DNA甲基转移酶3A在胰腺癌组织的表达及其调控因子筛选

Expression of DNA methyltransferase 3A in pancreatic cancer and screening of its regulatory factors

目的探讨胰腺癌组织和癌旁组织中DNA甲基转移酶3A的表达水平,并采用转录组学筛选调控DNA甲基转移酶3A的微小RNA(miRNA,miR)家族。方法选取2021年6月到2023年6月新乡医学院第一附属医院住院部手术切除的胰腺癌组织和对应癌旁组织作为研究对象。采用蛋白质免疫印迹分析DNA甲基转移酶3A的表达水平。随机选取6例癌旁组织和胰腺癌组织作为研究对象,采用转录组学分析癌旁组织和胰腺癌组织中差异表达的(microRNA,miRNA),并采用生物信息学和荧光素酶报告基因分析差异表达miRNA对DNA甲基转移酶3A的调控关系。结果癌旁组织中DNA甲基转移酶3A表达水平(0.77±0.16)明显低于胰腺癌组织(1.48±0.21),差异有统计学意义(t=19.690,P<0.05)。胰腺癌组织和癌旁组织共有390个miRNA差异表达,其中上调259个,下调131个。这些miRNA主要生物学过程包括细胞增殖、细胞发育、细胞分化等;分子功能主要包括肿瘤细胞运动、细胞迁移、细胞侵袭、细胞增殖和细胞间信号转导等。发现下调的miRNA与DNA甲基转移酶3A存在碱基互补,其中差异最为显著排序前10个的miRNA分别为miR-143-3p、miR-129-5p、miR-493-5p、miR-367-3p、miR-29a-3p、miR-212-3p、miR-323A-3p、miR-5195-3p、miR-4465和miR-532-5p。癌旁组织中miR-143-3p、miR-129-5p、miR-493-5p、miR-367-3p、miR-29a-3p、miR-212-3p、miR-323a-3p、miR-5195-3p、miR-4465和miR-532-5p表达水平(1.09±0.11、0.97±0.08、1.08±0.31、1.17±0.121、1.07±0.08、1.01±0.10、0.97±0.08、1.01±0.05、0.99±0.03、1.04±0.10)明显高于胰腺癌组织(0.51±0.10、0.46±0.11、0.47±0.15、0.62±0.12、0.37±0.07、0.56±0.10、0.66±0.04、0.68±0.06、0.73±0.05、0.66±0.13),差异有统计学意义(t=11.860、12.170、5.590、9.910、21.070、9.941、11.180、14.030、13.570、7.470,P<0.05)。miRNA对照和荧光素酶报告基因共转染细胞荧光素酶活性(0.97±0.02、0.96±0.02、0.97±0.02、0.98±0.02、0.97±0.02、0.97±0.04、0.95±0.03、0.96±0.03、0.97±0.02、0.96±0.02)明显高于miR-143-3p、miR-129-5p、miR-493-5p、miR-367-3p、miR-29a-3p、miR-212-3p、miR-323a-3p、miR-5195-3p、miR-4465和miR-532-5p模拟物和荧光素酶报告基因共转染细胞荧光素酶活性(0.32±0.07、0.33±0.06、0.35±0.09、0.50±0.04、0.50±0.05、0.53±0.07、0.58±0.03、0.63±0.03、0.65±0.05、0.81±0.05),差异有统计学意义(t=22.400,24.470,16.730,24.480、20.530,14.010,21.360,19.040,15.960,6.751,P<0.05)。结论DNA甲基转移酶3a在胰腺癌组织中表达水平显著增加,miR-143-3p、miR-129-5p、miR-493-5p、miR-367-3p、miR-29a-3p、miR-212-3p、miR-323a-3p、miR-5195-3p、miR-4465和miR-532-5p等miRNA参与调控DNA甲基转移酶3a在胰腺癌组织中的表达。

Objective To investigate the expression level of DNA methyltransferase 3A in pancreatic cancer tissues and adjacent tissues,and to screen the microRNA(miRNA,miR)family that regulates DNA methyltransferase 3A by transcriptomics.Methods From June 2021 to June 2023,the pancreatic cancer tissues and corresponding adjacent tissues resected in the inpatient department of the First Affiliated Hospital of Xinxiang Medical College were selected as the research objects.The expression level of DNA methyltransferase 3A was analyzed by Western blotting.A total of 6 cases of adjacent tissues and pancreatic cancer tissues were randomly selected as research objects.The differential expression(microRNA,miRNA)in adjacent tissues and pancreatic cancer tissues was analyzed by transcriptomics,and the regulation relationship of differential expression genes to DNA methyltransferase 3A was analyzed by bioinformatics and luciferase reporter genes.The comparison of quantitative data was done by t-test.Results The expression level of DNA methyltransferase 3A in adjacent tissues(0.77±0.16)was significantly lower than that in pancreatic cancer tissues(1.48±0.21,t=19.690,P<0.05).There were 390 differentially expressed miRNAs in pancreatic cancer tissues and adjacent tissues,of which 259 were up-regulated and 131 were down-regulated.The main biological processes of these miRNAs included cell proliferation,cell development,cell differentiation.Molecular functions mainly included tumor cell movement,cell migration,cell invasion,cell proliferation,and intercellular signal transduction.It was found that downregulated miRNAs were complementary to DNA methyltransferase 3A,with the top 10 miRNAs with the most significant differences being miR-143-3p,miR-129-5p,miR-493-5p,miR-367-3p,miR-29a-3p,miR-212-3p,miR-323a-3p,miR-5195-3p,miR-4465,and miR-532-5p,respectively.The expression levels of miR-143-3p,miR-129-5p,miR-493-5p,miR-367-3p,miR-29a-3p,miR-212-3p,miR-323a-3p,miR-5195-3p,miR-4465 and miR-532-5p in adjacent cancerous tissues(1.09±0.11,0.97±0.08,1.08±0.31,1.17±0.121,1.07±0.08,1.01±0.10,0.97±0.08,1.01±0.05,0.99±0.03,1.04±0.10)were significantly higher than those of pancreatic cancer tissues(0.51±0.10,0.46±0.11,0.47±0.15,0.62±0.12,0.37±0.07,0.56±0.10,0.66±0.04,0.68±0.06,0.73±0.05,0.66±0.13,t=11.860,12.170,5.590,9.910,21.070,9.941,11.180,14.030,13.570,7.470,P<0.05).The luciferase activity of cells co-transfected with miRNA control and luciferase reporter genes(0.97±0.02,0.96±0.02,0.97±0.02,0.98±0.02,0.97±0.02,0.97±0.04,0.95±0.03,0.96±0.03,0.97±0.02,0.96±0.02)was significantly higher than that of miR-143-3p,miR-129-5p,miR-493-5p,miR-367-3p,miR-29a-3p,miR-212-3p,miR-323a-3p,miR-5195-3p miR-4465 and miR-532-5p mimetics with luciferase reporter(0.32±0.07;0.33±0.06;0.35±0.09;0.50±0.04;0.50±0.05;0.53±0.07;0.58±0.03;0.63±0.03;0.65±0.05;0.81±0.05,t=22.400,24.470,16.730,24.480,20.530,14.010,21.360,19.040,15.960,6.751,P<0.05).Conclusion The expression level of DNA methyltransferase 3a in pancreatic cancer tissue is significantly increased.miR-143-3p,miR-129-5p,miR-493-5p,miR-367-3p,miR-29a-3p,miR-212-3p,miR-323a-3p,miR-5195-3p,miR-4465 and miR-532-5p are involved in regulating the expression of DNA methyltransferase 3A in pancreatic cancer tissue.