PDX1, suggesting that the expression of the HSP s was post-translationally regulated by the PDX1 protein. Finally, co-immunoprecipitation failed to identify any direct interaction between PDX1 and HSP70 proteins.CONCLUSION This study demonstrates the potential involvement of HSPs in PDX1-mediated effects on the genesis of gastric cancer.

老年重症急性胰腺炎继发胰腺感染的病原菌分布及其与血清PCT、PA、FIB、HSP70和HMGB1的相关性

目的探讨老年重症急性胰腺炎继发胰腺感染患者的病原菌分布及其与血清降钙素原(PCT)、前白蛋白(PA)、纤维蛋白原(FIB)、热休克蛋白70(HSP70)、高迁徙率族蛋白1(HMGB1)水平的相关性。方法选择2019年1月至2022年12月东莞东华医院收治的62例老年重症急性胰腺炎患者作为研究对象,根据是否继发胰腺感染分为感染组20例和未感染组42例,另选择同期健康体检的80例健康者作为对照组。记录感染组患者病原菌分布情况,比较三组受试者及感染组不同病原菌感染类型患者的血清PCT、PA、FIB、HSP70和HMGB1水平,并采用Pearson相关性分析法分析血清PCT、PA、FIB、HSP70及HMGB1水平和胰腺感染的相关性。结果感染组患者病原菌感染类型主要为细菌感染,占90.00%,其中细菌感染类型中革兰阴性菌占55.00%,革兰阳性菌占35.00%;感染组患者血清PCT、HSP70、HMGB1水平分别为(2.49±0.53)μg/L、(9.12±1.86)ng/mL、(18.33±2.61)ng/mL,明显高于未感染组的(1.56±0.41)μg/L、(5.73±1.45)ng/mL、(13.62±2.47)ng/mL和对照组的(0.23±0.06)μg/L、(0.97±0.13)ng/mL、(0.73±0.15)ng/mL,PA、FIB分别为(0.17±0.02)g/L、(2.06±0.21)g/L,明显低于未感染组的(0.23±0.04)g/L、(2.75±0.30)g/L和对照组的(0.30±0.03)g/L、(3.23±0.36)g/L,且未感染组患者的血清PCT、HSP70、HMGB1水平均高于对照组,PA、FIB均低于对照组,差异均有统计学意义(P<0.05);感染组细菌感染患者的血清PCT、HSP70、HMGB1水平分别为(2.74±0.31)μg/L、(11.06±1.43)ng/mL、(20.68±2.05)ng/mL,明显高于真菌感染患者的(2.13±0.25)μg/L、(7.93±1.05)ng/mL、(16.27±1.76)ng/mL,PA、FIB水平分别为(0.11±0.02)g/L、(1.91±0.24)g/L,明显低于真菌感染患者的(0.17±0.04)g/L、(2.58±0.34)g/L,差异均有统计学意义(P<0.05);经Pearson相关性分析结果显示,血清PCT、HSP70、HMGB1与胰腺感染呈正相关(r=0.581、0.613、0.567,P<0.05),与PA、FIB呈负相关(r=-0.463、-0.531,P<0.05)。结论老年重症急性胰腺炎继发胰腺感染患者的病原菌类型主要为细菌感染,患者血清PCT、HSP70、HMGB1水平明显升高,PA、FIB明显降低,且与胰腺感染的发生有明显相关性,临床上应予以密切关注。

Mannogalactoglucan from mushrooms protects pancreatic islets via restoring UPR and promotes insulin secretion in TIDM mice

Type 1 diabetes mellitus(T1DM) lacks insulin secretion due to autoimmune deficiency of pancreaticβ-cells.Protecting pancreatic islets and enhancing insulin secretion has been therapeutic approaches.Mannogalactoglucan is the main type of polysaccharide from natural mushroom,which has potential medicinal prospects.Nevertheless,the antidiabetic property of mannogalactoglucan in T1DM has not been fully elucidated.In this study,we obtained the neutral fraction of alkali-soluble Armillaria mellea polysaccharide(AAMP-N) with the structure of mannogalactoglucan from the fruiting body of A.mellea and investigated the potential therapeutic value of AAMP-N in T1DM.We demonstrated that AAMP-N lowered blood glucose and improved diabetes symptoms in T1DM mice.AAMP-N activated unfolded protein response(UPR) signaling pathway to maintain ER protein folding homeostasis and promote insulin secretion in vivo.Besides that,AAMP-N promoted insulin synthesis via upregulating the expression of transcription factors,increased Ca^(2+) signals to stimulate intracellular insulin secretory vesicle transport via activating calcium/calmodulin-dependent kinase Ⅱ(CamkⅡ) and cAMP/PKA signals,and enhanced insulin secretory vesicle fusion with the plasma membrane via vesicle-associated membrane protein 2(VAMP2).Collectively,these studies demonstrated that the therapeutic potential of AAMP-N on pancreatic islets function,indicating that mannogalactoglucan could be natural nutraceutical used for the treatment of T1DM.

ACLP、COL11A1在胰腺癌组织中的表达及其与临床病理特征和预后的关系

目的研究胰腺癌中主动脉羧肽酶类样蛋白(ACLP)、Ⅺ型胶原α1(COL11A1)表达及临床预后价值。方法回顾性选择2019年1月—2021年1月陕西省肿瘤医院中西医科和陕西中医药大学附属医院肿瘤科手术治疗的胰腺癌患者88例为研究对象。采用实时荧光定量PCR和免疫组织化学检测癌组织及癌旁组织中ACLP、COL11A1 mRNA表达和蛋白水平;Pearson相关分析ACLP mRNA与COL11A1 mRNA的相关性;Kaplan-Meier法分析ACLP、COL11A1表达对胰腺癌患者生存预后的影响;Cox回归分析胰腺癌预后的影响因素。结果胰腺癌患者癌组织ACLP、COL11A1 mRNA的相对表达量高于癌旁组织(t/P=31.058/<0.001,27.642/<0.001);胰腺癌患者癌组织中ACLP、COL11A1阳性率分别为69.32%(61/88)、70.45%(62/88),高于癌旁组织的9.09%(8/88)、6.82%(6/88),差异有统计学意义(χ^(2)=66.963、75.155,P均<0.001);胰腺癌组织中ACLP mRNA与COL11A1 mRNA表达呈正相关(r=0.642,P<0.001);TNM分期ⅡB~Ⅲ期、有淋巴结转移的胰腺癌组织中ACLP、COL11A1蛋白阳性率高于TNM分期Ⅰ~ⅡA期、无淋巴结转移(ACLP:χ^(2)/P=19.704/<0.001、12.908/<0.001;COL11A1:χ^(2)/P=22.440/<0.001、14.569/<0.001)。ACLP阳性组3年总生存率为22.95%(14/61),低于阴性组44.44%(12/27),差异有统计学意义(Log rankχ^(2)=5.433,P=0.020);COL11A1阳性组3年总生存率为20.97%(13/62),低于阴性组50.00%(13/26),差异有统计学意义(Log rankχ^(2)=7.281,P=0.007)。TNM分期ⅡB~Ⅲ期、淋巴结转移、ACLP阳性、COL11A1阳性是影响胰腺癌患者预后的独立危险因素[HR(95%CI)=1.781(1.199~2.646),1.962(1.172~3.285),1.505(1.066~2.125),1.568(1.117~2.201)]。结论胰腺癌组织中ACLP、COL11A1 mRNA表达和蛋白水平均明显上调,二者在胰腺癌的发生和进展中发挥促进作用,是新的临床上评估胰腺癌患者预后的标志物。

MicroRNA-105-5p/PPM1A对胰腺癌PANC-1细胞增殖、迁移及侵袭的机制研究

目的探讨microRNA-105-5p(miR-105-5p)/PPM1A对胰腺癌PANC-1细胞增殖、迁移、侵袭及上皮细胞向间质转化(EMT)进程的影响及其潜在作用机制。方法实时荧光定量聚合酶链反应(qRT-PCR)检测miR-105-5p在人胰腺导管上皮细胞hTRET-HPNE和胰腺癌细胞PANC-1、AsPC-1、Bxpc-3中的表达。利用Kaplan-Meier Plotter在线工具探讨miR-105-5p与胰腺癌患者预后的关系。在PANC-1细胞中分别转染mimic NC、miR-105-5p mimic、inhibitor NC、miR-105-5p inhibitor。CCK-8法、划痕实验、Transwell实验分别检测各组细胞的增殖、迁移及侵袭能力;qRT-PCR检测miR-105-5p对E-cadherin、N-cadherin、Vimentin、ZEB1表达的影响。生物信息学方法预测miR-105-5p的候选靶基因,并对候选靶基因进行GO和KEGG富集分析。双萤光素酶实验检测miR-105-5p与PPM1A的靶向关系。qRT-PCR检测在PANC-1细胞中分别转染mimic NC、miR-105-5p mimic、inhibitor NC、miR-105-5p inhibitor后PPM1A的表达。免疫荧光实验检测PPM1A在人胰腺导管上皮细胞hTRET-HPNE和胰腺癌细胞PANC-1、AsPC-1、Bxpc-3中的表达。在PANC-1细胞中分别转染mimic NC+pcDNA3.1、mimic NC+pcDNA3.1-PPM1A、miR-105-5p mimic+pcDNA3.1-PPM1A后,通过挽救实验进一步研究miR-105-5p inhibitor与PPM1A在胰腺癌细胞中的相互作用关系。结果胰腺癌细胞PANC-1、AsPC-1、Bxpc-3中miR-105-5p mRNA相对表达量高于hTRET-HPNE细胞中miR-105-5p mRNA相对表达量(P<0.05),以PANC-1细胞中的相对表达量最高。miR-105-5p高表达与胰腺癌患者的不良预后有关(P<0.05)。miR-105-5p mimic组细胞增殖、迁移及侵袭能力均高于mimic NC组(P<0.05)。与mimic NC比较,miR-105-5p mimic下调E-cadherin mRNA表达,上调N-cadherin、Vimentin、ZEB1 mRNA表达(P<0.05)。转染miR-105-5p inhibitor后得到相反的结果。双萤光素酶实验证实miR-105-5p与PPM1A存在靶向关系。免疫荧光实验显示在胰腺癌细胞PANC-1、AsPC-1、Bxpc-3中PPM1A的荧光强度低于人胰腺导管上皮细胞hTRET-HPNE(P<0.05)。挽救实验表明miR-105-5p可部分挽救PPM1A对PANC-1细胞增殖、迁移和侵袭的抑制作用(P<0.05)。结论miR-105-5p靶向PPM1A促进胰腺癌PANC-1细胞的增殖、迁移及侵袭。

高迁移率族蛋白B1在临床相关疾病中的作用研究进展

高迁移率族蛋白B1(HMGB1)是由巨噬细胞释放的一种具有晚期促炎作用的核蛋白。近年来,HMGB1作为一种新型炎症因子,国内外学者均对其进行了广泛研究,发现其在许多疾病中均充当炎症介质,发挥细胞因子活性,从而参与机体损伤。该文主要对HMGB1在心血管系统、肾脏、肝脏及胰腺等相关疾病中的损伤作用,以及其可能的机制进行了综述,以便为临床诊断和研究相关疾病提供依据。

Zinc finger E-box-binding homeobox 1 mediates aerobic glycolysis via suppression of sirtuin 3 in pancreatic cancer

AIM TO uncover the roles of tumor-promoting gene ZEB1 in aerobic glycolysis regulation and shed light on the underlying molecular mechanism.METHODS Endogenous zinc finger E-box binding homeobox-1 （ZEB1） was silenced using a and the impact of ZEB1 and lentivirus-mediated method, methyI-CpG binding domain protein 1 （MBD1） on aerobic glycolysis was measured using seahorse cellular flux analyzers, reactive oxygen species quantification, and mitochondrial membrane potential measurement. The interaction between ZEB1 and MBD1 was assessed by co-immunoprecipitation and immunofluorescence assays. The impact of ZEB1 and MBD1 interaction on sirtuin 3 （SIRT3） expression was confirmed by quantitative polymerase chain reaction, western blotting, and dual-luciferase and chromatinimmunoprecipitation assays.RESULTS ZEB1 was a positive regulator of aerobic glycolysis in pancreatic cancer. ZEB1 transcriptionally silenced expression of SIRT3, a mitochondrial-localized tumor suppressor, through interaction with MBD1.CONCLUSION ZEB1 silenced SIRT3 expression via interaction with MBD1 to promote aerobic glycolysis in pancreatic cancer.

Emerging role of the KRAS-PDK1 axis in pancreatic cancer

Pancreatic cancer is a highly aggressive tumour that is very resistant to treatments and it is rarely diagnosed early because of absence of specific symptoms. Therefore, the prognosis for this disease is very poor and it has the grim supremacy in terms of unfavourable survival rates. There have been great advances in survival rates for many types of cancers over the past few decades but hardly any change for pancreatic cancer. Mutations of the Ras oncogene are the most frequent oncogenic alterations in human cancers. The frequency of KRAS mutations in pancreatic cancer is around 90%. Given the well-established role of KRAS in cancer it is not surprising that it is one of the most attractive targets for cancer therapy. Nevertheless, during the last thirty years all attempts to target directly KRAS protein have failed. Therefore, it is crucial to identify downstream KRAS effectors in order to develop specific drugs able to counteract activation of this pathway. Among the different signalling pathways activated by oncogenic KRAS, the phosphoinositide 3-Kinase(PI3K) pathway is emerging as one of the most critical KRAS effector. In turn, PI3 K activates several parallel pathways making the identification of the precise effectors activated by KRAS/PI3 K more difficult. Recent data identify 3-phosphoinositide-dependent protein kinase 1 as a key tumour-initiating event downstream KRAS interaction with PI3 K in pancreatic cancer.

Scolopendra subspinipes mutilans protected the ceruleininduced acute pancreatitis by inhibiting high-mobility group box protein-1

AIM:To evaluate the inhibitory effects of Scolopendra subspinipes mutilans(SSM) on cerulein-induced acute pancreatitis(AP) in a mouse model.METHODS:SSM water extract(0.1,0.5,or 1 g/kg) was administrated intraperitoneally 1 h prior to the first injection of cerulein.Once AP developed,the stable cholecystokinin analogue,cerulein was injected hourly,over a 6 h period.Blood samples were taken 6 h later to determine serum amylase,lipase,and cytokine levels.The pancreas and lungs were rapidly removed for morphological examination,myeloperoxidase assay,and real-time reverse transcription polymerase chain reaction.To specify the role of SSM in pancreatitis,the pancreatic acinar cells were isolated using collagenase method.Then the cells were pre-treated with SSM,then stimulated with cerulein.The cell viability,cytokine productions and high-mobility group box protein-1(HMGB-1) were measured.Furthermore,the regulating mechanisms of SSM action were evaluated.RESULTS:The administration of SSM significantly attenuated the severity of pancreatitis and pancreatitis associated lung injury,as was shown by the reduction in pancreatic edema,neutrophil infiltration,vacuolization and necrosis.SSM treatment also reduced pancreatic weight/body weight ratio,serum amylase,lipase and cytokine levels,and mRNA expression of multiple inflammatory mediators such as tumor necrosis factor-α and interleukin-1β.In addition,treatment with SSM inhibited HMGB-1 expression in the pancreas during AP.In accordance with in vivo data,SSM inhibited the cerulein-induced acinar cell death,cytokine,and HMGB-1 release.SSM also inhibited the activation of c-Jun NH2-terminal kinase,p38 and nuclear factor(NF)-κB.CONCLUSION:These results suggest that SSM plays a protective role during the development of AP and pancreatitis associated lung injury via deactivating c-Jun NH2-terminal kinase,p38 and NF-κB.

重症胰腺炎患者血清载脂蛋白B/载脂蛋白A1、微管相关蛋白1-轻链3和细胞间黏附分子-1水平在预测并发感染性胰腺坏死中的价值

目的观察重症胰腺炎(SAP)患者血清载脂蛋白B与载脂蛋白A1比值(ApoB/ApoA1)、微管相关蛋白1-轻链3(MAP1-LC3)及细胞间黏附分子-1(ICAM-1)水平,并分析其与患者并发感染性胰腺坏死(IPN)的关系和预测价值。方法选取2019年1月至2022年1月于该院收治的172例SAP患者作为SAP组。收集临床资料及外周静脉血标本,检测患者血清ApoB/ApoA1、MAP1-LC3和ICAM-1水平,同期选取该院70例体检健康者作为对照组。比较SAP患者与体检健康者的血清ApoB/ApoA1、MAP1-LC3和ICAM-1水平差异;根据SAP患者后续有无并发IPN分为IPN组和非IPN组。采用单因素及多因素分析比较两组患者临床资料,分析血清ApoB/ApoA1、MAP1-LC3、ICAM-1水平及其他相关因素与SAP患者并发IPN的关系;并通过绘制受试者工作特征(ROC)曲线,分析血清ApoB/ApoA1、LC3和ICAM-1水平用于预测SAP患者并发IPN的价值。结果SAP组血清ApoB/ApoA1、MAP1-LC3、ICAM-1水平均高于对照组(均P<0.05);单因素分析显示,IPN组ApoB/ApoA1、MAP1-LC3及ICAM-1水平均高于非IPN组(均P<0.05),多因素分析显示,ApoB/ApoA1(β=2.309,P=0.027)、MAP1-LC3(β=5.447,P=0.037)及ICAM-1(β=0.039,P=0.045)水平均是SAP患者并发IPN的影响因素。血清ApoB/ApoA1、MAP1-LC3及ICAM-1水平预测SAP患者并发IPN的曲线下面积(AUC)分别为0.761(95%CI:0.683~0.840)、0.765(95%CI:0.681~0.848)、0.882(95%CI:0.829~0.935);灵敏度分别为76.1%、68.7%、71.6%,特异度分别为61.0%、89.5%、91.4%。联合预测的AUC为0.957,灵敏度为85.1%,特异度为96.2%。结论血清ApoB/ApoA1、MAP1-LC3、ICAM-1水平是SAP患者并发IPN的影响因素,并发IPN的患者ApoB/ApoA1、MAP1-LC3和ICAM-1水平更高,这些指标对于预测SAP患者并发IPN具有一定的价值。

血清可溶性神经调节蛋白-1、G蛋白偶联雌激素受体-1在急性胰腺炎患者病情及预后评估中临床价值研究

目的探讨血清可溶性神经调节蛋白-1(sNRG-1)、G蛋白偶联雌激素受体-1(GPER-1)在急性胰腺炎(AP)患者病情及预后评估中的临床价值。方法选取自2019年6月至2022年6月邯郸市第一医院收治的106例AP患者为研究对象,根据AP病情严重程度分为轻症组(n=49)、中重症组(n=36)及重症组(n=21);根据预后生存情况分为存活组(n=83)与死亡组(n=23)。采用酶联免疫吸附法检测血清sNRG-1、GPER-1水平。采用受试者工作特性(ROC)曲线评估血清sNRG-1、GPER-1及两者联合检测对AP患者预后的评估价值。采用多因素Logistic回归分析探讨AP患者预后影响因素。结果中重症组、重症组患者血清sNRG-1水平低于轻症组,且重症组低于中重症组;中重症组、重症组患者血清GPER-1水平高于轻症组,且重症组高于中重症组,差异均有统计学意义(P<0.05)。存活组与死亡组病情严重程度、入院急性生理与慢性健康评分(APACHEⅡ)、白细胞计数、C反应蛋白、血肌酐、乳酸脱氢酶、血淀粉酶、血脂肪酶、sNRG-1、GPER-1比较,差异有统计学意义(P<0.05)。APACHEⅡ评分≥12分、sNRG-1≤17.74 pg/ml、GPER-1≥4.82 pg/ml是影响AP患者预后的独立危险因素(P<0.05)。血清sNRG-1、GPER-1预测AP患者预后的ROC曲线下面积分别为0.846、0.753。两者联合预测AP患者预后的ROC曲线下面积为0.917,特异度为86.75%,灵敏度为86.96%。结论血清sNRG-1低表达、GPER-1高表达与AP患者的病情严重程度及预后有关,有望作为评估AP患者预后的生物学指标。

基于生物信息学分析NUSAP1在胰腺癌中的表达及临床预后分析

目的 研究核仁纺锤体相关蛋白1 (Nucleolar and spindle associated protein 1,NUSAP1)在胰腺癌组织中的表达及临床预后分析。方法 利用GEPIA数据库检索NUSAP1基因在胰腺癌组织和正常组织中的表达水平,TCGA数据库及组织芯片研究及验证NUSAP1在胰腺癌的临床预后分析。结果 GEPIA数据库分析结果显示胰腺癌组织中NUSAP1的表达高于正常对照组(P<0.05)。TCGA数据库结果显示,高表达NUSAP1的胰腺癌患者生存时间较低表达NUSAP1的患者更短(HR=2.02,P<0.05)。在病理分级G3-G4组、残余瘤R1和R2组、胰腺癌治疗进展组中NUSAP1的表达均分别高于病理分级G1-G2组、残余瘤R0组、治疗缓解组(P<0.05)。在死亡组中,NUSAP1的表达高于存活组(P<0.05)。组织芯片免疫组化结果显示,与癌旁组织相比,NUSAP1在胰腺癌中呈高表达(χ^(2)=44.10,P<0.001),NUSAP1高表达的患者生存时间更短,预后越差。单因素回归分析结果显示NUSAP1的表达量、性别、病理分级、肿瘤大小、T分期、N分期、TNM分期是影响胰腺癌患者总生存期的危险因素(P<0.05)。多因素回归分析结果显示NUSAP1的表达量、TNM分期是影响总生存期的独立危险因素(P<0.05)。结论 NUSAP1在胰腺癌组织中高表达,与胰腺癌患者不良预后相关。

胰腺神经内分泌肿瘤和实性假乳头状肿瘤中INSM1和SOX11的表达及意义

目的:探讨胰岛素瘤相关蛋白1(insulinoma associated protein 1,INSM1)和SRY相关高迁移率族盒蛋白11(SRY-related high-mobility group box 11,SOX11)在胰腺神经内分泌肿瘤(pancreatic neuroendocrine tumor,PNET)和实性假乳头状肿瘤(solid pseudopapillary neoplasm,SPN)中的表达及意义。方法:采用免疫组织化学方法在56例PNET、42例SPN、16例导管腺癌(ductal adenocarcinoma,DACC)和8例腺泡细胞癌(acinar cell carcinoma,ACC)中检测INSM1、SOX11、Syn、CgA、CD56、β-catenin和CD99,比较INSM1和SOX11的组合与传统标记物(Syn、CgA、CD56、β-catenin和CD99)在诊断和鉴别诊断PNET和SPN中的应用价值。结果:(1)56例PNET中,INSM1表达于肿瘤细胞及胰岛细胞核,其在肿瘤组织中的阳性表达率为91.07%(51/56);42例SPN、16例DACC和8例ACC均未见INSM1的阳性信号。INSM1在PNET中的阳性表达率与SPN、DACC和ACC相比,差异均有统计学意义(P<0.001)。(2)42例SPN中,SOX11的阳性信号位于肿瘤细胞核,其阳性表达率为92.86%(39/42);56例PNET中,SOX11的阳性表达率为8.93%(5/56),其阳性信号位于3例G 1型和2例G 3型PNET;16例DACC、8例ACC及肿瘤旁的正常胰腺组织均未见SOX11阳性信号;SOX11在SPN中的阳性表达率与PNET、DACC和ACC相比,差异均有统计学意义(P<0.001)。(3)INSM1(+)/SOX11(-)免疫表型对PNET的敏感度为85.71%,与CD56(57.14%)相比差异有统计学意义(P=0.001),与Syn(80.36%)和CgA(71.43%)相比差异无统计学意义(P>0.05);其特异度为100.00%,与Syn(42.86%)和CD56(47.62%)相比差异有统计学意义(P<0.001),与CgA(92.86%)相比差异无统计学意义(P>0.05)。INSM1(-)/SOX11(+)免疫表型对SPN的敏感度为92.86%,与β-catenin(90.48%)和CD99(85.71%)相比差异无统计学意义(P>0.05);其特异度为96.43%,与CD99(48.21%)相比差异有统计学意义(P<0.001),与β-catenin(100.00%)相比差异无统计学意义(P>0.05)。(4)在PNET和SPN中,INSM1和SOX11的表达情况与临床病理参数(患者年龄、性别、肿瘤大小、部位、分级及转移)无关(P均>0.05)。结论:在PNET和SPN中,INSM1和SOX11分别呈阳性的表达模式有助于区分两种肿瘤,两者组合在敏感度和特异度方面优于一些传统的免疫组织化学标记物。