Presence of High-Risk HPV mRNA in Relation to Future High-Grade Lesions among High-Risk HPV DNA Positive Women with Minor Cytological Abnormalities

Objective

Continuous expression of E6- and E7-oncogenes of high-risk human papillomavirus (HPV) types is necessary for the development and maintenance of the dysplastic phenotype. The aim of the study was to determine the sensitivity and specificity of the APTIMA HPV mRNA assay (Hologic) in predicting future development of high-grade cervical intraepithelial neoplasia (CIN) among high-risk HPV-DNA-positive women with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous epithelial lesion (LSIL) cytology.

Methods

Archived SurePath cervical samples of women ≥ 35 years of age with high-risk HPV DNA-positive ASCUS (n = 211) or LSIL, (n = 131) were tested for the presence of high-risk HPV E6/E7 mRNA using the APTIMA HPV assay, and the women were monitored for development of histopathologically verified CIN2+.

Results

Twenty-nine percent (61/211) of the women in the ASCUS group, and 34.3% (45/131) in the LSIL group developed CIN2+ within 4.5 years of follow-up. The prevalence of HPV mRNA was 90.0% (95% CI 85.9-94.0) among women with ASCUS and 95.4% (95% CI 91.8-99.0) among women with LSIL. The presence of HPV E6/E7 mRNA was associated with future development of CIN2+ among women with ASCUS and LSIL (p=0.02). The mRNA assay demonstrated high sensitivity in predicting future CIN2+ and CIN3 for index ASCUS (96.7%; 95% CI 87.6-99.4 and 100%; 95% CI 82.2-100, respectively) and LSIL (97.8%, 95% CI 86.8-99.9 and 100%, 95% CI 79.9-100, respectively). The corresponding specificity was low, 12.7% (95% CI 7.9-19.3) and 5.8% (95% CI 2.2-13.6), for future CIN2+, respectively. The negative predictive value of the HPV mRNA assay for detecting future CIN3 was 100%, since no mRNA-negative woman developed CIN3 (0/27) as compared to 13.6% (43/315) of the mRNA-positive women (p = 0.03).

Conclusion

The APTIMA mRNA assay demonstrated high sensitivity but low specificity in predicting future CIN2+ among women with minor cytological abnormalities. The assay had high negative predictive value for future CIN3, indicating that HPV-mRNA-negative women are at low risk of progression to high grade CIN.     

DNA甲基化抑制鼻咽癌细胞系膜联蛋白A1基因表达

为了研究不同分化程度和转移潜能鼻咽癌(NPC)细胞系膜联蛋白A1(ANXA1)mRNA和蛋白质表达情况及其与基因甲基化的关系．培养NPC细胞系CNE1、CNE2、5-8F、6-10B和永生化非癌性人鼻咽黏膜上皮细胞NP69细胞用于实验,用甲基化特异性聚合酶链反应(MSP)方法检测ANXA1基因甲基化状态,同时利用逆转录-聚合酶链反应(RT-PCR)方法检测ANXA1基因的mRNA表达水平．然后用不同浓度的5-杂氮-2′-脱氧胞苷(5-aza-2dC)对NPC细胞进行去甲基化处理,MSP和RT-PCR方法检测处理组和对照组细胞ANXA1基因甲基化状况和mRNA表达水平,并用Western-blotting方法检测ANXA1基因蛋白质表达水平．结果发现,NP69细胞ANXA1基因无甲基化,4株NPC细胞系ANXA1基因都存在不同程度的甲基化,甲基化程度与细胞的分化程度和转移潜能相关．NPC细胞ANXA1基因mRNA表达水平降低,低于NP69细胞,其降低的程度与基因的甲基化程度相关．5-aza-2dC能够剂量依赖性地引起ANXA1基因去甲基化,经去甲基化处理后,NPC细胞系ANXA1基因的mRNA和蛋白质的表达水平相应提高．研究证明,NPC细胞系ANXA1基因的mRNA和蛋白质表达水平出现下调,甲基化是导致表达下调的主要原因,5-aza-2dC去甲基化处理能够恢复ANXA1基因的表达水平．     

血清ANXA2、ANXA6、ANXA7与乳腺癌患者聚乙二醇多柔比星脂质体相关新辅助化疗方案治疗疗效的关系研究

摘要 目的：探讨血清膜联蛋白A2（ANXA2）、膜联蛋白A6（ANXA6）、膜联蛋白A7（ANXA7）与乳腺癌患者聚乙二醇多柔比星脂质体（PLD）相关新辅助化疗方案治疗疗效的关系。方法：选择2019年11月至2022年12月于山西医科大学第一医院95例行PLD相关新辅助化疗的乳腺癌患者。新辅助化疗前检测所有乳腺癌患者的血清ANXA2、ANXA6、ANXA7水平。单因素和多因素Logistic回归分析影响乳腺癌患者PLD相关新辅助化疗疗效的因素。受试者工作特征（ROC）曲线分析血清ANXA2、ANXA6、ANXA7预测乳腺癌患者PLD相关新辅助化疗疗效的价值。结果：无效组新辅助化疗前血清ANXA2、ANXA6、ANXA7水平均高于有效组（P＜0.05）。TNM分期IIIA期、ER阳性、高水平ANXA2、高水平ANXA6、高水平ANXA7是乳腺癌PLD相关新辅助化疗无效的危险因素（P＜0.05）。血清ANXA2、ANXA6、ANXA7单独检测预测乳腺癌PLD相关新辅助化疗无效的曲线下面积分别为0.783、0.774、0.821,三指标联合检测预测的曲线下面积为0.923,高于各指标单独预测效能。结论：高ANXA2、ANXA6、ANXA7水平及TNM分期ⅢA期、ER阳性均是乳腺癌患者PLD相关新辅助化疗无效的危险因素,联合检测血清ANXA2、ANXA6、ANXA7水平可提高对乳腺癌患者PLD相关新辅助化疗疗效的预测效能。     

Correlative Analysis of miRNA Expression and Oncotype Dx Recurrence Score in Estrogen Receptor Positive Breast Carcinomas

Altered expression of miRNAs has been observed in many types of cancer, including breast cancer, and shown to contribute to cancer growth, aggressiveness, and response to therapies. In this pilot study, we investigated the possible correlation of miRNAs with risk of recurrence of estrogen receptor positive, lymph node-negative mammary carcinomas as determined by the Oncotype DX^® Breast Cancer assay. To accomplish this, we extracted RNA from a collection of breast carcinomas that had previously been analyzed by Oncotype DX^®. Multiple Let-7 family members were negatively correlated with the recurrence score (RS), which is consistent with their tumor suppressor properties. Additional miRNAs were found to positively correlate with RS, including miR-377-5p, miR-633b, miR-548t and miR-3648. Pathway analysis of putative and validated targets suggests that these miRNAs may have a diverse range of functions that may contribute to tumor recurrence. Taken together, these findings provide evidence that a miRNA expression signature can be developed to aid existing methods to determine the risk of recurrence for women with estrogen receptor positive breast cancers treated with endocrine therapy.     

The Expression of SIRT1 and DBC1 in Laryngeal and Hypopharyngeal Carcinomas

Rationale and Objective

Sirtuin 1 (SIRT1) plays an important role in tumorigenesis and is increased in many human tumors. DBC1 is a negative regulator of SIRT1 via promotion of p53-mediated apoptosis. It is necessary to investigate the expression of SIRT1 and DBC1 in laryngeal and hypopharyngeal squamous cell carcinomas (LSCC and HSCC) and its correlation with available clinical parameters.

Methods

The mRNA levels of SIRT1 and DBC1 were measured in 54 paired LSCC or HSCC tumors and corresponding adjacent noncancerous mucosae using quantitative RT-PCR (qRT-PCR). The protein levels of SIRT1 and DBC1 were also evaluated in 120 cases of patients with LSCC or HSCC using immunohistochemical staining. The correlation between SIRT1 and DBC1 expression and clinical parameters was analyzed with Pearson chi-square test.

Results

qRT-PCR assay showed that, compared with the paired adjacent noncancerous mucosae, SIRT1 mRNA was significantly decreased in tumors. The immunohistochemical results indicated that the SIRT1 protein was also downregulated in tumors compared with noncancerous mucosae. Moreover, decreased SIRT1 was significantly correlated with the tumor clinical stage and lymph node metastasis. Additionally, DBC1 mRNA was significantly increased in tumors compared with noncancerous mucosae. The immunohistochemical results indicated that the DBC1 protein was downregulated in tumors, which is inconsistent with the results obtained by qRT-PCR. Finally, decreased DBC1 protein was significantly correlated with tumor differentiation, lymph node metastasis, and p53 expression.

Conclusions

SIRT1 and DBC1 might be involved in the pathophysiology of laryngeal and hypopharyngeal squamous cell carcinomas and are associated with lymph node metastasis and p53 positive staining in LSCCs and HSCCs.     

血清ANXA2、ANXA3与转移性结直肠癌患者化疗疗效的关系

摘要 目的：探讨血清膜联蛋白（ANX）A2、ANXA3与转移性结直肠癌（mCRC）患者化疗疗效的关系。方法：选取2019年1月～2020年10月徐州医科大学附属医院收治的90例mCRC患者,根据mFOLFOX6化疗联合西妥昔单抗治疗后的疗效分为未缓解组和缓解组。采用酶联免疫吸附法检测血清ANXA2、ANXA3水平。分别采用单因素、多因素Logistic回归分析和受试者工作特征（ROC）曲线分析mCRC患者化疗未缓解的影响因素和血清ANXA2、ANXA3对mCRC患者化疗未缓解的预测价值。结果：90例mCRC患者客观缓解率为58.89%（53/90）。单因素分析显示,两组年龄、回盲部肿瘤、TNM分期、Zubrod-东部肿瘤协助组-世界卫生组织（ZPS）评分、治疗目标、ANXA2、ANXA3组间比较存在统计学差异（P＜0.05）。多因素Logistic回归分析显示,回盲部肿瘤、TNM分期Ⅳ期、ZPS评分1～2分和ANXA2、ANXA3升高为mCRC患者化疗未缓解的独立危险因素（P＜0.05）。ROC曲线分析显示,血清ANXA2预测mCRC患者化疗未缓解的曲线下面积（AUC）为0.787,ANXA3预测mCRC患者化疗未缓解的AUC为0.791,血清ANXA2、ANXA3联合预测为0.904,二者联合预测的AUC最大（P＜0.05）。结论：血清ANXA2、ANXA3水平升高为mCRC患者化疗未缓解的独立危险因素,可影响mCRC患者的化疗疗效,二者联合预测mCRC患者化疗未缓解的价值较高。     

Overexpression of GLT1 in fps1DeltagpdDelta mutant for optimum ethanol formation by Saccharomyces cerevisiae

Glycerol is the main byproduct produced under anaerobic ethanol fermentations by Saccharomyces cerevisiae and consumes a considerable amount of substrate. To verify the metabolic phenotype predications for increasing ethanol formation, two engineered S. cerevisiae KAM-14, KAM-15 strains were constructed for possible redirection of glycerol carbon flux into ethanol by overexpression of GLT1 in the fps1DeltagpdDelta mutant. The engineered strains KAM-14 and KAM-15 compared to the control strain KAM-2, produced 12.24% and 10.42% higher ethanol, 39.72% and 31.03% lower glycerol yield during anaerobic batch fermentations, respectively. The maximum specific growth rates of KAM-14 and KAM-15 were found to be relatively lower than that of KAM-2 during the exponential growth phase. In the meantime, the biomass concentrations of both KAM-14 and KAM-15 were similar to KAM-2. Acetate and pyruvate concentrations of KAM-14 and KAM-15 were greatly decreased comparing to those of KAM-2, respectively. These experimental results approved the metabolic pathway strategies to improve ethanol formation.     

Overexpression of CmSOS1 confers waterlogging tolerance in Chrysanthemum

正Plants experiencing hypoxia (a shortage of oxygen)are unable to maintain aerobic respiration, which leads to an energy and carbohydrate deficit. The pervasive and rapid accumulation of ethylene is an early and reliable response to hypoxic stress(Sasidharan and Voesenek 2015), producing an uptick in the accumulation of reactive oxygen species (ROS).This in turn triggers apoptosis in root cortex cells,eventually leading to the formation of lysigenous aerenchyma, a tissue from which ethylene is readily     

Overexpression of HEMA1 encoding glutamyl-tRNA reductase

5-Aminolevulinic acid (ALA) synthesis has been shown to be the rate limiting step of tetrapyrrole biosynthesis. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of plant ALA synthesis and is controlled by interacting regulators, such as heme and the FLU protein. Induced inactivation of the HEMA1 gene encoding GluTR by RNAi expression in tobacco resulted in a reduced activity of Mg chelatase and Fe chelatase indicating a feed-forward regulatory mechanism that links ALA synthesis posttranslationally with late enzymes of tetrapyrrole biosynthesis (Hedtke et al., 2007). Here, the regulatory impact of GluTR was investigated by overexpression of AtHEMA1 in Arabidopsis and tobacco plants. Light-dependent ALA synthesis cannot benefit from an up to 7-fold induced expression of GluTR in Arabidopsis. While constitutive AtHEMA1 overexpression in tobacco stimulates ALA synthesis by 50-90% during light-exposed growth of seedlings, no increase in heme and chlorophyll contents is observed. HEMA1 overexpression in etiolated and dark-grown Arabidopsis and tobacco seedlings leads to additional accumulation of protochlorophyllide. As excessive accumulation of GluTR does not correlate with increased ALA formation, it is hypothesized that ALA synthesis is additionally limited by other effectors that balance the allocation of ALA with the activity of enzymes of chlorophyll and heme biosynthesis.     

食管鳞癌MLH1基因改变及其与微卫星不稳定的关系

MLH1是位于3p21．3上的一个DNA错配修复基因,其异常与多种肿瘤相关。为探索食管鳞癌中MLH1基因改变及其与微卫星不稳定(MSI)的关联情况,采用微卫星分析和RT—PCR方法检测了14个微卫星标志在食管癌中的状况及MLH1转录水平的表达,发现35％的食管癌出现至少一个微卫星的不稳定,66．7％的肿瘤在MLH1基因内标志D3S1611位点表现为杂合性丢失,但是MLH1没有明显的mRNA表达下调。MSI与食管癌分期、分级、淋巴结转移、患者年龄和性别等参数及MLH1基因杂合性丢失(LOH)之间无统计学意义的相关性。这些结果表明：食管癌中MLH1存在较高频率的等位基因丢失,但其mRNA表达水平并无明显异常;所测微卫星标志的不稳定是食管癌的频发事件,与MLH1基因LOH不存在必然联系。     

Overexpression of Aquaporin-1 in lung adenocarcinomas and pleural mesotheliomas

Aquaporin-1 (AQP1) is the main water channel responsible for water transport through many epithelia and endothelia. The latest evidence pointed toward an important role of this protein also in gas permeation, angiogenesis, cell proliferation and migration. In the present work we studied the expression of AQP1 by immunohistochemical staining of 92 lung biopsies from patients diagnosed with a pleuro-pulmonary tumor (71 lung and 21 pleural neoplasms). AQP1 expression was analyzed comparing the results among the different histological patterns and against 9 control cases (5 parenchyma and 4 healthy pleura). Clear staining of AQP1 was detected in 39 of the 92 tumors analyzed. In parenchyma, AQP1 was more frequently detected in primary lung adenocarcinomas (55%, P<0.001); in contrast, small cell carcinomas were the least AQP1 expressive tumors studied (93% of negative staining, P<0.05). Carcinomas analyzed in pleura (mesotheliomas and metastatic adenocarcinomas) also revealed strong expression of AQP1. High expression of this protein was detected in small capillaries in areas near or surrounding the tumor, and novel intense AQP1 immunostaining was detected over thicker alveolar walls in alveoli inside or next to the tumoral tissue regardless of the tumor type. An important role of AQP1 in tumor angiogenesis is sustained by the abundant expression of this protein in the endothelia of tumor capillaries. Further studies are necessary to elucidate the potential pathophysiological role of this protein in pleuro-pulmonary neoplasms.     

RAN1基因过表达抑制嗜热四膜虫大核无丝分裂

Ran GTPase通过RanGTP/RanGDP循环的形式,参与调控多种细胞增殖方式：包括有丝分裂和减数分裂.敲减RAN1基因可导致嗜热四膜虫大核内微管组装紊乱,从而抑制大核无丝分裂.为进一步分析Ran1在无丝分裂中的功能,本研究将野生型Ran1以及模拟GTP（Ran1Q70L）和GDP（Ran1T25N）锁定形式的Ran1突变体在嗜热四膜虫中过量表达,均导致四膜虫细胞增殖速率下降,并引起大核无丝分裂异常,且这种核异常细胞比率与Ran1过表达量呈正相关.免疫荧光定位结果显示,过表达的HA-Ran1在整个细胞中弥散分布,破坏了正常的Ran1分布形式;而过表达的HA-Ran1Q70L明显集中在大核核膜和胞质中,HA-Ran1T25N则主要定位在大核和小核内,分别与Ran1GTP/Ran1GDP循环的辅助调节因子定位模式一致.以上结果表明,过表达Ran1及其突变体可能影响嗜热四膜虫细胞中正常的Ran1GTP/Ran1GDP循环,进而导致大核无 丝分裂异常.