Emerging role of cystic fibrosis transmembrane conductance regulator- an epithelial chloride channel in gastrointestinal cancers

Cystic fibrosis transmembrane conductance regulator(CFTR), a glycoprotein with 1480 amino acids, has been well established as a chloride channel mainly expressed in the epithelial cells of various tissues and organs such as lungs, sweat glands, gastrointestinal system, and reproductive organs. Although defective CFTR leads to cystic fibrosis, a common genetic disorder in the Caucasian population, there is accumulating evidence that suggests a novel role of CFTR in various cancers, especially in gastroenterological cancers, such as pancreatic cancer and colon cancer. In this review, we summarize the emerging findings that link CFTR with various cancers, with focus on the association between CFTR defects and gastrointestinal cancers as well as the underlying mechanisms. Further study of CFTR in cancer biology may help pave a new way for the diagnosis and treatment of gastrointestinal cancers.

Low Level of Cystic Fibrosis Transmembrane Conductance Regulator Is Associated with Human Sperm Autophagy and Vitality

Low sperm motility is one of the main causes of male infertility. Cystic fibrosis transmembrane conductance regulator (CFTR, an anion channel protein) is related to the progressive motility of sperm. CFTR disruptor CFTRinh-172 or forskolin (FSK) in this study were used to treat human sperm separately, and the rates of sperm autophagy and progressive motility, mitochondrial membrane potential (MMP) and ATP concentration, and the expression levels of related factors were detected to explore their relationship. It was showed that sperms treated with CFTRinh-172 or FSK reduced the levels of cAMP, CFTR and PKA, but increased sperm autophagy rate, expression levels of AMPK and LC3B. However, reactive oxygen species content had no significant difference. It was indicated that low level of CFTR performed with cAMP and its downstream effectors such as PKA and AMPK to regulate mitochondrial structure and function, leading to increased autophagy rate and reduced vitality of sperm.

Cystic fibrosis transmembrane conductance regulator chloride channel blockers:Pharmacological,biophysical and physiological relevance

Dysfunction of the cystic fibrosis transmembrane con-ductance regulator(CFTR) chloride channel causes cys-tic fibrosis, while inappropriate activity of this channeloccurs in secretory diarrhea and polycystic kidney dis-ease. Drugs that interact directly with CFTR are there-fore of interest in the treatment of a number of diseasestates. This review focuses on one class of small mol-ecules that interacts directly with CFTR, namely inhibi-tors that act by directly blocking chloride movementthrough the open channel pore. In theory such com-pounds could be of use in the treatment of diarrheaand polycystic kidney disease, however in practice allknown substances acting by this mechanism to inhibitCFTR function lack either the potency or specificity forin vivo use. Nevertheless, this theoretical pharmaco-logical usefulness set the scene for the developmentof more potent, specific CFTR inhibitors. Biophysically,open channel blockers have proven most useful as ex-perimental probes of the structure and function of theCFTR chloride channel pore. Most importantly, the useof these blockers has been fundamental in developing afunctional model of the pore that includes a wide innervestibule that uses positively charged amino acid sidechains to attract both permeant and blocking anionsfrom the cell cytoplasm. CFTR channels are also subjectto this kind of blocking action by endogenous anionspresent in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physio-logical control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease states, of CFTR chan-nel structure and function, and of how CFTR activity is controlled by its local environment.

Tetramethylpyrazine stimulates cystic fibrosis transmembrane conductance regulator-mediated anion secretion in distal colon of rodents

AIM: To investigate the effect of tetramethylpyrazine (TMP), an active compound from Ligustiun Wollichii Franchat, on electrolyte transport across the distal colon of rodents and the mechanism involved.METHODS: The short-circuit current (ISC) technique in conjunction with pharmacological agents and specific inhibitors were used in analyzing the electrolyte transport across the distal colon of rodents. The underlying cellular signaling mechanism was investigated by radioimmunoassay analysis (RIA) and a special mouse model of cystic fibrosis.RESULTS: TMP stimulated a concentration-dependent rise in ISC, which was dependent on both Cl- and HCO3-, and inhibited by apical application of diphenylamine-2,2'-dicarboxylic acid (DPC) and glibenclamide, but resistant to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS). Removal of Na+ from basolateral solution almost completely abolished the ISC response to TMP, but it was insensitive to apical Na+ replacement or apical Na+channel blocker, amiloride. Pretreatment of colonic mucosa with BAPTA-AM, a membrane-permeable selective Ca2+chelator, did not significantly alter the TMP-induced ISC. No additive effect of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was observed on the TMP-induced ISc, but it was significantly reduced by a protein kinase A inhibitor, H89.RIA results showed that TMP (1 mmol/L) elicited a significant increase in cellular cAMP production, which was similar to that elicited by the adenylate cyclase activator, forskolin (10 μmol/L). The TMP-elicited ISC as well as forskolin- or IBMX-induced ISC were abolished in mice with homozygous mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) presenting defective CFTR functions and secretions.CONCLUSION: TMP may stimulate cAMP-dependent and CFTR-mediated Cl- and HCO3- secretion. This may have implications in the future development of alternative treatment for constipation.

Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Rat Ovary

The protein expression of cystic fibrosis transmembrane conductance regulator （CFTR）, a cAMP-activated Cl- channel, in ovarian stimulated premature female rat ovary during a cycle of follicle development and corpus luteum formation was investigated. Animals were injected with 10 U pregnant Mare＇s serum gonadotropin （PMSG） and subsequently 10 U hCG 48 h later. Time-dependent immunohistochemistry and Western blotting experiments were performed before and 24, 48, 72 h after hCG treatment. The immunohistochemistry revealed that administration of PMSG stimulated the CFTR expression in thecal cell layer and granulosa cell layer of mature follicles 48 h post injection, coincident with the PMSG-induced peak in follicular estradiol. However, the expression of CFTR in the granulose lutein cell layer and thecal lutein cell layer was time-dependently reduced following hCG injection, in accordance with the gradually increased progestogen level during luteum corpus formation. Western blotting analysis demonstrated that rat ovarian tissue expressed the special CFTR band at 170 kD. It is concluded that cAMP-dependent Cl- channels are involved in regulation of follicle development and luteum formation.

Synthesis and Characterization of A Small Molecule CFTR Chloride Channel Inhibitor

A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of 1H NMR and functionally in a CFTR-expressing cell line FRT/hCFTR/EYFP-H148Q by both fluorescent and electrophysiological methods. A large amount(100 g) of high-quality small molecule thiazolidinone CFTR chloride channel inhibitor,CFTR_ inh-172 ,can be produced with this simple three-step synthetic procedure. The structure of the final product 2-thioxo-3-(3-trifluromethylphenyl)-5-[4-carboxyphenyl- methylene]-4-thiazolidinone was confirmed by 1H NMR. The overall yield was 58% with a purity over 99% as analyzed by HPLC. The synthesized CFTR_ inh-172 specifically inhibited CFTR chloride channel function in a cell-based fluorescence assay( K _d≈1.5 μmol/L) and in a Ussing chamber-based short-circuit current assay( K _d≈0.2 μmol/L),indicating better quality than that of the commercial combinatorial compound. The synthesized inhibitor is nontoxic to cultured cells at a high concentration and to mouse at a high dose. The synthetic procedure developed here can be used to produce a large amount of the high-quality CFTR_ inh-172 suitable for antidiarrheal studies and for creation of cystic fibrosis models in large animals. The procedure can be used to synthesize radiolabled CFTR_ inh-172 for in vivo pharmacokinetics studies.

Identification of Herbal Compound Imperatorin with Adverse Effects on ANO1 and CFTR Chloride Channels

Calcium-activated chloride channels（CaCCs） are the crucial regulators of transepithelial fluid secretion, smooth muscle contraction and sensory transduction. Recently, compelling evidence has indicated that TMEM16A（ANO1 or anoctamin-1） is a bona fide calcium-acvtivated chloride channel. A few small molecule CaCCs regulators are available for functional and therapeutic studies. We screened 126 natural compounds from Chinese herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells to coexpress ANO1 and an iodide-sensitive fluorescent indicator（EYFP-H148Q/I152L）. Imperatorin, a coumarin compound, was identified to inhibit ANO1-mediated chloride transport activated by multiple calcium-elevating agonists. The inhibitory effect is dose-dependent with IC50～14.63 μmol/L. Interestingly, imperatorin activated CFTR chloride channel with EC50～35.52 μmol/L. The adverse effects of imperatorin on CaCC and CFTR chloride channels will make it useful in pharmacological dissection of chloride transport in airway and intestinal epithelium. Further studies are required to evaluate the therapeutic effects of imperatorin on hypertension, asthma and certain tumors.

Direct Activation of CFTR Chloride Channel by Natural Compound Theophylline

Theophylline has been widely used in the＇treatment of airway disease but molecular mechanism has not been clearly elucidated. Previous studies have manifested that theophylline increases intracellular cAMP concentra- tion. Because cystic fibrosis transmembrane conductance regulator（CFTR） is a cAMP-dependent Cl- channel that plays key roles in fluid secretion in vivo, we postulated that theophylline activates CFTR channel gating. We found （1） theophylline stimulated CFTR-mediated anion transport in a concentration-dependent manner, and CFTR specific blocker completely reversed the effect; （2） theophylline had no effect on △F508 or G551D mutant CFTR chloride channel activity; （3） theophylline had additive effect with forskolin（FSK） and 3-isobutyl-xanthin（IBMX）, thus a direct binding activation mechanism was suggested. In conclusion, the results may provide a clue to elucidating the molecular mechanism of theophylline activities and theophylline may present a novel lead drug in treating CFTR-related disease.

Cystic fibrosis transmembrane conductance regulator prevents ischemia/reperfusion induced intestinal apoptosis via inhibiting PI3K/AKT/NF-κB pathway

BACKGROUND Intestinal ischemia/reperfusion(I/R)injury is a fatal syndrome that occurs under many clinical scenarios.The apoptosis of intestinal cells caused by ischemia can cause cell damage and provoke systemic dysfunction during reperfusion.However,the mechanism of I/R-induced apoptosis remains unclear.Cystic fibrosis transmembrane conductance regulator(CFTR)is a cAMP-activated chloride channel.Few researchers have paid attention to its role in intestinal I/R injury,or the relationship between CFTR and intestinal apoptosis induced by hypoxia/reoxygenation(H/R).AIM To investigate the effects of CFTR on I/R-induced intestinal apoptosis and its underlying molecular mechanisms.METHODS An intestinal I/R injury model was established in mice with superior mesenteric artery occlusion, and Caco2 cells were subjected to H/R for the simulation of I/R in vivo.RESULTSThe results suggested that CFTR overexpression significantly increased the Caco2 cell viability anddecreased cell apoptosis induced by the H/R. Interestingly, we found that the translocation of p65,an NF-κB member, from the cytoplasm to the nucleus after H/R treatment can be reversed by theoverexpression of CFTR, the NF-κB P65 would return from the nucleus to the cytoplasm asdetermined by immunostaining. We also discovered that CFTR inhibited cell apoptosis in theH/R-treated cells, and this effect was significantly curbed by the NF-κB activator BA, AKTinhibitor GSK690693 and the PI3K inhibitor LY294002. Moreover, we demonstrated that CFTRoverexpression could reverse the decreased PI3K/AKT expression induced by the I/R treatment invivo or H/R treatment in vitro.CONCLUSIONThe results of the present study indicate that the overexpression of CFTR protects Caco2 cells fromH/R-induced apoptosis;furthermore, it also inhibits H/R-induced apoptosis through thePI3K/AKT/NF-κB signaling pathway in H/R-treated Caco2 cells and intestinal tissues.

Cystic fibrosis transmembrane conductance regulator functional evaluations in a G542X+/-IVS8Tn:T7/9 patient with acute recurrent pancreatitis

BACKGROUND Acute recurrent pancreatitis(ARP)is characterized by episodes of acute pancreatitis in an otherwise normal gland.When no cause of ARP is identifiable,the diagnosis of"idiopathic"ARP is given.Mutations in the cystic fibrosis transmembrane conductance regulator(CFTR)gene increase the risk of ARP by 3-to 4-times compared to the general population,while cystic fibrosis(CF)patients present with a 40-to 80-times higher risk of developing pancreatitis.CASE SUMMARY In non-classical CF or CFTR-related disorders,CFTR functional tests can help to ensure a proper diagnosis.We applied an individualized combination of standardized and new CFTR functional bioassays for a patient referred to the Verona CF Center for evaluation after several episodes of acute pancreatitis.The CFTR genotype was G542X+/-with IVS8Tn:T7/9 polymorphism.The sweat(Cl-)values were borderline.Intestinal current measurements were performed according to the European Cystic Fibrosis Society Standardized Operating Procedure.Recent nasal surgery for deviated septum did not allow for nasal potential difference measurements.Lung function and sputum cultures were normal;azoospermia was excluded.Pancreas divisum was excluded by imaging but hypoplasia of the left hepatic lobe was detected.Innovative tests applied in this case include sweat rate measurement by image analysis,CFTR function in monocytes evaluated using a membrane potential-sensitive fluorescent probe,and the intestinal organoids forskolin-induced swelling assay.CONCLUSION Combination of innovative CFTR functional assays might support a controversial diagnosis when CFTR-related disorders and/or non-classical CF are suspected.

Epithelial Sodium and Chloride Channels and Asthma

Objective：To focus on the asthmatic pathogenesis and clinical manifestations related to epithelial sodium channel （ENaC）/chlorine ion channel.Data Sources：The data analyzed in this review were the English articles from 1980 to 2015 from journal databases,primarily PubMed and Google Scholar.The terms used in the literature search were：（1） ENaCs;cystic fibrosis （CF） transmembrane conductance regulator （CFTR）;asthma/asthmatic,（2） ENaC/sodium salt;CF;asthma/asthmatic,（3） CFTR/chlorine ion channels;asthma/asthmatic,（4） ENaC/sodium channel/scnn1a/scnn1b/scnn1g/scnn1d/amiloride-sensitive/amiloride-inhibtable sodium channels/sodium salt;asthma/asthmatic,lung/pulmonary/respiratory/tracheal/alveolar,and （5） CFTR;CF;asthma/asthmatic （ti）.Study Selection：These studies included randomized controlled trials or studies covering asthma pathogenesis and clinical manifestations related to ENaC/chlorine ion channels within the last 25 years （from 1990 to 2015）.The data involving chronic obstructive pulmonary disease and CF obtained from individual studies were also reviewed by the authors.Results：Airway surface liquid dehydration can cause airway inflammation and obstruction.ENaC and CFTR are closely related to the airway mucociliary clearance.Ion transporters may play a critical role in pathogenesis of asthmatic exacerbations.Conclusions：Ion channels have been the center of many studies aiming to understand asthmatic pathophysiological mechanisms or to identify therapeutic targets for better control of the disease.

c.753＿754delAG, a novel CFTR mutation found in a Chinese patient with cystic fibrosis: A case report and review of the literature

BACKGROUND Cystic fibrosis(CF)is rare in Asian populations relative to the Caucasian population.In this paper,we report the cystic fibrosis transmembrane conductance regulator(CFTR)variation in a family of Chinese CF patients,and systematically review the previous literature.CASE SUMMARY Here we report a 30-month-old Chinese girl who was diagnosed with CF based on her history and symptoms such as recurrent productive cough,wheezing with repeated infection of Pseudomonas aeruginosa,and parasinusitis.Chest computed tomography(CT)scanning revealed obvious exudative lesions and bilateral bronchiectasis.Liver CT scanning revealed a low-density lesion in the left lobe of the liver.A diagnosis of CF was made based upon CFTR gene tests.The CFTR gene was sequenced using the blood samples of her and her parents and showed a heterozygous novel missense mutation of c.753_754delAG in exon 7.In addition,a heterozygous c.1240 C>T mutation was found in exon 10 of the CFTR.The mutation c.753_754delAG was verified to have been inherited from her mother,and the c.1240 C>T mutation was from her father who was diagnosed with congenital absence of vas deferens.CONCLUSION A novel mutation of CFTR,c.753_754delAG,was found in a Chinese CF child.c.2909G>A is the most common mutation among Chinese CF patients.

Cystic fibrosis associated liver disease in children

Cystic fibrosis(CF)is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator gene.CF liver disease develops in 5%-10%of patients with CF and is the third leading cause of death among patients with CF after pulmonary disease or lung transplant complications.We review the pathogenesis,clinical presentations,complications,diagnostic evaluation,effect of medical therapies especially CF transmembrane conductance regulator modulators and liver transplantation in CF associated liver disease.

Lumacaftor/ivacaftor therapy is associated with reduced hepatic steatosis in cystic fibrosis patients

BACKGROUND Hepatic steatosis is a common form of cystic fibrosis associated liver disease(CFLD)seen in an estimated 15%-60%of patients with cystic fibrosis(CF).The pathophysiology and health implications of hepatic steatosis in cystic fibrosis remain largely unknown.In the general population,hepatic steatosis is strongly associated with insulin resistance and type 2 diabetes.Cystic fibrosis related diabetes(CFRD)impacts 40%-50%of CF adults and is characterized by both insulin insufficiency and insulin resistance.We hypothesized that patients with CFRD would have higher levels of hepatic steatosis than cystic fibrosis patients without diabetes.AIM To determine whether CFRD is associated with hepatic steatosis and to explore the impact of lumacaftor/ivacaftor therapy on hepatic steatosis in CF.METHODS Thirty patients with CF were recruited from a tertiary care medical center for this cross-sectional study.Only pancreatic insufficient patients with CFRD or normal glucose tolerance(NGT)were included.Patients with established CFLD,end stage lung disease,or persistently elevated liver enzymes were excluded.Mean magnetic resonance imaging(MRI)proton density fat fraction(PDFF)was obtained for all participants.Clinical characteristics[age,sex,body mass index,percent predicted forced expiratory volume at 1 s(FEV1),lumacaftor/ivacaftor use]and blood chemistries were assessed for possible association with hepatic steatosis.Hepatic steatosis was defined as a mean MRI PDFF>5%.Patients were grouped by diabetes status(CFRD,NGT)and cystic fibrosis transmembrane conductance regulator(CFTR)modulator use(lumacaftor/ivacaftor,no lumacaftor/ivacaftor)to determine between group differences.Continuous variables were analyzed with a Wilcoxon rank sum test and discrete variables with a Chi square test or Fisher’s exact test.RESULTS Twenty subjects were included in the final analysis.The median age was 22.3 years(11.3-39.0)and median FEV1 was 77%(33%-105%).Twelve subjects had CFRD and 8 had NGT.Nine subjects were receiving lumacaftor/ivacaftor.The median PDFF was 3.0%(0.0%-21.0%).Six subjects(30%)had hepatic steatosis defined as PDFF>5%.Hepatic fat fraction was significantly lower in patients receiving lumacaftor/ivacaftor(median,range)(2.0%,0.0%-6.4%)than in patients not receiving lumacaftor/ivacaftor(4.1%,2.7-21.0%),P=0.002.Though patients with CFRD had lower PDFF(2.2%,0.0%-14.5%)than patients with NGT(4.9%,2.4-21.0%)this did not reach statistical significance,P=0.06.No other clinical characteristic was strongly associated with hepatic steatosis.CONCLUSION Use of the CFTR modulator lumacaftor/ivacaftor was associated with significantly lower hepatic steatosis.No association between CFRD and hepatic steatosis was found in this cohort.

Cystic fibrosis-related diabetes:The unmet need

Cystic fibrosis(CF)is a common autosomal recessive disease.Life expectancy of patients with CF continues to improve mainly driven by the evolving therapies for CF-related organ dysfunction.The prevalence of CF-related diabetes(CFRD)increases exponentially as patients’age.Clinical care guidelines for CFRD from 2010,recommend insulin as the mainstay of treatment.Many patients with CFRD may not require exogenous insulin due to the heterogeneity of this clinical entity.Maintenance of euglycemia by enhancing endogenous insulin production,secretion and degradation with novel pharmacological therapies like glucagonlike peptide-1 agonist is an option that remains to be fully explored.As such,the scope of this article will focus on our perspective of glucagon-like peptide-1 receptor agonist in the context of CFRD.Other potential options such as sodiumglucose cotransporter-2 and dipeptidyl peptidase 4 inhibitors and their impact on this patient population is limited and further studies are required.

Ginkgolide C Stimulates CFTR-mediate Anion Conductance in Distal Colon:Implication for Therapy of Gastrointestinal Diseases

The effects and the mechanisms of natural compounds ginkgolides on CFTR-mediate anion transport were investigated. The CFTR-mediate iodide influx rates were studied via a cell-based fluorescence assay done for FRT cells stably transfected by CFTR; transepithelial short-circuit current recordings of FRT cells and rat distal colon mucosa were respectively obtained. Cellular cAMP concentrations were measured via a radioimmunoassay analysis kit. Ginkgolide C dose-dependently increases CFTR-mediate anion transport, whereas ginkgolide A and B show no effect. The activation is sensitive to CFTR specific activator CFTRinh-172. Ginkgolide C stimulated amiloride and indomethacin pre-treated Cl currents in rat distal colon mucosa. Studies on FRT cells also manifest that ginkgolide C had additive effect with FSK/IBMX mixture and didn＇t elevate intracellular cAMP concentration, which implies it works through a direct binding mechanism. In conclusion, Ginkgolide C directly stimulates CFTR-mediate anion transport. Ginkgolide C may be a promising drug for the prevention and treatment of CFTR-related diseases such as idiopathic chronic pancreatitis（ICP）, habitual constipation, and kcratoconjunctivitis sicca（KCS）.

获得性囊性纤维化跨膜传导调节因子功能障碍及炎性因子在儿童鼻-鼻窦炎中的表达特征分析

目的探讨获得性囊性纤维化跨膜传导调节因子(cystic fibrosis transmembrane conductance regulator,CFTR)功能障碍及炎性细胞因子在儿童鼻-鼻窦炎发展中的作用。方法纳入2021年1—12月在首都医科大学附属北京儿童医院耳鼻咽喉头颈外科因急慢性鼻-鼻窦炎行鼻内镜手术治疗31例患儿,其中慢性鼻窦炎(chronic rhinosinusitis,CRS)患儿17例(CRS组),急性细菌性鼻窦炎(acute bacterial rhinosinusitis,ABRS)患儿14例(ABRS组)。收集所有患儿的全血、鼻黏膜及鼻息肉样本,应用全外显子二代测序技术检测全血样本中CFTR基因的表达情况,q RT-PCR检测息肉和黏膜组织中CFTR mRNA的表达水平,微量样本多重蛋白定量技术(cytometric bead array,CBA)检测息肉和黏膜组织中炎性细胞因子的表达水平。结果根据CFTR基因检测结果,所有入组患儿均排除囊性纤维化。ABRS组鼻黏膜组织CFTR mRNA的表达水平明显低于CRS组,而CRS组鼻息肉组织CFTR mRNA的表达水平明显低于鼻黏膜组织。ABRS组鼻黏膜组织γ干扰素(interferon-γ,IFN-γ)、白细胞介素-4(interleukin-4,IL-4)、IL-13、IL-6、IL-8的表达水平明显高于CRS组(P<0.05),而CRS组鼻息肉组织IL-6、IL-8、单核细胞趋化因子-1(monocyte chemoattractant protein-1,MCP-1)的表达水平明显高于鼻黏膜组织(P<0.01)。结论获得性CFTR功能障碍及与中性粒细胞趋化相关的炎性因子在ABRS中的作用更为明显,在CRS息肉的发生发展中也可能起到一定作用。

LINC01089下调miR-1246对胰腺癌细胞增殖和迁移侵袭的影响

目的阐明长基因间非蛋白质编码RNA 1089(LINC01089)调控微小RNA-1246(miR-1246)在胰腺癌细胞增殖、迁移和侵袭的作用。方法采用实时定量PCR(qPCR)检测LINC01089和miR-1246在人胰腺癌细胞(CFPAC-1、PANC-1、AsPC-1、SW1990、BxPC-3)的表达。选择BxPC-3细胞并分为对照组、pcDNA3.1组、LINC01089过表达组(转染pcDNA3.1-LINC01089)和LINC01089+miR-1246过表达组(转染pcDNA3.1-LINC01089和miR-1246模拟物mimics)。活细胞计数试剂盒-8、划痕和Transwell小室实验评价细胞增殖、迁移和侵袭能力。双荧光素酶报告基因实验和qPCR验证miR-1246与LINC01089和囊性纤维化跨膜传导调节因子(CFTR)的靶向关系,qPCR和Western blot检测基质金属蛋白酶(MMP)3、Snail和CFTR的表达情况。结果与人正常胰腺导管细胞HPDE相比,胰腺癌细胞的LINC01089低表达而miR-1246高表达(P<0.05)。与对照组相比,LINC01089过表达组细胞的增殖、迁移和侵袭能力均受到抑制,且MMP3和Snail表达水平降低(P<0.05)。与LINC01089过表达组相比,LINC01089+miR-1246过表达组细胞的增殖、迁移和侵袭能力增强,且MMP3和Snail表达水平升高(P<0.05)。LINC01089可靶向调控miR-1246的表达而CFTR为miR-1246的靶基因。结论LINC01089可能通过miR-1246介导CFTR表达下调来抑制胰腺癌细胞的增殖、迁移和侵袭,LINC01089/miR-1246轴有望成为胰腺癌防治的靶点。

ADAMTS9-AS2调控miRNA-1290和CFTR的表达及对三阴性乳腺癌细胞增殖、迁移与侵袭的影响

目的本研究旨在探究长链非编码RNA(lncRNA)ADAMTS9反义RNA 2(ADAMTS9-AS2)靶向调控微小RNA-1290(miR-1290)和囊性纤维化跨膜传导调节因子(CFTR)的表达及对三阴性乳腺癌细胞增殖、迁移和侵袭的影响。方法实时荧光定量PCR(qRT-PCR)检测ADAMTS9-AS2在乳腺癌组织和细胞系(MCF-7、MDA-MB-453、T47D、MDA-MB-468、MDA-MB-231和SK-BR-3)中的表达情况。培养MDA-MB-231细胞,分为:对照组(空白)、pcDNA组(阴性对照)和ADAMTS9-AS2过表达组。MTT法、划痕实验和Transwell实验分别检测MDA-MB-231细胞的增殖、迁移和侵袭。双荧光素酶报告基因实验、qRT-PCR和Western blot验证ADAMTS9-AS2和miR-1290靶向关系。结果ADAMTS9-AS2在乳腺癌组织(0.444±0.045)中的相对表达水平低于癌旁组织(1.000±0.062)(P<0.01)。与癌旁正常乳腺组织相比(1.000±0.092),miR-1290在乳腺癌组织中相对表达水平更高(1.504±0.104)(P<0.01)。ADAMTS9-AS2在乳腺癌细胞MCF-7、MDA-MB-453、T47D、MDA-MB-468、MDA-MB-231和SK-BR-3相对表达量低于正常乳腺上皮细胞(MCF-10A),miR-1290在乳腺癌细胞MCF-7、MDA-MB-453、T47D、MDA-MB-468、MDA-MB-231和SK-BR-3中相对表达量高于MCF-10A细胞,其中MDA-MB-231的ADAMTS9-AS2相对表达量最低,miR-1290相对表达量最高。ADAMTS9-AS2过表达组MDA-MB-231细胞增殖(48 h和72 h)、迁移(12 h和24 h)和侵袭(24 h)能力均低于对照组和pcDNA组,CFTR的mRNA和蛋白表达水平均高于对照组和pcDNA组(均P<0.05)。ADAMTS9-AS2可靶向结合miR-1290。结论ADAMTS9-AS2可能调控miR-1290和CFTR表达,抑制乳腺癌细胞的迁移和侵袭,提示ADAMTS9-AS2可能是三阴性乳腺癌治疗的潜在靶点。

CFTR在原发性肝细胞癌中的表达及临床意义

目的:探究细胞囊性纤维化跨膜转导调节因子(CFTR)在原发性肝细胞癌(HCC)中的表达水平及其与患者临床特征及预后的关联。方法:收集HCC患者癌组织和癌旁组织,检测样本中CFTR的基因表达,分析CFTR表达水平与HCC患者临床病理特征及预后的关联。结果:CFTR在HCC癌组织中的表达水平低于癌旁组织(P<0.05);患者的各种临床病理特征在CFTR低表达组与CFTR高表达组间的分布比较,差异均无统计学意义(P>0.05);生存分析显示,CFTR低表达患者的平均生存时间低于CFTR高表达患者,但两组比较差异无统计学意义(P>0.05);Cox回归模型分析显示,癌组织中CFTR的表达与HCC患者死亡风险无统计学意义(P>0.05)。结论:CFTR在HCC中可能是一个抑癌基因,但尚未发现CFTR表达水平与HCC患者的临床病理特征及预后的关联。