Isolation of Zizania latifolia Species-specific DNA Sequences and Their Utility in Identification of Z. latifolia DNA Introgressed into Rice

根据两个植物抗病基因N和RPS2中核酸结合位点 (NBS)和富亮氨酸重复区 (LRR)中的保守序列设计了一对特异引物 ,用PCR从具有水稻 (OryzasativaL .)改良所需要的许多优良性状的水稻近缘野生种菰 (Zizanialatifolia(Griseb .)Turcz.exStapf)的基因组DNA中扩增同源片段。PCR产物经克隆后 ,分别以菰和水稻的基因组DNA为探针 ,通过点杂交对所得克隆进行了分析。点杂交结果表明 ,在所分析的 6 0个克隆中有 2个克隆是菰专化的序列 ,即它们与水稻无杂交信号。基因组DNA的Southern杂交进一步证实了这 2个克隆的专化性。为了验证一些可能的“水稻_菰”渐渗杂交系是否确实含有源自供体菰的DNA ,以这 2个克隆为探针 ,与经EcoRⅠ酶切的 5个可能的渐渗杂交系进行了Southern杂交。结果表明 ,这 2个克隆均能检测出其中的一个系含有其同源序列。这一结果为曾经报道的经一种非常规有性杂交方法将菰DNA导入水稻提供了确凿的证据。

Microdissection of Haynaldia villosa Telosome 6VS and Cloning of Species-specific DNA Sequences

The material T240_6 derived from SC 2 young embryo of the combination CA9211/RW15 (6D/6V alien substitution) was telosomic substitution line of 6VS identified by GISH (genomic in situ hybridization) analysis. The 6VS was microdissected with a needle and transferred into a 0.5 mL Ep tube. In the 'single tube', all the subsequence steps were conducted. After two round of LA (Linker adaptor)_PCR amplification, the size of PCR bands ranged from 100 to 3 000 bp, with predominate bands 600-1 500 bp. The products were confirmed by Southern blotting analysis using Haynaldia villosa (L.) Schur. genomic DNA labeled with 32 P as probe. The PCR products were purified and ligated into clone vector-pGEM_T easy vector. Then, the plasmids were transformed into competence E. coli JM109 with cool CaCl 2. It was estimated that there were more than 17 000 white clones in the library. The size of insert fragments distributed from 100-1 500 bp, with average of 600 bp. Using H. villosa genomic DNA as probe, dot blotting results showed that 37% clones displayed strong and medium positive signals, and 63% clones had faint or no signals. It is demonstrated that there were about 37% repeat sequence clones and 67% single/unique sequence clones in the library. Eight H. villosa_specific clones were screened from the library, and two clones pHVMK22 and pHVMK134 were used for RFLP analysis and sequencing. Both of them were H. villosa specific clones. The pHVMK22 was a unique sequence clone, and the pHVMK134 was a repeat sequence clone. When the pHVMK22 was used as a probe for Southern hybridization, all the powdery mildew resistance materials showed a special band of 2 kb, while all the susceptible ones not. The pHVMK22 may be applied to detect the existence of Pm21.

基于活产建立体外受精-胚胎移植精子DNA碎片指数的参考阈值及子代短期安全性

背景:精子DNA碎片指数与受精、胚胎发育潜能、胚胎植入、流产及子代安全性等存在显著的相关性。然而,其临床参考值受多种因素的影响,导致临床意义极其有限,该研究以活产为结局,通过倾向评分匹配校正其他混杂因素后,构建精子DNA碎片指数与活产的最佳临床截断值,并对其进行内外部验证,具有较好的预测价值及临床应用效能。目的:探讨基于活产建立体外受精-胚胎移植精子DNA碎片指数的参考阈值及子代短期安全性。方法:选取2019年5月至2021年5月于常州市妇幼保健院接受体外受精-胚胎移植患者1921例,以倾向匹配容差0.02为标准,1∶1进行倾向评分匹配,结果活产组与非活产组各成功匹配540例,以此建立模型组;通过选取同时期广西壮族自治区南溪山医院接受体外受精-胚胎移植患者135例作为外部验证组;采用受试者工作曲线探求精子DNA碎片指数对活产的临床最佳截断值,分别采用限制性立方样条曲线、标准曲线、临床决策曲线、临床影响曲线及内外部验证等方法,对该截断值的准确性及临床应用效能进行评估。结果与结论:(1)非活产组精子DNA碎片指数显著高于活产组且与活产存在显著的负相关性(r=-0.444,P<0.001);(2)受试者工作曲线结果显示,DNA碎片指数对活产的最佳截断值为24.33%,曲线下面积为0.775(0.746,0.804),特异度为72.60%,敏感度为78.90%,准确度为75.70%;(3)限制性立方样条曲线拟合Logistic回归结果显示,当精子DNA碎片指数大于24.57%时,临床非活产的风险呈趋势性增涨;(4)Logistic回归概率分析结果显示,精子DNA碎片指数为活产的危险因素[OR(95%CI)=0.916(0.904,0.928),P<0.001],且当精子DNA碎片指数大于27.78%时,临床活产发生的概率将小于50%,随着精子DNA碎片指数每增高1个单位,活产的概率下降8.4%;(5)内外部对该临床截断值的验证均显示,该截点具有一定的临床预测价值及准确性;(6)临床决策曲线与临床影响曲线显示,以该临床截断值建立的预测模型在阈概率为0.22-0.73时具有临床最大净获益值,且在该阈概率范围内损失与获益的比值始终小于1,证实该预测模型具有较好的临床应用效能;(7)精子DNA碎片指数与子代短期安全性分析结果显示,精子DNA碎片指数与出生儿早产、体质量、畸形、性别差异无显著性;(8)结果表明,精子DNA碎片指数对体外受精-胚胎移植活产的最佳临床截断值为24.33%,以此建立的临床预测模型具有较好的区分度、准确度与临床应用效能,精子DNA碎片指数对子代短期安全性影响并不显著,但仍需大样本及长期的追踪评估。

Sequence Analysis of Mitochondrial DNA D-loop Region in Xinjiang Goose

[Objective] The sequences of mitochondrial DNA D-loop region of Xinjiang Goose with three different colors of plumage were analyzed in order to study the genetic diversity of Xinjiang Goose, as well as the phylogeny and evolution. [Method] Ten geese were selected randomly from the core populations of grey-, mosaic- and white-plumaged Xinjiang Goose respectively with a total number of thirty as experi- mental materials, of which the blood samples were collected from the largest vein under the wing （brachial vein） for DNA extraction. Sequences of mitochondrial DNA D-loop regions were determined using DNA sequencing technology to analyze the polymorphism. In addition, the genetic distances among different populations were estimated through the comparison with the reference sequences. [Resull] The con- tents of A, G, C and T nucleotides in the D-loop region of Xinjiang Goose were 28.85%, 17.05%, 25.38% and 28.72%, respectively. The average haplotype diversity and nucleotide diversity of Xinjiang Goose were 0.583 and 0.056. Xinjiang Goose and Greylag Goose were clustered into the same group. [Conclusion] The results showed that Xinjiang Geese with three different colors of plumage all descend from Greylag Goose （Anser anser）.

Coseismic Coulomb stress changes induced by a 2020-2021 M_(W)>7.0 Alaska earthquake sequence in and around the Shumagin gap and its influence on the Alaska-Aleutian subduction interface

Three M_(W)>7.0 earthquakes in 2020-2021 occurred in the Shumagin seismic gap and its adjacent area of the Alaska-Aleutian subduction zone,including the Mw7.8 Simeonof thrust earthquake on July 22,2020,the M_(W)7.6 Sand Point strike-slip earthquake on October 19,2020,and the M_(W)8.2 Chignik thrust earthquake on July 29,2021.The spatial and temporal proximity of these three earthquakes prompts us to probe stress-triggering effects among them.Here we examine the coseismic Coulomb stress change imparted by the three earthquakes and their influence on the subduction interface.Our results show that:(1)The Simeonof earthquake has strong loading effects on the subsequent Sand Point and Chignik earthquakes,with the Coulomb stress changes of 3.95 bars and 2.89 bars,respectively.The Coulomb stress change caused by the Sand Point earthquake at the hypocenter of the Chignik earthquake is merely around 0.01 bars,suggesting the negligible triggering effect on the latter earthquake;(2)The triggering effects of the Simeonof,Sand Point,and Chignik earthquakes on aftershocks within three months are not well pronounced because of the triggering rates of 38%,14%,and 43%respectively.Other factors may have played an important role in promoting the occurrence of these aftershocks,such as the roughness of the subduction interface,the complicated velocity structure of the lithosphere,and the heterogeneous prestress therein;(3)The three earthquakes caused remarkable coseismic Coulomb stress changes at the subduction interface nearby these mainshocks,with an average Coulomb stress change of 3.2 bars in the shallow region directly inwards the trench.

Pse-in-One 2.0: An Improved Package of Web Servers for Generating Various Modes of Pseudo Components of DNA, RNA, and Protein Sequences

Pse-in-One 2.0 is a package of web-servers evolved from Pse-in-One (Liu, B., Liu, F., Wang, X., Chen, J. Fang, L. & Chou, K.C. Nucleic Acids Research, 2015, 43:W65-W71). In order to make it more flexible and comprehensive as suggested by many users, the updated package has incorporated 23 new pseudo component modes as well as a series of new feature analysis approaches. It is available at http://bioinformatics.hitsz.edu.cn/Pse-in-One2.0/. Moreover, to maximize the convenience of users, provided is also the stand-alone version called “Pse-in-One-Analysis”, by which users can significantly speed up the analysis of massive sequences.

Chaos game representation(CGR)-walk model for DNA sequences

Chaos game representation （CGR） is an iterative mapping technique that processes sequences of units, such as nucleotides in a DNA sequence or amino acids in a protein, in order to determine the coordinates of their positions in a continuous space. This distribution of positions has two features： one is unique, and the other is source sequence that can be recovered from the coordinates so that the distance between positions may serve as a measure of similarity between the corresponding sequences. A CGR-walk model is proposed based on CGR coordinates for the DNA sequences. The CGR coordinates are converted into a time series, and a long-memory ARFIMA （p, d, q） model, where ARFIMA stands for autoregressive fractionally integrated moving average, is introduced into the DNA sequence analysis. This model is applied to simulating real CGR-walk sequence data of ten genomic sequences. Remarkably long-range correlations are uncovered in the data, and the results from these models are reasonably fitted with those from the ARFIMA （p, d, q） model.

A novel color image encryption scheme using fractional-order hyperchaotic system and DNA sequence operations

In this paper, Adomian decomposition method （ADM） with high accuracy and fast convergence is introduced to solve the fractional-order piecewise-linear （PWL） hyperchaotic system. Based on the obtained hyperchaotic sequences, a novel color image encryption algorithm is proposed by employing a hybrid model of bidirectional circular permutation and DNA masking. In this scheme, the pixel positions of image are scrambled by circular permutation, and the pixel values are substituted by DNA sequence operations. In the DNA sequence operations, addition and substraction operations are performed according to traditional addition and subtraction in the binary, and two rounds of addition rules are used to encrypt the pixel values. The simulation results and security analysis show that the hyperchaotic map is suitable for image encryption, and the proposed encryption algorithm has good encryption effect and strong key sensitivity. It can resist brute-force attack, statistical attack, differential attack, known-plaintext, and chosen-plaintext attacks.

Rice bicoid-related cDNA sequence and its expression during early embryogenesis

Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation. To clone and characterize the rice bicoid-related genes, one cDNA clone, Rb24 (EMBL accession number: AJ2771380), was isolated by screening of rice unmature seed cDNA library. Sequence analysis indicates that Rb24 contains a putative amino acid sequence, which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity, 75% similarity) and involves a lys-9 in putative helix 3. Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner. The transcript was detected strongly in young panicles, but less in young leaves and roots. This results are further confirmed with paraffin section in situ hybridization. The signal is intensive in rice globular embryo and located at the apical tip of the embryo, then, along with the development of embryo, the signal is getting reduced and transfers into both sides of embryo. The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

LONG-RANGE CORRELATIONS IN DNA SEQUENCES USING TWO-DIMENSIONAL DNA WALKS

The characterization of long-range correlations and fractal properties of DNA sequences has proved to be adifficult though rewarding task mainly due to the mosaic character of DNA consisting of many patches of various lengthswith different nucleotide constitutions.In this paper we investigate statistical correlations among different positions in DNAsequences using the two-dimensional DNA walk.The root-mean-square fluctuation F(l)is described by a power law.Theautocorrelation function C(l),which is used to measure the linear dependence and periodicity,exists a power law ofC(l)-l^(-μ).We also calculate the mean-square distance<R^2(l)>along the DNA chain,and it may be expressed as<R^2(l)>-l^(?)with 2>γ>1.Our investigations can provide some insights into long-range correlations in DNA sequences.

Active motif finder-a bio-tool based on mutational structures in DNA sequences

Active Motif Finder （AMF） is a novel algorithmic tool, designed based on mutations in DNA sequences. Tools available at present for finding motifs are based on matching a given motif in the query sequence. AMF describes a new algorithm that identifies the occurrences of patterns which possess all kinds of mutations like insertion, deletion and mismatch. The algorithm is mainly based on the Alignment Score Matrix （ASM） computation by com paring input motif with full length sequence. Much of the effort in bioinformatics is directed to identify these motifs in the sequences of newly discovered genes. The proposed bio-tool serves as an open resource for analysis and useful for studying polymorphisms in DNA sequences. AMF can be searched via a user-friendly interface. This tool is intended to serve the scientific community working in the areas of chemical and structural biology, and is freely available to all users, at http：//www.sastra.edu/scbt/amf/.

Construction of Agropyrum intermedium 2Ai-2 Chromosome DNA Library and Cloning of Species-Specific DNA Sequences

The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) wasidentified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes weremicroisolated and collected. After two rounds of PCR amplification, the PCR products wereranged from 150-3 000 bp,with predominant fragments at about 200-2 000 bp. Using Ag.intermedium genomic DNA as a probe, Southern blotting analysis confirmed the products originatedfrom Ag. intermedium genome. The products were purified, ligated to pUC18 and then transformedinto competence E.coli DH5αto produce a 2Ai-2 chromosome DNA library. The microcloningexperiments produced approximately 5 ×105 clones, the size range of the cloned inserts was 200-1 500 bp, with an average of 580 bp. Using Ag.intermedium genomic DNA as a probe, dot blottingresults showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences inthe library. Four Ag. intermedium clones were screened from the library by RFLP, and threeclones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitivesequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitiveDNA sequence.

Pseudo DNA Sequence Generation of Non-Coding Distributions Using Variant Maps on Cellular Automata

In a recent decade, many DNA sequencing projects are developed on cells, plants and animals over the world into huge DNA databases. Researchers notice that mammalian genomes encoding thousands of large noncoding RNAs (lncRNAs), interact with chromatin regulatory complexes, and are thought to play a role in localizing these complexes to target loci across the genome. It is a challenge target using higher dimensional tools to organize various complex interactive properties as visual maps. In this paper, a Pseudo DNA Variant MapPDVM is proposed following Cellular Automata to represent multiple maps that use four Meta symbols as well as DNA or RNA representations. The system architecture of key components and the core mechanism on the PDVM are described. Key modules, equations and their I/O parameters are discussed. Applying the PDVM, two sets of real DNA sequences from both the sample human (noncoding DNA) and corn (coding DNA) genomes are collected in comparison with two sets of pseudo DNA sequences generated by a stream cipher HC-256 under different modes to show their intrinsic properties in higher levels of similar relationships among relevant DNA sequences on 2D maps. Sample 2D maps are listed and their characteristics are illustrated under a controllable environment. Various distributions can be observed on both noncoding and coding conditions from their symmetric properties on 2D maps.

Identification of <i>Angelica acutiloba</i>and Related Species by Analysis of Inter- and Intra-Specific Sequence Variations in Chloroplast and Nuclear DNA Sequences

Japanese Angelica Root prepared from Angelica acutiloba var. acutiloba and A. acutiloba var. sugiyamae, known in Japan as “Toki” and “Hokkai Toki”, is an important crude drug used in Kampo medicine (traditional Japanese medicine). However, since these Angelica varieties have recently outcrossed with each other, it is unclear whether Japanese Angelica Root sold for use in Kampo medicine is a pure variety. Here, we describe DNA sequence polymorphisms that can be used to distinguish between A. acutiloba var. acutiloba and A. acutiloba var. sugiyamae. In our analyses, differences in the trnK region of chloroplast DNA distinguished among some A. acutiloba varieties and related species, but not between A. acutiloba var. acutiloba and A. acutiloba var. iwatensis. One geographical strain of A. acutiloba var. acutiloba and A. acutiloba var. sugiyamae showed identical sequences in three regions of chloroplast DNA, but differences in the internal transcribed spacer region of nuclear ribosomal DNA. One strain of A. acutiloba var. iwatensis and A. acutiloba var. sugiyamae had identical sequences in all of the chloroplast and nuclear ribosomal DNA regions examined. These findings show that A. acutiloba var. acutiloba has hybridized with A. acutiloba var. sugiyamae and that the “Hokkai Toki” variety resulted from outcrossing with A. acutiloba var. iwatensis. Molecular authentication based on analyses of chloroplast and nuclear ribosomal DNA sequences of A. acutiloba and related species is an efficient method to authenticate Japanese Angelica Root at the variety level. Therefore, these analyses can determine whether a product is derived from A. acutiloba var. acutiloba or A. acutiloba var. sugiyamae.

Fuzzy splicing in precursor-mRNA sequences: prediction of aberrant splice-junctions in viral DNA context

RNA splicing normally generates stable splice- junction sequences in viruses that are important in the context of virus mimicry. Potential variability in envelop proteins may occur with point-mutations inducing cryptic splice-junctions, which would remain unrecognized by T-memory cells of higher organisms in vaccine trials. Such aberrant splice- junctions result from evolution-specific non-conser- vation of actual splice-junction sites due to mutations;as such, locations of splice-junctions in a test DNA sequence could only be imprecisely specified. Such impreciseness of splice-junction locations (or cryptic sites) in a sequence is evaluated in this study via “noisy” attributes (with associated stochastics) to the mutated subspace;and, relevant fuzzy considerations are invoked with membership attributes expressed in terms of a spatial signal-to-noise ratio (SSNR). That is, SSNR adopted as a membership function expresses the belongingness of a site-region to exon/intron subspaces. An illustrative example with actual (Dengue 1 viral) DNA data is furnished demonstrating the pursuit developed in predicting aberrant splice-junctions at cryptic sites in the test sequence.

The “3 Genomic Numbers” Discovery: How Our Genome Single-Stranded DNA Sequence Is “Self-Designed” as a Numerical Whole

This article proves the existence of a hyper-precise global numerical meta-architecture unifying, structuring, binding and controlling the billion triplet codons constituting the sequence of single-stranded DNA of the entire human genome. Beyond the evolution and erratic mutations like transposons within the genome, it’s as if the memory of a fossil genome with multiple symmetries persists. This recalls the “intermingling” of information characterizing the fractal universe of chaos theory. The result leads to a balanced and perfect tuning between the masses of the two strands of the huge DNA molecule that constitute our genome. We show here how codon populations forming the single-stranded DNA sequences can constitute a critical approach to the understanding of junk DNA function. Then, we suggest revisiting certain methods published in our 2009 book “Codex Biogenesis”. In fact, we demonstrate here how the universal genetic code table is a powerful analytical filter to characterize single-stranded DNA sequences constituting chromosomes and genomes. We can then show that any genomic DNA sequence is featured by three numbers, which characterize it and its 64 codon populations with correlations greater than 99%. The number “1” is common to all sequences, expressing the second law of Chargaff. The other 2 numbers are related to each specific DNA sequence case characterizing life species. For example, the entire human genome is characterized by three remarkable numbers 1, 2, and Phi = 1.618 the golden ratio. Associated with each of these three numbers, we can match three axes of symmetry, then “imagine” a kind of hyperspace formed by these codon populations. Then we revisit the value (3-Phi)/2 which is probably universal and common to both the scale of quarks and atomic levels, balancing and tuning the whole human genome codon population. Finally, we demonstrate a new kind of duality between “form and substance” overlapping the whole human genome: we will show that—simultaneously with the duality between genes and junk DNA—there is a second layer of embedded hidden structure overlapping all the DNA of the whole human genome, dividing it into a second type of duality information/redundancy involving golden ratio proportions.

THE ABNORMAL EXPRESSION OF CLONED REPEATED SEQUENCE DNA, L5B-4, IN RAT HEPATOMA BERH-2

A repeated sequence DNA fragment, L5B-4, was cloned from the 5 kb BamHI DNA fragments of rat genomic DNA. The expressions of the L5B-4 DNA fragment are different in liver and hepatoma cells. The amounts of transcripts in hepatoma cells are lower in nucleus and higher in cytoplasm, especially in polysomal RNA, as compared with that in liver cells. The alteration shown in polysomal RNA of hepatoma cells seems to be specific. These results are discussed with respect to the possible function of this repeated DNA and its variation in hepatoma cells.

Genetic Polymorphism of Eighteen Lycium barbarum Resources Based on nrDNA ITS Sequence

[Objective] The study aimed to investigate the genetic polymorphism of eighteen Lycium barbarum resources via nrDNA ITS sequencing. [Method] The genomic DNAs from Lycium barbarum leaves were isolated by modified CTAB method for PCR amplification on the nrDNA ITS region using specifically synthesized primers; the amplified fragments were cloned and sequenced, then the sequencing results were clustered. [Result] nrDNA ITS sequences of the tested eighteen Lycium barbarum were firstly obtained in the present study. For all eighteen tested materials, the variation range of whole ITS region was 559-634 bp, with an average of 612 bp; alignment analyses showed that the whole length of internal transcribed spacer (ITS1+ITS2) was 480 bp, within which there are 194 variation sites (accounting for 40.4%) and 286 conserved sites (accounting for 59.6%). The cluster results showed that the eighteen tested materials could be grouped into three classes. [Conclusion] Analysis of nrDNA ITS sequence may avail to identify the Lycium barbarum germplasm resources.

血清CYFRA21-1、CA125联合HPV DNA检测在宫颈癌早期筛查中的价值及病理特征研究

目的分析血清细胞角蛋白19片段(CYFRA21-1)、糖类抗原125(CA125)联合HPV DNA检测在宫颈癌早期筛查中的价值及病理特征。方法选取2019年8月至2023年6月沧州市中心医院收治的宫颈癌患者152例为观察组,另选取同期在本院行体检且各项正常女性80名为对照组;对比两组血清CYFRA21-1、CA125水平以及HPV DNA检测结果;对比观察组不同手术病理结果及血清CYFRA21-1、CA125表达水平以及HPV DNA检测结果;以病理学检查为金标准,分析血清CYFRA21-1、CA125水平以及HPV DNA检测单独以及联合诊断宫颈癌的一致性;绘制ROC曲线,分析血清CYFRA21-1、CA125联合HPV DNA单独检测及联合检测宫颈癌的效能。结果152例患者中,鳞状细胞癌104例,腺癌患者48例;其中轻度不典型增生66例、中度不典型增生57例、重度不典型增生29例。观察组血清CYFRA21-1、CA125水平以及HPV DNA阳性率均高于对照组,差异有统计学意义(P<0.05);血清CYFRA21-1、CA125水平:鳞状细胞癌<腺癌,轻度不典型增生<中度不典型增生<重度不典型增生,差异均有统计学意义(P<0.05)。观察组不同分期、不同肿瘤增生类型的HPV DNA阳性率比较,差异无统计学意义(P>0.05);血清CYFRA21-1、CA125水平、HPV DNA检测单独以及联合诊断宫颈癌与病理学检查结果的一致性Kappa值分别为0.677、0.731、0.756、0.902;CA125+CYFRA21-1+HPV DNA联合检测的AUC为0.894,高于CA125、CYFRA21-1、HPV DNA单独检测(P>0.05)。结论血清CYFRA21-1、CA125联合HPV DNA检测的诊断效能高于单一检测,提示三指标联合检测可显著提高宫颈癌早期筛查的诊断价值,可为制定临床治疗方案提供参考资料。

miR-214-5p通过DNMT1介导的AXIN2基因DNA甲基化修饰在皮肤基底细胞癌中的作用机制

目的探讨皮肤基底细胞癌中轴抑制蛋白2(Axis inhibition protein 2,AXIN2)基因启动子甲基化对基因转录的影响及miR-214-5p通过靶向DNA甲基转移酶1(DNA methyltransferase1,DNMT1)对AXIN2甲基化率的调控机制。方法收集2022年1月-2023年6月在广州市皮肤病防治所就诊治疗的102例皮肤基底细胞癌(Cutaneous basal cell carcinoma,BCC)患者作为研究对象,提取癌组织和癌旁正常组织标本及基线资料。焦磷酸测序法检测AXIN2基因启动子区甲基化率。实时荧光定量PCR检测AXIN2、DNMT1基因mRNA和miR-214-5p的表达水平。将miR-214-5p模拟物(mimic)、抑制物(inhibitor)及其阴性对照(mimic NC和inhibitor NC)分别对基底细胞癌A431细胞进行转染,48 h后检测DNMT1基因mRNA表达水平和AXIN2基因甲基化率。结果BCC癌组织的AXIN2基因甲基化率显著高于癌旁正常组织(t=5.128,P<0.001),AXIN2基因mRNA相对表达水平显著低于癌旁正常组织(t=7.826,P<0.001),DNMT1基因mRNA表达水平显著高于癌旁正常组织(t=4.838,P<0.001),miR-214-5p表达水平显著低于癌旁正常组织(t=5.426,P<0.001)。BCC癌组织的AXIN2基因甲基化率与其mRNA表达水平呈负相关(r=-0.793,P<0.001),DNMT1基因mRNA水平与AXIN2基因甲基化率呈正相关(r=0.814,P<0.001),miR-214-5p表达水平与DNMT1基因mRNA水平呈负相关(r=-0.747,P<0.001)。双荧光素酶报告基因实验结果证实,DNMT1是miR-214-5p的靶基因。细胞转染后,与mimic NC、inhibitor和inhibitor NC比较,mimic的DNMT1基因mRNA水平、AXIN2基因甲基化率显著降低(P<0.001);而inhibitor的DNMT1基因mRNA水平和AXIN2基因甲基化率相较于其他三组明显上升(P<0.001)。结论miR-214-5p可通过调控下游靶蛋白DNMT1表达,影响AXIN2基因的DNA甲基化率,调控AXIN2基因的表达水平,参与皮肤基底细胞癌的发生机制。