棉铃虫叉头框蛋白A类似蛋白基因HarmFoxAl的克隆及表达谱分析

【目的】本研究旨在克隆并分析一种棉铃虫Helicoverpa armigera叉头框蛋白A (forkhead box protein A, FoxA)类似蛋白基因HarmFoxAl,探讨2-十三烷酮胁迫下棉铃虫中肠中HarmFoxAl的表达情况,为进一步明确棉铃虫FoxA的功能和参与棉铃虫生长发育的调控通路提供依据。【方法】从棉铃虫幼虫中肠中扩增得到HarmFoxAl的cDNA序列,并对其氨基酸序列和蛋白结构进行分析。将HarmFoxAl的ORF序列连接至pET32a载体并转化大肠杆菌Escherichia coli Transetta菌株,IPTG诱导后检测目的蛋白的表达形式,并利用镍柱亲和层析法纯化融合蛋白。通过qPCR检测棉铃虫不同发育阶段(1-6龄幼虫和预蛹),6龄幼虫不同组织(脂肪体、中肠、体壁和头部)以及10 mg/g 2-十三烷酮处理6龄幼虫不同时间后中肠中HarmFoxAl的表达谱。【结果】HarmFoxAl(GenBank登录号:XM021331806)的开放阅读框为669 bp,编码222个氨基酸,蛋白的相对分子质量和等电点分别为25.03 kD和6.34。氨基酸序列分析表明,HarmFoxAl单体蛋白无信号肽、跨膜区和二硫键,核心区域是由4个α螺旋和3个β折叠组成的球状结构。将重组的Transetta (pET32a-HarmFoxAl)菌株用0.5 mmol/L IPTG在25℃条件下诱导5 h,约45 kD的融合蛋白His-HarmFoxAl能以可溶的形式存在于重组菌中,这与预测的分子量(42.8 kD)相一致。发育阶段特异性表达谱表明,HarmFoxAl在棉铃虫1-3龄幼虫期、6龄幼虫期和预蛹期均有表达,且预蛹期的表达量最高。组织表达谱结果表明,该基因在6龄幼虫的脂肪体、中肠和体壁中表达,且脂肪体内的表达量最高,而在头部中不表达。10 mg/g 2-十三烷酮处理棉铃虫6龄幼虫后中肠中HarmFoxAl的表达量显著降低,但随着时间延长其表达量逐渐升高,处理48 h后表达量显著高于对照。【结论】棉铃虫HarmFoxAl在预蛹期和幼虫脂肪体中表达量最高,2-十三烷酮处理幼虫后HarmFoxAl的表达量急速降低后逐渐升高,推测其在棉铃虫变态发育和解毒代谢过程中发挥重要作用。     

自噬基因PULK、PI3KC3在宫颈病变的表达及相关性研究

目的探讨自噬基因PULK、PI3KC3在正常宫颈组织及病变宫颈组织的表达及相关性。方法随机抽取正常宫颈组织30例、CIN20例及宫颈癌45例,采用qRT-PCR法检测组织中PULK和PI3KC3基因表达水平,免疫组织化学染色方法定性检测PULK、PI3KC3在正常宫颈组织及病变宫颈组织中的表达情况。结果免疫组化结果显示,PULK和PI3KC3在CIN及宫颈癌组织中的阳性表达显著低于正常宫颈组织,CIN及宫颈癌组织的PULK和PI3KC3表达水平均低于正常宫颈组织(P0.05或P0.01)。PULK与PI3KC3表达呈正相关(r=0.862,P0.01)。结论 PULK和PI3KC3可能参与宫颈病变的恶性转变过程。     

CEACAM1 in cervical cancer and precursor lesions: association with human papillomavirus infection.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is an adhesion molecule expressed in a wide variety of tissues including epithelial cells, leukocytes, and tumors that may establish both homotypic and heterotypic interactions. The aim of this work was to study the protein expression pattern of CEACAM1 in cervical cancer and precursor lesions in the context of human papillomavirus (HPV) infection. We used immunohistochemistry to analyze CEACAM1 expression in formalin-fixed, paraffin-embedded cervical tissues from 15 healthy women, 15 patients with low-grade squamous intraepithelial lesions (SIL), 15 patients with high-grade SIL, and 15 patients with squamous carcinomas. HPV types were identified by PCR. CEACAM1 was either undetectable (13/15) or low (2/15) in normal cervical tissues. By contrast, CEACAM1 expression was increased in high-grade SIL (10 samples staining intermediate/high and 4 samples staining low) as compared with low-grade SIL with undetectable (n=3) or low (n=12) expression. CEACAM1 expression was undetectable or low in cervical carcinoma. Our results suggest that CEACAM1 may be an interesting progression marker in SIL and cervical cancer, in particular due to reported immunoregulatory properties.     

FoxP3 genetic variants and risk of non-small cell lung cancer in the Chinese Han population

CD4⁺CD25⁺ regulatory T cell-mediated immunosuppression is one of the crucial mechanisms that tumor cells use to evade the immune system. The forkhead box P3 (FoxP3) gene regulates regulatory T-cell development and function and may modulate the susceptibility to non-small cell lung cancer (NSCLC). Because a single nucleotide polymorphism (SNP) within the FoxP3 gene (rs3761548 in the promoter region) is associated with susceptibility to Graves' disease, this study detected rs3761548 in a hospital-based case–control study. A total of 192 NSCLC patients and 259 healthy subjects were recruited for the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis of FoxP3 SNP. The data showed that the A allele of rs3761548 significantly increased NSCLC risk (P = 0.000, OR = 2.32, 95%CI = 1.736–3.102). The AC genotype, AA genotype, and the combined A variant genotype (AA + AC) were also associated with a higher risk of NSCLC (OR [95%CI] = 2.147[1.419–3.247], 4.413[2.359–8.255], and 2.563[1.746–3.761], respectively). Moreover, a significantly higher frequency of AA + AC genotype was observed in patients with stage II NSCLC (OR, 2.053; 95%CI, 1.033–4.078). In conclusion, the data from the current study demonstrated for the first time the association of the FoxP3 SNP with a risk of developing NSCLC in the Chinese Han population.     

Elevated glucose-6-phosphate dehydrogenase expression in the cervical cancer cases is associated with the cancerigenic event of high-risk human papillomaviruses

The most important etiologic agent in the pathogenesis of cervical cancers (CCs) is human papillomavirus (HPV), while the mechanisms underlying are still not well known. Glucose-6-phosphate dehydrogenase (G6PD) is reported to elevate in various tumor cells. However, no available references elucidated the correlation between the levels of G6PD and HPV-infected CC until now. In the present study, we explored the possible role of G6PD in the pathology of CC induced by HPV infection. Totally 48 patients with HPV + CC and another 63 healthy women enrolled in the clinical were employed in the present study. Overall, prevalence of cervical infection with high-risk-HPV (HR-HPV) type examined was HPV-16, followed by HPV-18. The expressions of G6PD in CC samples were also detected by immunohistochemistry (IHC), qRT-PCR, and Western blot. Regression analysis showed elevated G6PD level was positively correlated with the CC development in 30–40 aged patients with HR-HPV-16/18 infection. The HPV16 + Siha, HPV18 + Hela, and HPV-C33A cell lines were employed and transfected with G6PD deficient vectors developed in vitro. MTT and flow cytometry were also employed to determine the survival and apoptosis of CC cells after G6PD expressional inhibition. Our data revealed that G6PD down-regulation induced poor proliferation and more apoptosis of HPV18 + Hela cells, when compared with that of HPV16 + Siha and HPV-C33A cells. These findings suggest that G6PD expressions in the HR-HPV + human CC tissues and cell lines play an important role in tumor growth and proliferation.     

宫颈癌患者人乳头瘤病毒感染情况及STING、ZEB1蛋白水平分析

分析宫颈癌患者人乳头瘤病毒（HPV）感染情况及癌灶组织内干扰素基因刺激因子（STING）、E盒锌指结合蛋白1（ZEB1）表达情况,为该类患者的治疗提供参考。

收集我院病理检验科2019年1月至2022年1月63例宫颈癌标本（宫颈癌组）、55例宫颈上皮内瘤变（CIN）标本（CIN组）及50例正常宫颈标本（正常组）,采用免疫组化法检测各标本内STING及ZEB1表达情况,采用原位杂交法检测各标本内高危HPV感染情况,分析STING及ZEB1蛋白表达情况与宫颈癌病理特征及高危HPV感染之间的关系。

宫颈癌组标本STING及ZEB1表达量均高于CIN组及正常组,CIN组标本ZEB1表达量高于正常组,差异均有统计学意义（均P＜0.05）;CIN组标本STING表达量与正常组比较差异无统计学意义（P＞0.05）。宫颈癌组标本HPV16、18检出率高于CIN组及正常组,CIN组标本HPV16、18检出率高于正常组,差异均有统计学意义（均P＜0.05）。宫颈癌患者FIGO分期及浸润深度与其组织内STING及ZEB1蛋白阳性情况均存在相关性;此外,宫颈癌淋巴结转移及肿瘤分化程度与组织内STING蛋白阳性情况存在相关性,差异均有统计学意义（均P＜0.05）。宫颈癌患者STING及ZEB1蛋白表达与其HPV16、18检出率间均呈正相关（均P＜0.05）。

宫颈癌组标本STING及ZEB1蛋白表达量均高于正常组及CIN组,且其表达量还与宫颈癌浸润、FIGO分期相关。STING和ZEB1可能与HPV16、18感染共同参与宫颈癌的发生及进展。

     

北京地区宫颈癌HPV16上游调控序列、E6、E7癌基因序列初步分析

为检测HPV16上游调控序列(Upstream regulatory region, URR)、E6、E7癌基因变异在北京地区宫颈癌患者癌组织中的分布特征, 探讨该地区宫颈癌发生同HPV16变异株间的相关性, 文章以提取的31例HPV16检测阳性宫颈癌组织DNA为模板, 设计针对性引物扩增URR、E6、E7 3个目的片段, PCR产物直接测序并通过GenBank对比分析变异和分支鉴定情况。在所分析的宫颈癌组织中, URR是突变频率最高的片段, 其次为E7, 最保守的序列为E6。共发现热突变位点8个, 分别为URR序列上G7521A(100%)、C7435G(96.77%)、C24T(45.16%)、A7729C(45.16%)、G7839A(45.16%); E6序列上T178G(41.94%); E7序列上A647G(45.16%)、T846C(45.16%)。HPV16分支分布频率最广的是As型(54.84%), 其次为E型(45.16%)。研究结果提示, HPV16URR序列上G7521A、A7729C、G7839A, E6序列上T178G、T350G, E7序列上A647G、G658A等位点的变异可能与病毒致癌潜能及宫颈癌的发生相关。北京地区宫颈癌患者中As和E型可能是两种最主要的HPV16分支, 这有可能会为HPV疫苗的研制和感染治疗提供有价值的信息。As型和E型病毒在不同年龄组和不同肿瘤分期组的患者中分布频率有差异, 这可能会为揭示宫颈癌年轻化趋势提供新的线索。     

Functional characterization of human myosin-binding protein C3 variants associated with hypertrophic cardiomyopathy reveals exon-specific cardiac phenotypes in zebrafish model

Myosin-binding protein C 3 (MYBPC3) variants are the most common cause of hypertrophic cardiomyopathy (HCM). HCM is a complex cardiac disorder due to its significant genetic and clinical heterogeneity. MYBPC3 variants genotype–phenotype associations remain poorly understood. We investigated the impact of two novel human MYBPC3 splice-site variants: V1: c.654+2_654+4dupTGG targeting exon 5 using morpholino MOe5i5; and V2: c.772+1G>A targeting exon 6 using MOe6i6; located within C1 domain of cMyBP-C protein, known to be critical in regulating sarcomere structure and contractility. Zebrafish MOe5i5 and MOe6i6 morphants recapitulated typical characteristics of human HCM with cardiac phenotypes of varying severity, including reduced cardiomyocyte count, thickened ventricular myocardial wall, a drastic reduction in heart rate, stroke volume, and cardiac output. Analysis of all cardiac morphological and functional parameters demonstrated that V2 cardiac phenotype was more severe than V1. Coinjection with synthetic human MYBPC3 messenger RNA (mRNA) partially rescued disparate cardiac phenotypes in each zebrafish morphant. While human MYBPC3 mRNA partially restored the decreased heart rate in V1 morphants and displayed increased percentages of ejection fraction, fractional shortening, and area change, it failed to revert the V1 ventricular myocardial thickness. These results suggest a possible V1 impact on cardiac contractility. In contrast, attempts to rescue V2 morphants only restored the ventricular myocardial wall hypertrophy phenotype but had no significant effect on impaired heart rate, suggesting a potential V2 impact on the cardiac structure. Our study provides evidence of an association between MYBPC3 exon-specific cardiac phenotypes in the zebrafish model providing important insights into how these genetic variants contribute to HCM disease.     

ABC3 Part I: a review of the guidelines for terminology,classification and management of cervical cytology in England

J. H. F. Smith ABC3 Part I: a review of the guidelines for terminology, classification and management of cervical cytology in England The provision of guidance on cytology reporting and evaluation, first outlined in 1995 with the publication of Achievable Standards, Benchmarks for Reporting, and Criteria for Evaluating Cervical Cytopathology (ABC), and subsequently revised and expanded in a second edition in 2000, has been pivotal to the success of the National Health Service Cervical Screening Programme (NHSCSP), ensuring that standards are upheld, and that rigorous evaluation and quality assurance take place. In the last decade, major changes to the NHSCSP, notably the adoption of revised age ranges and screening intervals for all women in England, implementation of liquid‐based cytology and, most recently, the decision to introduce high‐risk human papillomavirus (HR‐HPV) testing for triage of low‐grade and borderline (equivalent to 'atypical') cytological abnormalities and test of cure after treatment of cervical intraepithelial neoplasia (CIN) determined that an updated version of ABC was required. The third edition of ABC recommends adoption, with minor modification, of the revised British Society for Clinical Cytology terminology and provides guidance on the management of abnormal cytology results linked to this terminology taking account of HR‐HPV testing. To accommodate these changes, expanded result codes, which are electronic codes used to transfer management information to central computers for follow‐up, call and recall of individual women, have been developed. Further guidance on specimen adequacy is also provided. Revised performance indicators are described and explained in a separate article by R. Blanks in this issue of Cytopathology. All the changes in ABC3 are designed to support the mission statement of the NHSCSP that ‘the objective of cervical screening is to reduce cervical cancer incidence and mortality by screening with a high sensitivity for the detection of CIN2 or worse, whilst maintaining a high specificity’.     

Review of negative and low‐grade cervical smears in women with invasive cervical cancer after the first 3 years of the national cervical screening programme in Slovenia

A. Repše‐Fokter, A. Pogačnik, V. Snoj, M. Primic‐Žakelj and M. S. Fležar
Review of negative and low‐grade cervical smears in women with invasive cervical cancer after the first 3 years of the national cervical screening programme in Slovenia Objective: The purpose of the study was to perform a national review of negative, low‐grade and inadequate smears reported during the latest screening period before cervical cancer diagnosis in 2006, after the first 3 years of the screening programme. Methods: Among 162 new cervical cancer cases there were 47 (29%) without previous cytology, 47 (29%) with one high‐grade smear prior to diagnosis and 68 (42.0%) with at least one previous negative, low‐grade, atypical or inadequate smear 1–40 months before diagnosis. Of the latter 68 cases, 37 patients with 59 smears (together with 118 control slides) were included in the review as 31 had smears reported at laboratories no longer operating. Findings were related to the last cytology report before diagnosis as well as to histological type and stage of the cancer. Results: In our study group, 19 (51%) of 37 patients had squamous cell carcinoma, 15 (41%) adenocarcinoma and 3 (8%) adenosquamous carcinoma, compared with 121 (75%), 26 (16%), 12 (7%), respectively, and 3 (2%) other types, for all carcinomas. Twenty‐one of 37 women also had high‐grade cytology prior to diagnosis of cancer. Women with previous cytology (with or without recent high‐grade smears) were more likely to have stage I cancers than those without cytology (P < 0.0001). The expert group upgraded 17/33 smears in the patients with squamous carcinomas, which was more than in those with adeno‐ and adenosquamous carcinomas (5/24, P < 0.05). Conclusion: As expected, a higher proportion of smears preceding adenocarcinomas were true negative. Under‐diagnosed smears were not related to cancer stage or last cytology report before diagnosis.     

3p14.1 de novo microdeletion involving the FOXP1 gene in an adult patient with autism,severe speech delay and deficit of motor coordination

Interstitial deletion of chromosome region 3p14.1, including FOXP1 gene, is relatively rare and, until recently, there were no strong evidences to support the hypothesis that this microdeletion could play a role in the etiology of genomic disorders. Here, we report on an adult patient with a recognizable phenotype of autism, severe speech delay, deficit of motor coordination and typical dysmorphic features. Analysis of a dense whole genome single-nucleotide polymorphism (SNP) array showed a 1 Mb interstitial deletion of chromosome region 3p14.1 including the entire coding region of FOXP1 (MIM 605515) gene. In order to study the parental origin of the deletion, we analyzed selected SNPs in the deleted area in the proband and his parents showing Mendelian incompatibilities suggesting a de novo deletion on the chromosome of paternal origin. Despite the frequency of this genomic alteration has not been estimated, our patient confirm the hypothesis that microdeletion of 3p14.1 seems to be a rare cause of cognitive disorders and that haploinsufficiency of FOXP1 may play a role in neurological and language deficits in patients carrying a 3p14.1 deletion. Finally, our patient is also important because useful to further delineate the clinical spectrum secondary to the 3p14.1 microdeletions.     

Human bone marrow mesenchymal stem cell-derived extracellular vesicles impede the progression of cervical cancer via the miR-144-3p/CEP55 pathway

Cervical cancer is the most common gynaecological malignancy, with a high incidence rate and mortality rate in middle-aged women. Human bone marrow mesenchymal stem cells (hBMSCs) have been implicated in the initiation and subsequent development of cancer, along with the involvement of extracellular vesicles (EVs) mediating intracellular communication by delivering microRNAs (miRNAs or miRs). This study is aimed at investigating the physiological mechanisms by which EVs-encapsulated miR-144-3p derived from hBMSCs might mediate the progression of cervical cancer. The expression profiles of centrosomal protein, 55 Kd (CEP55) and miR-144-3p in cervical cancer cell lines and tissues, were quantified by RT-qPCR and Western blot analysis. The binding affinity between miR-144-3p and CEP55 was identified using in silico analysis and luciferase activity determination. Cervical cancer cells were co-cultured with EVs derived from hBMSCs that were treated with either miR-144-3p mimic or miR-144-3p inhibitor. Cervical cancer cell proliferation, invasion, migration and apoptosis were detected in vitro. The effects of hBMSCs-miR-144-3p on tumour growth were also investigated in vivo. miR-144-3p was down-regulated, whereas CEP55 was up-regulated in cervical cancer cell lines and tissues. CEP55 was targeted by miR-144-3p, which suppressed cervical cancer cell proliferation, invasion and migration and promoted apoptosis via CEP55. Furthermore, similar results were obtained by hBMSCs-derived EVs carrying miR-144-3p. In vivo assays confirmed the tumour-suppressive effects of miR-144-3p in hBMSCs-derived EVs on cervical cancer. Collectively, hBMSCs-derived EVs-loaded miR-144-3p impedes the development and progression of cervical cancer through target inhibition of CEP55, therefore providing us with a potential therapeutic target for treating cervical cancer.     

香菇C91-3菌丝发酵液提取蛋白抗小鼠宫颈癌U14的初步探讨

目的 探讨香菇C91-3菌丝发酵液提取蛋白对小鼠宫颈癌的作用.方法 观察香菇C91-3菌丝发酵液提取蛋白对小鼠宫颈癌U14荷瘤小鼠生存期的影响和对体外培养的小鼠宫颈癌U14细胞的抑杀作用.结果 香菇C91-3菌丝发酵液提取蛋白能明显延长小鼠宫颈癌U14荷瘤小鼠的生存期并能对体外培养的小鼠宫颈癌U14细胞有直接抑杀作用.结论 香菇C91-3菌丝发酵液提取蛋白对机体有调节、增强机体免疫系统功能的作用.