Mucins in neoplasms of pancreas,ampulla of Vater and biliary system

Tumors of the pancreas,the ampulla of Vater,and the extrahepatic and intrahepatic bile ducts have significant histological similarities due to the common embryonic origin of the pancreatobiliary system.This obviates the need for discovery of biomarkers with diagnostic and prognostic value for these tumors.Mucins,especially MUC-1,-2,-4 and-5AC,are important candidates for developing into such reliable biomarkers.Increased expression of MUC1 occurs in pancreatic ductal adenocarcinomas and is associated with increased degrees of dysplasia in pancreatic intraepithelial neoplasia(Pan IN).Positive expression of MUC2 in intraductal papillary mucinus neoplasms(IPMN) of the intestinal type indicates high potential progression to invasive carcinoma with de novo expression of MUC1,while absence of MUC2 expression in IPMNs of gastric type implies low potential to malignant evolution.De novo MUC4 expression correlates to the severity of dysplasia in Pan IN and is associated with a poor prognosis in patients with pancreatic ductal adenocarcinomas.In biliary intraepithelial neoplasia(Bil IN),increased expression of MUC1 is associated with higher degrees of dysplasia.Intrahepatic cholangiocarcinomas(ICC) are characterized by increased expression of all glycoforms of MUC1.Positive MUC2 expression in intraductal papillary neoplasm of the bile ducts(IPNB) of the intestinal type indicates high malignant potential with de novo expression of MUC1 in the invasive element.Absent MUC2 expression in any degree of Bil IN may prove useful in differentiating them from IPNB.De novo expression of MUC4 is associated with poor prognosis in patients with ICC or carcinoma of the extrahepatic bile ducts(EHBDC).High de novo expression of MUC5 AC is found in all degrees of Bil IN and all types of IPNB and ICC.The MUC5 AC is useful in the detection of neoplastic lesions of the bile duct at an early stage.Increased expression of mucin MUC1 in carcinoma of the ampulla of Vater associated with unfavorable behavior of the tumor,such as lymph node metastasis,infiltration of the pancreas and duodenum,advanced TNM classification and worse prognosis.Patients withintra-ampullary papillary-tubular neoplasm(IAPN) of the pancreatobiliary immunophenotype did not show MUC2,while those of the intestinal immunophenotype are MUC2 positive.The expression of MUC4 is associated with poor prognosis in patients with carcinoma of the ampulla of Vater favoring metastasis and making them resistant to apoptosis.Moreover,it appears that MUC4 positivity correlates with recurrence of the tumor.Expression of MUC5 AC is associated with the invasive potential of the tumor.

CHARACTERISTICS OF MUCINS AND CEA IN GLOBOID DYSPLASTIC CELLS OF HUMAN STOMACH AND ITS RELATIONSHIP WITH SIGNET RING CELL CARCINOMA

Fourty-three cases of globoid dysplasia and signet ring cell carcinoma were stained by mucin and CEA (ABC method). It was found that there were three kinds of mucins (neutral, sialo and sulphomucin in both globoid dysplastic cells and signet ring cells. The percentages of the three kinds of mucins seen in the two kinds of cells were not much different. It was indicated that the altered mucins in the gastric epithelial cells must be a sign of dedifferentiation of the cells and the results of malfunction. The globoid dysplasia type I mainly contained neutral mucin, whereas type II, the acid mucin was predominant, especially the sulphomucin. The CEA positive reaction became stronger as the atypia being remarkable, and the characteristics of distribution of CEA positive particles were similar in the two kinds of cells namely, randomly or disorderly in the cells. Based on the analysis of the results, a conclusion can be made that the variety of mucins in globoid dysplastic cells can be used as a reference point in classification and is not much significant in grading, but the amount of CEA positive matter can be a reference point in grading. The globoid dysplasia is such a lesion with special features in morphology and function manifested in the process of de-differentiation towards signet ring cell carcinoma following the successive action of carcinogens upon the cells of gastric epithelium.

Membrane-bound mucins and mucin terminal glycans expression in idiopathic or Helicobacter pylori, NSAID associated peptic ulcers

AIM: To determine the expression of membrane-bound mucins and glycan side chain sialic acids in Helicobacter pylori (H. pylori)-associated, non-steroidal inflammatory drug (NSAID)-associated and idiopathic-gastric ulcers.

Interactions of Human T Cell Immunoglobin Mucins with Apoptotic Cells

T cell immunoglobulin mucin (TIM) family plays a key role in regulating immune re-sponses.In this study,the interactions of human TIM family with apoptotic cells were evaluated in order to provide a foundation for further study on the roles of human TIM genes in apoptosis.Nine kinds of pEGFP-N1 eukaryotic expression vectors containing different lengths of the three members of human TIM genes for the expression of TIM-EGFP and the vectors for the expression of TIM-Fc fusion pro-teins were constructed.It was found that human TIM proteins could recognize and bind to apoptotic cells directly,but not to viable cells.The interactions of sTIM-1-EGFP,sTIM-3-EGFP and sTIM-4-EGFP with apoptotic cells were blocked by TIM-1-Ig,TIM-3-Ig and TIM-4-Ig fusion proteins respectively.In addition,human TIM proteins mediated the recognition of apoptotic cells and bound to apoptotic cells directly via the IgV domains.In conclusion,the TIM family may play a key role in the regulation of apoptosis.Our data also suggest that human TIM proteins probably serve as novel proteins for the detection of the early cellular apoptosis.

QUALITATIVE STUDY OF SIALOMUCINS CHANGES DURING N－METHYL－N－NITROSOUREA－INDUCED C OLONIC CARCINOGENESIS IN MICE

The sequential histological and sialomucins histochemical changes in colonic epithelium during the N-methyl-N-nitrosourea (MNU) induced colonic carcinogenesis were studied in female ICR mice. The sialomucin histochemical changes were assessed by use of high iron diamine-alcian blue staining (HID-AB) at PH 2.5. In the untreated controls, the epithelial cells in the distal colon are characterized by a predominance of sulfomucins, and the cells in the proximal site stain for abundant sialomucins, with occasioual sulfomucins in the base of the crypt. The earlier changes detected at the 4th week are focal areas of hyperplasia in which sulfomucins still predominant as in controls. As the experimental progress, the number of lesions with severe dysplasia increase, with carcinoma in situ and invasive carcinoma appeariug from the 10th week. The areas of mild or moderate dysplasia show marked changes in the composition of mucin, characterized by the progressively increased of staining of sialomucins and accompanied in general by a decrease of sulfomucins, similar to those changes described in transitional mucosa (TM) adjacent to colorectal cancer in the human. It is suggested the morphological malignant transformation of colonic epithelium were paralleled qualitatively enhanced sialomucin staining in concert with reduced sulfomucin staining of epithelial cells, providing further evidence that the mucin changes in TM characterized by an increase in sialomucins might reflect early primary premalignant transformation.

D-Psicose intake exacerbates dextran sulfate sodium-induced colitis in mice through alteration in the gut microbiota and dysfunction of mucosal barrier

D-Psicose,as a low-calorie rare sugar,has attracted a lot of attention in recent years for alternating to sucrose.The anti-obesity effect of D-psicose has been extensively confirmed in previous studies,however,the impact of D-psicose on colitis remains vague.Here,we firstly evaluated the effect of the D-psicose prophylactic intervention on dextran sulfate sodium-induced colitis in C57BL/6 mice.The pathological symptoms,inflammatory cytokines levels,gut microbiota composition,short chain fatty acids(SCFAs)production and colonic barrier integrity were comprehensively evaluated.The results confirmed that D-psicose intervention aggravated colitis,characterized by the exacerbation of colon shortening,increase of colonic inflammatory infiltration,and marked exaltation of disease activity indices and IL-6,IL-1βand TNF-αlevels.Further,the dysfunction of gut microbiota was identified in the psicose group.The abundance of pro-inflammatory bacteria Lachnospiraceae_NK4A136_group was significantly up-regulated while the abundance of probiotics Akkermansia and Lactobacillus were significantly down-regulated in the psicose group compared to the model group.Moreover,the production of SCFAs was suppressed in the psicose group,accompanied by a decrease in the level of mucin 2(Muc-2).Collectively,the underlying mechanism of the exacerbation of colitis by D-psicose intervention might be attributed to microbiota dysfunction accompanied by the reduction of SCFAs,which leads to the damage of the mucosal barrier and the intensifi cation of inflammatory invasion.

Progress on immuno-microenvironment and immune-related therapies in patients with pseudomyxoma peritonei

Pseudomyxoma peritonei(PMP) is an indolent malignant syndrome. The standard treatment for PMP is cytoreductive surgery combined with intraperitoneal hyperthermic chemotherapy(CRS + HIPEC). However, the high recurrence rate and latent clinical symptoms and signs are major obstacles to further improving clinical outcomes. Moreover, patients in advanced stages receive little benefit from CRS + HIPEC due to widespread intraperitoneal metastases. Another challenge in PMP treatment involves the progressive sclerosis of PMP cell-secreted mucus, which is often increased due to activating mutations in the gene coding for guanine nucleotide-binding protein alpha subunit(GNAS). Consequently, the development of other PMP therapies is urgently needed. Several immune-related therapies have shown promise, including the use of bacterium-derived non-specific immunogenic agents, radioimmunotherapeutic agents, and tumor cell-derived neoantigens, but a well-recognized immunotherapy has not been established. In this review the roles of GNAS mutations in the promotion of mucin secretion and disease development are discussed. In addition, the immunologic features of the PMP microenvironment and immune-associated treatments are discussed to summarize the current understanding of key features of the disease and to facilitate the development of immunotherapies.

Downregulation of MUC1 Inhibits Proliferation and Promotes Apoptosis by Inactivating NF-κB Signaling Pathway in Human Nasopharyngeal Carcinoma

Objective To investigate the effect of mucin 1(MUC1)on the proliferation and apoptosis of nasopharyngeal carcinoma(NPC)and its regulatory mechanism.Methods The 60 NPC and paired para-cancer normal tissues were collected from October 2020 to July 2021 in Quanzhou First Hospital.The expression of MUC1 was measured by real-time quantitative PCR(qPCR)in the patients with PNC.The 5-8F and HNE1 cells were transfected with siRNA control(si-control)or siRNA targeting MUC1(si-MUC1).Cell proliferation was analyzed by cell counting kit-8 and colony formation assay,and apoptosis was analyzed by flow cytometry analysis in the 5-8F and HNE1 cells.The qPCR and ELISA were executed to analyze the levels of TNF-αand IL-6.Western blot was performed to measure the expression of MUC1,NFкB and apoptosis-related proteins(Bax and Bcl-2).Results The expression of MUC1 was up-regulated in the NPC tissues,and NPC patients with the high MUC1 expression were inclined to EBV infection,growth and metastasis of NPC.Loss of MUC1 restrained malignant features,including the proliferation and apoptosis,downregulated the expression of p-IкB、p-P65 and Bcl-2 and upregulated the expression of Bax in the NPC cells.Conclusion Downregulation of MUC1 restrained biological characteristics of malignancy,including cell proliferation and apoptosis,by inactivating NF-κB signaling pathway in NPC.

Diagnosis and treatment of biliary mucinous cystic neoplasms: A single-center experience

Background: Biliary mucinous cystic neoplasms(BMCNs) are rare hepatobiliary cystic tumors, which can be divided into noninvasive and invasive types. This study aimed to investigate the diagnosis, treatment, and prognosis of BMCNs in a large single center. Methods: We analyzed 49 patients with BMCNs confirmed by postoperative pathology at the First Afflliated Hospital, Zhejiang University School of Medicine between January 2007 and December 2021. Results: Among the 49 patients, 37 were female(75.5%), and the average age was 57.04 years. Common symptoms included abdominal discomfort, jaundice and fever, while 22 patients(44.9%) had no symptoms. Serum carbohydrate antigen(CA) 19-9 and CA125 concentrations were elevated in 34.8% and 19.6% of patients, respectively. Forty-eight patients had tumors in the intrahepatic bile ducts and only one had a tumor in the extrahepatic bile duct. Forty-eight patients with noninvasive intrahepatic BMCNs were further analyzed in terms of pathological features: 34(70.8%) had low-grade intraepithelial neoplasms(LGINs), and 14(29.2%) had high-grade intraepithelial neoplasms(HGINs). The potential immunohistochemical markers of BMCNs were cytokeratin(CK) 19, CK7, estrogen receptor and progesterone receptor. Follow-up data for 37 patients with intrahepatic BMCNs were obtained. The median overall survival(OS) of BMCNs was not reached. The longest survival time was 137 months.The 5-and 10-year OS rates were 100% and 85.4%, respectively. The 5-and 10-year recurrence-free survival(RFS) rates were 93.9% and 80.2%, respectively. Conclusions: BMCNs are rare cystic neoplasms that commonly occur in middle-aged females. BMCNs can only be diagnosed and classified by postoperative pathology, as there are no specific clinical presentations, serological indicators or imaging modalities for preoperative diagnosis. Complete surgical resection is necessary for BMCNs, and the postoperative prognosis is favorable.

Establishment of a cholangiocarcinoma risk evaluation model based on mucin expression levels

BACKGROUND Cholangiocarcinoma(CCA)is a highly malignant cancer,characterized by frequent mucin overexpression.MUC1 has been identified as a critical oncogene in the progression of CCA.However,the comprehensive understanding of how the mucin family influences CCA progression and prognosis is still incomplete.AIM To investigate the functions of mucins on the progression of CCA and to establish a risk evaluation formula for stratifying CCA patients.METHODS Single-cell RNA sequencing data from 14 CCA samples were employed for elucidating the roles of mucins,complemented by bioinformatic analyses.Subse-quent validations were conducted through spatial transcriptomics and immuno-histochemistry.The construction of a risk evaluation model utilized the least absolute shrinkage and selection operator regression algorithm,which was further confirmed by independent cohorts and diverse data types.RESULTS CCA tumor cells with elevated levels of MUC1 and MUC4 showed activated nucleotide metabolic pathways and increased invasiveness.MUC5AC-high cells were found to promote CCA progression through WNT signaling.MUC5B-high cells exhibited robust cellular oxidation activities,leading to resistance against antitumoral treatments.MUC13-high cells were observed to secret chemokines,recruiting and transforming macrophages into the M2-polarized state,thereby suppressing antitumor immunity.MUC16-high cells were found to promote tumor progression through interleukin-1/nuclear factor kappa-light-chain-enhancer of activated B cells signaling upon interaction with neutrophils.Utilizing the expression levels of these mucins,a risk factor evaluation formula for CCA was developed and validated across multiple cohorts.CCA samples with higher risk factors exhibited stronger metastatic potential,chemotherapy resistance,and poorer prognosis.CONCLUSION Our study elucidates the functional mechanisms through which mucins contribute to CCA development,and provides tools for risk stratification in CCA.

Asymptomatic low-grade appendiceal mucinous neoplasm:A case report

BACKGROUND Low-grade appendiceal neoplasms(LAMN)are characterized by low incidence and atypical clinical presentations,often leading to misdiagnosis as acute or chronic appendicitis before surgery.The primary diagnostic tool for LAMN is abdominal computed tomography(CT)imaging.Surgical resection remains the cornerstone of LAMN management,necessitating en bloc tumor excision to minimize the risk of iatrogenic rupture.Laparoscopy,known for its minimal invasiveness,reduced postoperative discomfort,and expedited recovery,is a safe and reliable approach for LAMN treatment.Despite the possibility of pseudomyxoma peritonei development,appendectomy and partial appendectomy generally result in negative tumor margins and favorable outcomes,which can be attributed to the disease’s slow growth and lower malignancy.CASE SUMMARY A 71-year-old male patient was admitted to our hospital with a pelvic spaceoccupying lesion detected 1 mo prior.Physical examination showed a soft abdomen without tenderness or rebound and no palpable masses.No shifting dullness was noted,and digital rectal examination revealed no palpable mass.Enteroscopy revealed a raised,smooth-surfaced mass measuring 3.0 cm in the cecum.Abdominal contrast-enhanced CT showed a markedly thickened and dilated appendix with visible cystic shadows.Laparoscopic surgery was performed and revealed a significantly dilated appendix,leading to laparoscopic resection of the appendix and part of the cecum.Post-surgical pathologic analysis confirmed LAMN.The patient received symptomatic and supportive post-operative care and was discharged on postoperative day 4 without complications such as abdominal bleeding,intestinal obstruction,or incision infection.No tumor recurrence was observed during a 7-mo follow-up period.CONCLUSION LAMN is a rare disease that lacks specific clinical manifestations.Abdominal CT plays a crucial role in diagnosing LAMN,and laparoscopic surgery is a safe and effective diagnostic and therapeutic approach.

Waist subcutaneous soft tissue metastasis of rectal mucinous adenocarcinoma: A case report

BACKGROUND Rectal mucinous adenocarcinoma(MAC)is a rare pathological type of rectal can-cer with unique pathological features and a poor prognosis.It is difficult to diag-nose and treat early because of the lack of specific manifestations in some aspects of the disease.The common metastatic organs of rectal cancer are the liver and lung;however,rectal carcinoma with metastasis to subcutaneous soft tissue is a rare finding.CASE SUMMARY In this report,the clinical data,diagnosis and treatment process,and postope-rative pathological features of a patient with left waist subcutaneous soft tissue masses were retrospectively analyzed.The patient underwent surgical treatment after admission and recovered well after surgery.The final pathological diagnosis was rectal MAC with left waist subcutaneous soft tissue metastasis.CONCLUSION Subcutaneous soft tissue metastasis of rectal MAC is rare,and it can suggest that the tumor is disseminated,and it can appear even earlier than the primary ma-lignant tumor,which is occult and leads to a missed diagnosis and misdiagnosis clinically.When a subcutaneous soft tissue mass of unknown origin appears in a patient with rectal cancer,a ma-lignant tumor should be considered.

Visualizing the landscape of appendiceal tumor research after 2010: A bibliometric study

BACKGROUND Despite the rarity of appendiceal tumors,research in this field has intensified,resulting in a growing number of studies and published papers.Surprisingly,no comprehensive bibliometric analysis has specifically addressed appendiceal tumors.AIM To offer a thorough analysis of the current landscape and future trends in appendiceal tumor research.METHODS In our bibliometric analysis studies,we explored the Web of Science Core Collection database.The bibliographic details of the chosen publications were automatically converted and analyzed using the bibliometric package in the R environment.Additionally,we employed VoSviewer to create cooperation network maps for countries,institutions,and authors,as well as clustering maps for keywords.Furthermore,CiteSpace,another software tool,was utilized to build dual-map overlays of journals and analyze references with citation bursts.RESULTS Our study included 780 English-language articles published after 2010.The number of related publications and citations has increased in the past decade.The United States leads in this area,but there is a need to improve cooperation and communication among countries and institutions.Co-occurrence analysis also revealed close collaboration among different authors.Annals of Surgical Oncology was the most influential journal in this field.Analysis of references with high cocitations and references with citation bursts,consistent with analysis of keywords and hotspots,indicated that current research primarily centers on the classification and management of appendiceal mucinous neoplasms and consequent pseudomyxoma peritonei.Despite the abundance of clinical studies,a greater number of in-depth basic research studies should be conducted.CONCLUSION Current research on appendiceal tumors focuses on classification and management of appendiceal mucinous neoplasms and pseudomyxoma peritonei.Enhanced collaboration and basic research are vital for further advancement.

Mucinous neoplasm of the appendix:A case report and review of literature

BACKGROUND Appendiceal mucinous neoplasms(AMNs),although not classified as rare,are relatively uncommon tumors most often discovered incidentally during colorectal surgery.Accurate identification of AMNs is difficult due to non-specific sym-ptoms,overlapping tumor markers with other conditions,and the potential for misdiagnosis.This underscores the urgent need for precision in diagnosis to pre-vent severe complications.CASE SUMMARY This case report describes the unexpected discovery and treatment of a low-grade AMN(LAMN)in a 74-year-old man undergoing laparoscopic hemicolectomy for transverse colon adenocarcinoma(AC).Preoperatively,non-specific gastroin-testinal symptoms and elevated tumor markers masked the presence of AMN.The tumor,presumed to be an AMN peritoneal cyst intraoperatively,was con-firmed as LAMN through histopathological examination.The neoplasm exhibited mucin accumulation and a distinct immunohistochemical profile:Positive for Ho-meobox protein CDX-2,Cytokeratin 20,special AT-rich sequence-binding protein 2,and Mucin 2 but negative for cytokeratin 7 and Paired box gene 8.This profile aids in distinguishing appendiceal and ovarian mucinous tumors.Postoperative recovery was uncomplicated,and the patient initiated adjuvant chemotherapy for the colon AC.CONCLUSION This case highlights the diagnostic complexity of AMNs,emphasizing the need for vigilant identification to avert potential complications,such as pseudomyxoma peritonei.

Beyond boundaries:Feasibility of curved linear array echoendoscope in appendiceal neoplasm detection

We recently read with great interest a study by Zhang et al in the World Journal of Gastroenterology.In our practice,we focus specifically on examining appendiceal mucinous neoplasms(AMNs)with endoscopic ultrasound(EUS)using different scopes.AMNs are rare neoplastic lesions characterized by an accumulation of mucin inside a cystic dilatation of the appendix.Clinically,they can present as nonspecific acute appendicitis.AMNs can turn into a life-threatening condition,termed pseudomyxoma peritonei,in which the ruptured appendix causes accumulation of mucin in the abdomen.Therefore,accurate and rapid diagnosis of AMN is essential.EUS is able to confirm and stage AMNs;although,EUS examination was once limited to the rectal and anal regions due to the conven-tional oblique-view scopes.With the emergence of new forward-view linear echoendoscopes and instruments like EUS miniprobes and overtubes,the scope of examination is changing.Herein,we discuss the feasibility of using the curved linear array echoendoscopes to examine cecal and appendiceal orifice lesions.

The Influence of Aerial Exposure on Sea Anemones Aulactinia veratra Mucin Genes Expression Using the RNA Sequencing

Mucin genes are the main component of mucus. The sea anemone species, Aulactinia veratra (Phylum Cnidaria) contains different types of mucin genes. In the intertidal zone, A. veratra is found to be exposed to air during the low tide and produces large quantities of mucus as an external covering. The relation between low tide and mucus secretion is still unclear, and what is the role of mucin during arial exposure is not yet investigated. This study hypothesised that the mucin genes in A. veratra would have significantly high expression in response to aerial exposure. Therefore, the aim of current study was to examine and analyses the response of A. veratra mucins in response to an experiment involving three hours of aerial exposure. To achieve this, aim the RNA-sequencing and bioinformatics analyses were used to examine the expression profile of A. veratra mucin genes in response to aerial exposure. The generated results have shown that, Mucin4-like and mucin5B-like were up-regulated in response to the three hours of aerial exposure in A. veratra. This finding shows a significant role of mucin5B-like and mucin4-like genes in response to air stress at low tide. The data generated from this study could be used in conjunction with future mucin gene studies of sea anemones and other cnidarians to compare A. veratra mucin gene expression results across time, and to extend our understanding of mucin stress response in this phylum.

Advances in the Diagnosis and Treatment of Appendiceal Mucinous Neoplasms

Primary appendiceal neoplasms represent a relatively low percentage of all gastrointestinal cancers. A subset of these neoplasms, those of epithelial origin, are characterised by the production of a considerable amount of mucus, which is referred to as appendiceal mucinous neoplasms (AMN). Appendiceal mucinous neoplasms (AMN) have a low incidence, are easily misdiagnosed, depend on postoperative examination for confirmation of the diagnosis, are prone to form a “diagnosis”, and have a high incidence of the disease. Furthermore, they are prone to form peritoneal pseudomyxoma peritonei (PMP), are controversial in surgical decision-making, are prone to recurring after surgery alone, and are tricky to manage clinically. In this paper, we review the pathological characteristics, diagnosis and treatment of appendiceal mucinous tumours in the light of recent literature reports, with a view to providing certain references for the clinical diagnosis and treatment of this disease. .

A Case of Diagnosis of Pseudomyxoma Peritonei

Objective: To report a case of pseudomyxoma peritonei accidentally discovered during preoperative examination and review relevant literature to improve the early diagnosis of the origin of pseudomyxoma peritonei. Methods: To analyze a case accidentally discovered due to abdominal distension in our hospital in October 2023. Combined with domestic and foreign literature, the etiology, clinical manifestations, diagnosis, differential diagnosis and treatment of PMP were discussed. Results: The patient was admitted due to abdominal distension with no other specific discomfort. Mucinous tumors were found during imaging examinations. Conclusion: PMP is a rare disease, and clinical manifestations often include abdominal distension. It is usually first discovered through imaging examinations such as ultrasound, CT or MRI. CRS and HIPEC are still recommended as the standard treatment for PMP.

miRNAs-MUC 13调控体系中与仔猪腹泻有关的基因与通路

为研究microRNAs-Mucin 13(miRNAs-MUC 13)调控体系中与仔猪腹泻相关的分子调控机制,对miRNAs-MUC 13调控体系中与仔猪腹泻有关的基因与通路进行了发掘与验证。用NCBI的blastn工具比对了小鼠MUC 13基因和猪基因库中的核酸序列,发现两者的相似性在74%~87%,同源性较高。以小鼠为模型,选择miRDB、mirTarBase、TargetScan数据库预测调控MUC 13基因的miRNA序列,通过StarBase数据库预测这些miRNA序列的靶基因,得到36个靶基因交集。用Cytoscape软件围绕MUC 13和这36个靶基因构建仔猪腹泻基因互作网络,该网络包含19个筛选到的靶基因,基于MCOMD算法分析获得4个分子复合物集团。通过DAVID平台对这4个分子复合物进行基因和通路富集分析,结果发现,靶基因SRF和STAG 2及其参与的有关通路与仔猪腹泻相关。以大肠埃希菌200和仔猪肠上皮细胞系(IPEC-1)作为试验材料,通过体外细胞试验设计仔猪腹泻模型进行验证,实时荧光定量PCR(qRT-PCR)检测结果显示,试验组MUC 13、SRF、IL-6、MAPK、STAG 2、NF-κB、TLR 7等基因的表达量均显著(P<0.05)高于对照组,由此推测,SRF与STAG 2基因在miRNAs-MUC 13调控体系中分别通过MAPK、IL6、Toll-like受体信号通路参与仔猪腹泻调控。围绕miRNAs-MUC 13调控体系发掘了4个分子复合物集团和2个核心基因(SRF和STAG 2)并进行了试验验证,这为靶向治疗仔猪腹泻提供了理论参考和科学依据。

中华蜜蜂肠粘蛋白AcMucin5AC-1的鉴定和定位分析

【目的】本研究旨在鉴定中华蜜蜂Apis cerana cerana肠粘蛋白AcMucin5AC-1的结构、分布及对中肠的影响,为解析蜜蜂中肠的生理功能提供理论依据。【方法】利用生物信息学比较分析中华蜜蜂AcMucin5AC蛋白的序列特征;利用RT-qPCR检测AcMucin 5 AC-1在中华蜜蜂4日龄幼虫中肠和表皮以及2日龄幼虫感染中华蜜蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)后0,12,24,48和72 h时中肠中的表达量。通过原核表达系统对中华蜜蜂AcMucin5AC-1进行表达,亲和层析柱纯化重组蛋白并制备相应多克隆抗体;Western blot检测AcMucin5AC-1在中华蜜蜂健康3-6日龄幼虫中以及4日龄幼虫中肠、表皮和围食膜中的表达,应用间接免疫荧光试验分析AcMucin5AC-1在中华蜜蜂4日龄幼虫中的定位。通过饲喂法利用RNAi沉默中华蜜蜂2日龄幼虫AcMucin 5 AC-1后12,24,48和72 h时分析RNAi干扰效率,利用苏木精-伊红(hematoxylin-eosin,HE)染色法探究RNAi干扰2日幼虫AcMucin 5 AC-1后24,48和72 h时对中华蜜蜂幼虫整体组织形态的影响。【结果】中华蜜蜂基因组中有8个AcMucin5AC基因,其氨基酸序列均含有粘蛋白结构域。RT-qPCR检测结果表明,AcMucin 5 AC-1在中华蜜蜂4日龄幼虫中肠中表达量较表皮中的高,且在感染CSBV病毒的12和72 h时的2日龄幼虫中肠中比对照组显著下调。成功获得约50 kD的重组蛋白AcMucin5AC-1和多克隆抗体,Western blot结果显示在中华蜜蜂3-6日龄幼虫以及4日龄幼虫中肠、表皮和围食膜的总蛋白中均能检测到AcMucin5AC-1。间接免疫荧光试验结果表明AcMucin5AC-1主要定位在4日龄幼虫中肠和围食膜。RNAi效率检测结果表明,2.0μg/头ds AcMucin 5 AC-1干扰24 h时中华蜜蜂AcMucin 5 AC-1较ds EGFP对照组表达量下调92%。HE染色检测结果表明,在AcMucin 5 AC-1的RNAi后72 h时中华蜜蜂幼虫整个肠腔的细胞间的致密程度减弱,形态结构紊乱。【结论】中华蜜蜂AcMucin5AC-1是位于幼虫的中肠和围食膜的粘蛋白,AcMucin 5 AC-1的下调影响中华蜜蜂幼虫中肠的形态,暗示该基因在幼虫中肠发育过程中可能发挥重要作用。