THE EXPRESSION OF CONNEXIN GENES IN NASOPHARYNGEAL CARCINOMA CELLS AND THE EFFECT OF RETINOIC ACID ON THE REGULATION OF THOSE GENES

Objective: To detect which members in the connexin gene family are expressed in nasopharyngeal carcinoma (NPC) cell line HNE1, and the mechanism by which those genes are specifically switched on and off during retinoic acid (RA) induction. Methods: Establishing the cell growth curves of NPC cells. Observing the effect of RA on connexin genes by Northern hybridization. Results: Two genes Cx46 and Cx37, belonging to the connexin gene family, were expressed in HNE1. The down-regulation of Cx46 and Cx37, up-regulation of RARa and growth inhibition was observed in HNE1 after exposure to RA. The gene expression and cell growth in HNE1 cells was restored after removal of RA. Conclusion: Two members of the connexin gene family: Cx37 and Cx46 were expressed in HNE1 cells, RA can inhibit the expression of those tow genes mediated by RARa, and the effects of RA on HNE1 are reversible.

Retinoic acid enhances expression of neural specific genes in Sca-1^+ cells of mouse fetal liver through activating protein kinase C

BACKGROUND： Interstitial stem cell is charactenzed by multiple differentiations, and retinoic acid （RA） can induce differentiation of stromal cells into nerve tissue cells in fetal liver of mice, so, its signal transduction pathway should be discussed to trigger differentiation. OBJECTIVE ： To study the effect of RA on expression of neural specific gene and its signal transduction in fetal liver of mice.DESIGN ： Paired controlled study on the basis of cell.SETTING ： Institute of Hematology, Medical College of Jinan University.MATERIALS： The experiment was completed in the Institute of Hematology, Medical College of Jinan University from April to December 2005. C57BL/6 mice, of clean grade, aged 8-10 weeks, weighting 20-35 g, 10 females and 4 males, were selected in this study.METHODS： Sca-1^＋ cells in fetal liver were prepared with MACS kit and cultured with DMEM ＋ 10% fetal bovine serum （FBS）. On the fourth day, it was added with or without protein kinase C （PKC） inhibitor chelerythrine chloride （3μmol/L） and 5×10^-7 mol/L RA for 24 hours, and then incubated in serum-free medium for 5 days. Expressions of genes were assayed by Westem blotting and semi-quantitative RT-PCR.MAIN OUTCOME MEASURES ： Expression of neural specific gene NF-L, NF-H, BF-1 and TH.RESULTS： Expression of neural specific gene NF-L, NF-H, BF-1 and TH was significantly increased after treatment with RA and they were increased 5.06, 5.15, 4.63 and 3.33 times, respectively. However, chelerythrine chloride could inhibit expression of neural specific gene NF-L, NF-H, BF-1 and TH induced by RA.CONCLUSION ： RA can promote the expression of neural specific genes in Sca-1^＋ cells of fetal liver, and its pathway may be related to PKC.

Molecular Cloning,Characterization and Expression Analysis of Woodchuck Retinoic Acid-Inducible GeneⅠ

Cytosolic retinoic acid-inducible gene I（RIG-I） is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β（IFN-β）. Innate immune response to hepatitis B virus（HBV） plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I（w RIG-I）, we analyzed the complete coding sequences（CDSs） of w RIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced w RIG-I protein was 106.847 k D with a theoretical isoelectric point（p I） of 6.07, and contained three important functional structures [caspase activation and recruitment domains（CARDs）, DEx D/H-box helicases, and a repressor domain（RD）]. In woodchuck fibroblastoma cell line（WH12/6）, w RIG-I-targeted small interfering RNA（si RNA） down-regulated RIG-I and its downstrean effector–IFN-β transcripts under RIG-I＇ ligand, 5＇-ppp double stranded RNA（ds RNA） stimulation. We also measured m RNA levels of w RIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus（WHV）-infected woodchucks. The basal expression levels of w RIG-I were abundant in the kidney and liver. Importantly, w RIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.

Effects of 9-cis retinoic acid on human homeobox gene NKX3.1 expression in prostate cancer cell line LNCaP

Aim:To study the regulatory effects of 9-cis retinoic acid(RA)on the expression of human homeobox gene NKX3.1 in prostate cancer cell line LNCaP.Methods:Flow cytometry,reverse transcriptase polymerase chain reaction and Western blotting were performed to evaluate the effects of 9-cis RA on NKX3.1 expression and cell cycle of LNCaP cells.To identify a regulatory region within the NKX3.1 promoter contributing to the regulation induced by 9-cis RA, we have constructed an NKX3.1 promoter-reporter plasmid,pGL_3-1040bp,and its 5′-deletion mutants,which were transfected into LNCaP cells with treatment of 9-cis RA in indicated concentrations.Results:With the treatment of 9-cis RA,the NKX3.1 promoter activity was increased in reporter gene assay and NKX3.1 expression was enhanced at both mRNA and protein levels in LNCaP cells.We found that the region between -936 and -921 in the upstream of NKX3.1 gene involved the inducible regulation by 9-cis RA treatment.In flow cytometry,9-cis RA treatment caused accumulation of cells in the G_1 phase of the cell cycle and a fewer cells pass through to G_2/M.Conclusion:Our results demonstrated that 9-cis RA as a differentiating agent can arrest prostate cancer cells in G_1 phase and reduce cell mitosis,and upregulate the expression of human homeobox gene NKX3.1,which is thought to play an important role in prostate differentiation and to act as a tumor suppressor gene in the prostate.(Asian J Androl 2006 Jul;8:435-441)

Nr2e1 Downregulation Is Involved in Excess Retinoic Acid-induced Developmental Abnormality in the Mouse Brain

Objective This study aimed to investigate the expression pattern and function of Nuclear receptor subfamily 2 group E member 1 （Nr2e1） in retinoic acid （RA）-induced brain abnormality. Methods The mouse model of brain abnormality was established by administering 28 mg/kg RA, and neural stem cells （NSCs） were isolated from the mouse embryo and cultured in vitro. Nr2e1 expression was detected by whole mount in situ hybridization, RT-PCR, and Western blotting. Nr2e1 function was determined by transducing Nr2e1 sh RNA into NSCs, and the effect on the sonic hedgehog （Shh） signaling pathway was assessed in the cells. In addition, the regulation of Nr2e1 expression by RA was also determined in vitro. Results Nr2e1 expression was significantly downregulated in the brain and NSCs of RA-treated mouse embryos, and knockdown of Nr2e1 affected the proliferation of NSCs in vitro. In addition, a similar expression pattern of Nr2e1 and RA receptor （RAR） α was observed after treatment of NSCs with different concentrations of RA. Conclusion Our study demonstrated that Nr2e1 could be regulated by RA, which would aid a better understanding of the mechanism underlying RA-induced brain abnormality.

MGMT is down-regulated independently of promoter DNA methylation in rats with all-trans retinoic acidinduced spina bifida aperta

O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.

The role of tazarotene-induced gene 1 in carcinogenesis:is it a tumor suppressor gene or an oncogene?

Tazarotene-induced gene 1(TIG1)is induced by a derivative of vitamin A and is known to regulate many important biological processes and control the development of cancer.TIG1 is widely expressed in various tissues;yet in many cancer tissues,it is not expressed because of the methylation of its promoter.Additionally,the expression of TIG1 in cancer cells inhibits their growth and invasion,suggesting that TIG1 acts as a tumor suppressor gene.However,in some cancers,poor prognosis is associated with TIG1 expression,indicating its protumor growth characteristics,especially in promoting the invasion of inflammatory breast cancer cells.This review comprehensively summarizes the roles of the TIG1 gene in cancer development and details the mechanisms through which TIG1 regulates cancer development,with the aim of understanding its various roles in cancer development.

Relationship between the Expression of Connexin43 and Bystander Effect of Suicide Gene Therapy in Ovarian Cancer

The relationship of connexin43 (Cx43) and bystander effect in ovarian tumor cells in herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy in vitro was explored and the effect of all-trans retinoic acid (RA) on the expression of Cx43 and bystander effect investigated. The Cx43 expression was detected by flowcytometry, Western blot, and immunofluorescence in two ovarian tumor cell lines OVCAR3, CaOV3 before and after RA treatment. Bystander effect was determined by the cells growth inhibitory rate with methyl thiazolyl tetrazolium. Following exposure to ganciclovir, there was much greater bystander killing in OVCAR3 than that in CaOV3 (P<0.05). The expression of Cx43 was detected in OVCAR3 by flowcytometry and Western blot, but it could not be detected in CaOV3. The expression of Cx43 in both cell lines could be induced by RA. Immunofluoresence staining showed that Cx43 protein of OVCAR3 was located on membrane surface, whereas CaOV3 in cytoplasm. RA could not change the location of Cx43 protein in both cell lines. There is relationship between Cx43 expression and HSV-TK/GCV bystander effect. HSV-TK/GCV bystander effect can be enhanced by RA in ovarian cancer.

Cell cycle-related genes p57kip2, Cdk5 and Spin in the pathogenesis of neural tube defects

In the field of developmental neurobiology, accurate and ordered regulation of the cell cycle and apoptosis are crucial factors contributing to the normal formation of the neural tube. Preliminary studies identified several genes involved in the development of neural tube defects. In this study, we established a model of developmental neural tube defects by administration of retinoic acid to pregnant rats. Gene chip hybridization analysis showed that genes related to the cell cycle and apoptosis, signal transduction, transcription and translation regulation, energy and metabolism, heat shock, and matrix and cytoskeletal proteins were all involved in the formation of developmental neural tube defects. Among these, cell cycle-related genes were predominant. Retinoic acid treat-ment caused differential expression of three cell cycle-related genes p57kip2, Cdk5 and Spin, the expression levels of which were downregulated by retinoic acid and upregulated during normal neural tube formation. The results of this study indicate that cell cycle-related genes play an im-portant role in the formation of neural tube defects. P57kip2, Cdk5 and Spin may be critical genes in the pathogenesis of neural tube defects.

Andrographolide as An Anti-H1N1 Drug and the Mechanism Related to Retinoic Acid-Inducible Gene-I-Like Receptors Signaling Pathway

Objective： To observe the anti-virus effects of andrographolide （AD） on the retinoic acid-inducible gene-I （RIG-I）-Iike receptors （RLRs） signaling pathway when immunological cells were infected with HIN1. Methods： Leukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line （16HBE）. The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium （MTT） assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma （IFN- ~, ） and interleukin-4 （IL-4） enzyme-linked immunosorbent assay （ELISA） detection while immunological cells for real-time polymerase chain reaction （RT-PCR）. Results： The optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-13 promoter stimulator-1 （IPS-1）, interferon regulatory factor （IRF）-7, IRF-3 and nuclear transcription factor K B （NF- K B） mRNA levels increased significantly （P〈0.05）. When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF- K B decreased significantly in each group with significant statistic differences （P〈0.05）. Conclusions： The RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.

Are Gene Expression Microarray Analyses Reliable? A Review of Studies of Retinoic Acid Responsive Genes

Microarray analyses of gene expression are widely used, but reports of the same analyses by different groups give widely divergent results, and raise questions regarding reproducibility and reliability. We take as an example recent published reports on microarray experiments that were designed to identify retinoic acid responsive genes. These reports show substantial differences in their results. In this article, we review the methodology, results, and potential causes of differences in these applications of microarrays. Finally, we suggest practices to improve the reliability and reproducibility of microarray experiments.

Identification of key genes and biological pathways in lung adenocarcinoma by integrated bioinformatics analysis

BACKGROUND The objectives of this study were to identify hub genes and biological pathways involved in lung adenocarcinoma(LUAD)via bioinformatics analysis,and investigate potential therapeutic targets.AIM To determine reliable prognostic biomarkers for early diagnosis and treatment of LUAD.METHODS To identify potential therapeutic targets for LUAD,two microarray datasets derived from the Gene Expression Omnibus(GEO)database were analyzed,GSE3116959 and GSE118370.Differentially expressed genes(DEGs)in LUAD and normal tissues were identified using the GEO2R tool.The Hiplot database was then used to generate a volcanic map of the DEGs.Weighted gene co-expression network analysis was conducted to cluster the genes in GSE116959 and GSE-118370 into different modules,and identify immune genes shared between them.A protein-protein interaction network was established using the Search Tool for the Retrieval of Interacting Genes database,then the CytoNCA and CytoHubba components of Cytoscape software were used to visualize the genes.Hub genes with high scores and co-expression were identified,and the Database for Annotation,Visualization and Integrated Discovery was used to perform enrichment analysis of these genes.The diagnostic and prognostic values of the hub genes were calculated using receiver operating characteristic curves and Kaplan-Meier survival analysis,and gene-set enrichment analysis was conducted.The University of Alabama at Birmingham Cancer data analysis portal was used to analyze relationships between the hub genes and normal specimens,as well as their expression during tumor progression.Lastly,validation of protein expression was conducted on the identified hub genes via the Human Protein Atlas database.RESULTS Three hub genes with high connectivity were identified;cellular retinoic acid binding protein 2(CRABP2),matrix metallopeptidase 12(MMP12),and DNA topoisomerase II alpha(TOP2A).High expression of these genes was associated with a poor LUAD prognosis,and the genes exhibited high diagnostic value.CONCLUSION Expression levels of CRABP2,MMP12,and TOP2A in LUAD were higher than those in normal lung tissue.This observation has diagnostic value,and is linked to poor LUAD prognosis.These genes may be biomarkers and therapeutic targets in LUAD,but further research is warranted to investigate their usefulness in these respects.

NCSTN Gene Silencing Inhibits the Retinoic Acid Signaling Pathway in Human Immortalized Keratinocytes

Objective:Acne inversa is a multifactorial chronic debilitating disease.Genetic factors are involved in 40%of patients,especially the nicastrin(NCSTN)gene.However,the role of the mutated NCSTN gene in the pathogenesis of acne inversa remains unclear.Retinoic acid is recommends to treat moderate to severe acne inversa,therefor we conduct this in vitro research to study the association between NCSTN gene mutation and the retinoic acid signaling pathway in human immortalized skin keratinocyte(HaCaT)cells.Methods:HaCaT cells were infected with a lentivirus-mediated short hairpin RNA(shRNA)expression plasmid specifically targeting the NCSTN gene.Real-time polymerase chain reaction(PCR)and Western blotting were used to detect the interference efficiency of NCSTN.RNA sequencing was used to detect differential genes in the NSCTN-deficient HaCaT cells.Based on bioinformatics analysis and clinical treatment data,the retinoic acid signal pathway was selected for screening.Quantitative PCR was used to verify the changes in the expressions of retinoic acid signaling pathway-related receptors and molecules in the HaCaT cell line after NCSTN silencing.The Student t test and one-way analysis of variance were used to evaluate intergroup differences.Results:Sequencing showed that the NCSTN-shRNA lentiviral recombinant expression plasmid was successfully constructed.After lentivirus infection of HaCaT cells,real-time PCR results showed significantly reduced NCSTN mRNA expression in the interference group compared with the negative control group,and the interference efficiency was 75.0%.Western blotting showed that the inhibition rate of NCSTN protein expression in the shRNA group was 71.7%.RNA sequencing revealed significant differential expression of some genes,and changes in signaling pathways.Compared with the control group,the group with the silenced NCSTN showed significantly decreased expression of retinoic acid receptors(RARα:F=23.482,RARβ:F=603.241,RXRα:F=69.689,and RARRES1:F=167.482,and all P<0.001),and peroxisome proliferator-activated receptorγ(F=8.138,P<0.01).Conclusion:Defective function of the NCSTN gene leads to an impaired retinoic acid signaling pathway in HaCaT cells,which suggests that the retinoic acid signaling pathway may play a role on the onset of acne inversa caused by NCSTN gene mutation.

Between Scylla and Charybdis:The role of the human immune system in the pathogenesis of hepatitis C

Hepatitis C virus(HCV)frequently elicits only mild immune responses so that it can often establish chronic infection.In this case HCV antigens persist and continue to stimulate the immune system.Antigen persistence then leads to profound changes in the infected host’s immune responsiveness,and eventually contributes to the pathology of chronic hepatitis.This topic highlight summarizes changes associated with chronic hepatitis C concerning innate immunity(interferons,natural killer cells),adaptive immune responses(immunoglobulins,T cells,and mechanisms of immune regulation(regulatory T cells).Our overview clarifies that a strong anti-HCV immune response is frequently associated with acute severe tissue damage.In chronic hepatitis C,however,the effector arms of the immune system either become refractory to activation or take over regulatory functions.Taken together these changes in immunity may lead to persistent liver damage and cirrhosis.Consequently,effector arms of the immune system will not only be considered with respect to antiviral defence but also as pivotal mechanisms of inflammation,necrosis and progression to cirrhosis.Thus,avoiding Scylla-a strong,sustained antiviral immune response with inital tissue damage-takes the infected host to virus-triggered immunopathology,which ultimately leads to cirrhosis and liver cancerthe realm of Charybdis.

沉默星形细胞上调基因-1联合全反式维甲酸抑制胶质瘤血管拟态形成的机制分析

目的研究沉默星形细胞上调基因-1(AEG-1)联合全反式维甲酸(ATRA)对胶质瘤血管拟态(VM)形成的影响及其可能的机制。方法构建AEG-1 shRNA慢病毒稳染U87胶质瘤细胞并用含20μmol/L全反式维甲酸(ATRA)的培养基干预,分组:空白对照组(U87细胞)、ATRA组(U87细胞+20μmol/L ATRA),siCon组(U87-siCon细胞)、siCon+ATRA组(U87-siCon细胞+20μmol/L ATRA),siAEG-1组(U87-siAEG-1细胞)和siAEG-1+ATRA组(U87-siAEG-1细胞+20μmol/L ATRA)。体外管腔形成实验评估U87细胞体外血管拟态形成能力,实时PCR及Western-Blot检测U87细胞中血管拟态形成相关基因表达情况。建立裸鼠胶质瘤皮下移植瘤模型并以10mg/kg剂量给予腹腔注射ATRA干预,分为:对照组(U87细胞造模)、ATRA组(U87细胞造模+ATRA干预),siCon组(U87-siCon细胞造模)、siCon+ATRA组(U87-siCon细胞造模+ATRA干预),siAEG-1组(U87-siAEG-1细胞造模)与siAEG-1+ATRA组(U87-siAEG-1细胞造模+ATRA干预)。定期测量皮下移植瘤大小并绘制肿瘤生长曲线,CD34-PAS双染检测移植瘤中血管拟态。结果沉默AEG-1联合ATRA干预(即siAEG-1+ATRA组)显著抑制胶质瘤细胞及胶质瘤模型中血管拟态形成及皮下移植瘤生长,干预后胶质瘤细胞中基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9),钙黏蛋白(VE-cadherin)表达明显下调。上述结果与对照组比较,差异均具有统计学意义(均P<0.05)。结论沉默AEG-1联合ATRA能显著抑制胶质瘤血管拟态形成及肿瘤生长,其机制可能与抑制MMP-2、MMP-9以及VE-cadherin表达有关。

All-trans retinoic acid as a single agent induces complete remission in a patient with acute leukemia of M_(2a) subtype

Objective To present a special case with the karyotype and molecular marker of acute myeloid leukemia (AML)-M 2 who was induced to complete remission by all-trans retinoic acid (ATRA) alone.Methods A recently hospitalized young female patient with acute leukemia was initially diagnosed as M 3 subtype based on morphological French-American-British (FAB) classification. Karyotype analysis using standard G and R banding techniques and RT-PCR were applied to further define the diagnosis. After primarily cultured bone marrow cells from the iliac aspiration were tested for in vitro induced differentiation, the patient was treated with oral all-trans retinoic acid alone, 60?mg per day until complete remission was achieved. Peripheral blood and bone marrow changes were monitored over the whole treatment course.Results The characteristic chromosomal aberration for M 3, the t(15;17) reciprocal translocation, was not found while a t(8;21) translocation was verified. Furthermore, an amplified product of the AML-1/ETO fusion gene instead of the PML/RARα fusion gene was detected by RT-PCR and the diagnosis was corrected from M 3 to M 2. Primary cultured bone marrow cells can be fully induced to terminal differentiation after 4 days exposure to ATRA. A hematological complete remission was achieved after 40 days treatment with ATRA as a single therapeutic agent, suggesting an alternative pathway mediating ATRA-induced myeloid differentiation. Conclusion A leukemia patient with a subtype other than M 3, such as M 2 in this case, may also be induced to complete remission by the mechanism of ATRA-induced terminal differentiation. This implies that there may be a pathway other than PML/RARα fusion gene product which mediates ATRA-induced myeloid maturation in leukemia cells.

视黄酸诱导的小鼠神经管畸形基因表达趋势分析

目的·探究小鼠胚胎在视黄酸(retinoic acid,RA)诱导下产生神经管畸形的分子调控机制,揭示小鼠神经管闭合阶段基因表达规律。方法·基于已获得的小鼠胚胎神经管闭合关键期[胚胎发育第8.5日(embryonic day 8.5,E8.5)、E9.5、E10.5]高质量脑泡转录组数据,利用短时间序列表达挖掘器(Short Time-series Expression Miner,STEM)软件分别得到RA处理组和正常组在3个时间点的基因表达趋势数据。对处理组与正常组基因表达趋势不一致的基因进行基因本体(Gene Ontology,GO)富集分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析,并随机筛选候选基因以验证测序数据可靠性。利用RA诱导构建神经管畸形小鼠模型,分为处理组和正常组,每组各9只。处理组和正常组孕鼠在E7.5分别接受28 mg/kg RA和香油灌胃处理,在E8.5、E9.5、E10.5收集胎鼠脑泡组织,对筛选的候选基因进行实时荧光定量PCR(quantitative real-time PCR,RT-qPCR)验证。结果·正常组共检测出18255个基因的表达量数据,处理组共检测出19037个基因的表达量数据;正常组基因可归纳至7个具有显著意义的表达模式中,处理组基因可归纳至6个具有显著意义的表达模式中;正常组和处理组检测到表达的基因数目足够、表达的模式相似,具有可比性。进一步分析发现正常组中呈现上升表达趋势但在处理组中呈现下降表达趋势的基因共有46个,在生物学过程层面富集在器官发育、神经元凋亡的正负调控、少突胶质细胞增殖、成纤维生长因子信号通路等;在细胞组分层面,主要参与组成细胞、神经元的基本结构;在分子功能层面,主要与成纤维细胞生长因子受体结合有关。正常组中呈现下降表达趋势而在处理组中呈现上升表达趋势的61个基因,在生物学过程层面富集在细胞溶解、氨基酸/离子转运等功能上;在细胞组分层面,富集在胞内分子、皮质颗粒、胞外区域、细胞间隙等;在分子功能层面,与一系列酶及转运蛋白的活性有关。RT-qPCR验证结果显示转录组测序数据真实可靠。结论·RA干预使小鼠胚胎发育过程中发生基因表达失调和应激反应,导致胚胎发育异常,机体自我保护相关信号通路激活,维持胚胎正常发育的基因受到抑制。

低叶酸和维甲酸诱导神经管畸形小鼠模型胎脑的转录物组学分析

神经管畸形(neural tube defects,NTDs)是一类中枢神经系统相关的重大出生缺陷型疾病。随着孕龄期妇女叶酸增补政策的推广,其发病率逐年下降致临床标本不易获取,因此,建立可靠的动物模型来研究NTDs的致病机制尤为重要。本研究分别通过低叶酸联合甲氨蝶呤(methotrexate,MTX)建立的诱导NTDs小鼠模型和维甲酸(retinoic acid,RA)诱导建立的NTDs小鼠模型,对NTDs胎鼠脑组织进行转录物组测序(transcriptome sequencing,RNA-seq)并分析差异表达谱,最后,通过实时荧光定量PCR(real-time quantitative polymerase chain reaction,RT-qPCR)进行结果验证。结果显示,低叶酸联合MTX诱导的胎鼠NTDs发生率为21.7%;RA诱导的胎鼠出现了强烈的致畸现象,畸形率为73.2%。相比正常胎鼠,低叶酸联合MTX诱导的NTDs小鼠组筛选出1443个差异表达基因(differentially expression genes,DEGs);RA诱导NTDs小鼠组筛选出3070个DEGs。对DEGs进行生物信息学分析,GO富集显示,上调基因主要参与前后轴发育、区域化和模式分化过程等生物学过程;KEGG富集显示,上调基因与心肌收缩、心肌病、神经活性配体-受体相互作用等信号通路相关。对2种模型鼠的DEGs进行Veen交集分析,结果显示,共有132个DEGs在2组模型中显著上调,其中包括Hox基因家族。通过RT-qPCR进行验证,发现与RNA-seq的结果一致。本研究对2种NTDs小鼠胎脑进行RNA-seq和差异表达谱分析,发现Hox的异常表达可能导致了NTDs的发生,为后续发病机制的研究提供了探索与思考。

CKMT1A、CRABP2、PAX8在子宫内膜癌患者血清中的表达水平及其与临床病理参数和预后的关系

目的探讨线粒体肌酸激酶1A(CKMT1A)、细胞视黄酸结合蛋白2(CRABP2)、配对盒基因8(PAX8)在子宫内膜癌患者血清中的表达水平及其与临床病理参数和预后的关系。方法选取2021年5月至2022年5月唐山市妇幼保健院收治的107例子宫内膜癌患者作为研究组。并在治疗后进行1年定期随访,根据患者复发情况分为复发组和未复发组。另选取同时期在该院进行体检的86例健康者作为健康组。采用酶联免疫吸附试验检测各组血清CKMT1A、CRABP2、PAX8水平,比较不同临床病理指标的子宫内膜癌患者血清CKMT1A、CRABP2、PAX8水平。采用Pearson相关分析子宫内膜癌患者血清CKMT1A、CRABP2、PAX8水平的相关性。绘制受试者工作特征(ROC)曲线分析血清CKMT1A、CRABP2、PAX8单独及三者联合检测对子宫内膜癌复发的预测价值。结果研究组血清CKMT1A、CRABP2、PAX8水平均高于健康组,差异均有统计学意义(P<0.05)。Pearson相关分析结果显示,血清CKMT1A与CRABP2水平呈正相关(r=0.437,P<0.001),CKMT1A与PAX8水平呈正相关(r=0.526,P<0.001),CRABP2与PAX8水平呈正相关(r=0.493,P<0.001)。不同肌层浸润、分化程度、淋巴结转移情况等的子宫内膜癌患者血清CKMT1A、CRABP2、PAX8水平比较,差异均有统计学意义(P<0.05),且血清CKMT1A、CRABP2、PAX8水平随着分化程度的降低及淋巴结的转移而升高(P<0.05)。复发组纳入12例患者,非复发组纳入95例患者。复发组血清CKMT1A、CRABP2、PAX8水平均高于非复发组,差异均有统计学意义(P<0.05)。ROC曲线分析结果显示,血清CKMT1A、CRABP2和PAX8单独预测子宫内膜癌复发的曲线下面积分别为0.886、0.850、0.811,均低于三者联合检测的0.978(Z三者联合-CKMT1A=2.318,P=0.020;Z三者联合-CRABP2=2.030,P=0.042;Z三者联合-PAX8=2.955,P=0.003)。结论子宫内膜癌患者血清CKMT1A、CRABP2和PAX8水平均高表达,三者表达与病理类型、分化程度、淋巴结转移情况等临床病理参数以及预后有关,可以作为评估子宫内膜癌患者预后的生物学指标。

全反式维甲酸调控K562细胞红系分化的表观遗传机制

全反式维甲酸(ATRA)是早幼粒细胞分化的有效诱导剂,其对红系分化过程的作用尚不完全清楚。为研究ATRA在红系分化进程中的作用及其表观遗传调控机制,本文以诱导白血病细胞K562向红系分化为模型,对ATRA干扰红系分化过程的调控机制进行研究。首先利用血红素(he-min)诱导K562细胞向红系分化;流式细胞术结果显示,ATRA影响细胞向红系分化过程中的谱系变化,阻滞细胞分化进程;ATRA处理分化中的细胞后,红系分化相关基因表达水平降低;而通过3C、FAIRE和ChIP技术对其中的表观遗传机制进行探究发现,ATRA处理细胞后,β-珠蛋白家族基因座位内的染色质可及性降低,LCR与其靶基因启动子之间的相互作用频率降低;而基因座位染色质可及性降低导致了红系相关转录因子GATA1、LDB1、LMO2和TAL1在LCR及珠蛋白家族基因座位的启动子区的富集频率降低。上述结果表明,ATRA处理分化中的细胞导致红系分化相关基因的染色质可及性降低,更加封闭的染色质结构阻碍了LCR招募转录因子与基因启动子区的结合,进而抑制β-珠蛋白家族基因表达,这种动态的变化过程阐明了ATRA调控红系分化的表观遗传机制。