Interaction between aromatic hydrocarbon receptor signaling pathway-related gene polymorphism and its mRNA in the development of endometriosis
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摘要:目的 探讨芳香烃受体(AhR)信号通路相关基因[AHR、AhR核异位蛋白(ARNT)、细胞色素P450 1A1(CYP1A1)、AhR阻遏蛋白(AhRR)、谷胱甘肽S转移酶1(GSTP1)、白细胞介素-1β(IL-1β)]多态性及mRNA在子宫内膜异位症(EMT)发病中的相互作用。方法 选择手术病理确诊为EMT的25例患者为病例组, 25例非EMT患者为对照组。实时定量PCR法检测mRNA表达，定量PCR为基础的高分辨率熔解曲线(PCR-HRM)法检测基因多态性，采用单纯病例研究方法对基因-基因交互作用进行分析。结果 病例组中AhR mRNA、GSTP1 mRNA和IL-1β mRNA水平较对照组显著增高(P < 0.01)。病例组中ARNT mRNA水平显著高于对照组(P < 0.05)。携带CYP1A1 rs4646903 CC突变基因型的个体发生EMT的危险度是TT纯合野生型的7.5倍(95%CI: 1.29~43.69), 其他基因多态性的分布组间比较无显著差异(P>0.05)。CYP1A1 6235 T/C突变型与ARNT 522 G/C突变型(OR=9.33, 95%CI: 1.47~59.48)、GSTP1313 A/G突变型(OR=13.50, 95%CI: 1.34~135.98)存在协同作用。AHR 1661 G/A突变型与AHRR 565 C/G突变型存在协同作用(OR=13.50, 95%CI: 1.34~159.98), 其他基因位点未见交互作用。结论 携带CYP1A1 rs4646903 CC基因型的个体发生EMT的风险性较高, CYP1A1 6235 T/C与ARNT 522 G/C突变型间的联合作用以及CYP1A1 6235 T/C与GSTP1313 A/G突变型间的联合作用与EMT的发病有关。Abstract:Objective To explore the interaction between aromatic hydrocarbon receptor (AhR) signaling pathway-related genes [AHR, AhR nuclear heterotopic protein (ARNT), cytochrome P450 1A1 (CYP1A1), AhR repressor protein (AhRR), glutathione S-transferase 1 (GSTP1), interleukin-1β (IL-1β)] polymorphism and messenger RNA (mRNA) in endometriosis pathogenesis (EMT).Methods A total of 25 patients with endometriosis confirmed by surgery and pathology were selected as case group (n=25), and 25 non-EMT patients were selected as control group (n=25). Gene polymorphism were detected by Polymerase Chain Reaction-High Resolution Melt (PCR-HRM) method, and expressions of mRNA were detected by real-time quantitative PCR in both groups. The gene-gene interactions were analyzed based on the simple-case-study method.Results The levels of AhR, GSTP1 and IL-1β mRNAs in case group were significantly higher than those in control group (P < 0.01). The level of ARNT mRNA in case group increased significantly when compared with that in control group (P < 0.05). The risk of EMT in patients with CYP1A1 rs4646903 CC mutation genotype was 7.5 times higher than patients with homozygous wild genotype TT (95%CI: 1.29 to 43.69), and there were no significant differences in frequencies of other genotypes between case group and control group (P>0.05). The synergistic role of mutated CYP1A1 6235 T/C and mutated ARNT 522 G/C gene increased the risk of endometriosis (OR=9.33, 95%CI: 1.47 to 59.48), and the risk of disease also increased when combined with mutated CYP1A1 6235 T/C and mutated GSTP1313 A/G gene (OR=13.50, 95%CI: 1.34 to 135.98). Moreover, combined with mutated AHR 1661 G/A and AHRR 565 C/G appeared to cause a significant increase in endometriosis (OR=13.50, 95%CI: 1.34 to 159.98). No interaction was found at other loci.Conclusion Patients with CYP1A1 rs4646903 CC gene has a high risk in developing endometriosis. The combined mutated genotypes (CYP1A1 6235 T/C and ARNT 522 G/C, CYP1A1 6235 T/C and GSTP1313 A/G) might be related with susceptibility to endometriosis.
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Keywords:
- endometriosis /
- gene polymorphism /
- gene-gene interaction /
- signaling pathway /
- gene mutation
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表 1 引物序列及PCR产物大小
引物 序列 长度 GAPDH 正义引物: CAATGACCCCTTCATTGACC; 106 bp 反义引物: GACAAGCTTCCCGTTCTCAG AHR 正义引物: ACATCACCTACGCCAGTCG; 94 bp 反义引物: CGCTTGGAAGGATTTGACTTGA ARNT 正义引物: TGACTCCTGTTTTGAACCAGC; 95 bp 反义引物: CTGCTCACGAAGTTTATCCACAT CYP1A1 正义引物: TCGGCCACGGAGTTTCTTC; 141 bp 反义引物: GGTCAGCATGTGCCCAATCA GSTP1 正义引物: CATCTACACCAACTATGAGGCG; 81 bp 反义引物: AGCAGGGTCTCAAAAGGCTTC IL-1β 正义引物: ATGATGGCTTATTACAGTGGCAA 132 bp 反义引物: GTCGGAGATTCGTAGCTGGA 
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表 2 病例组与对照组6个基因mRNA的表达水平(x±s)
组别 n AHR mRNA AHRR mRNA ARNT mRNA CYP1A1 mRNA GSTP1 mRNA IL-1β mRNA 病例组 25 3.05±1.00** 0.50±0.01 1.14±0.34* 0.94±0.33 4.26±0.28** 6.88±0.72** 对照组 25 0.89±0.26 0.53±0.03 1.00±0.33 0.56±0.15 0.49±0.02 0.41±0.01 与对照组比较, *P < 0.05, **P < 0.01。
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表 3 AHR、ARNT、AHRR、CYP1A1、GSTP1、IL-1β基因多态性在病例组与对照组中分布[n(%)]
基因型 病例组 对照组 OR(95%CI) P AHR 1661 G/A (rs2066853) GG 5(20.00) 5(20.00) Referent 1.00 GA 12(48.00) 18(72.00) 0.67(0.16~2.81) 0.58 AA 8(32.00) 2(8.00) 4.00(0.55~29.10) 0.17 GA, AA 20(80.00) 20(80.00) 1.00(0.25~4.00 1.00 ARNT 522 G/C (rs2228099) GG 5(20.00) 8(32.00) Referent CG 10(40.00) 12(48.00) 2.40(0.61~9.38) 0.21 CC 10(40.00) 5(20.00) 3.20(0.68~15.07) 0.14 CG, CC 20(80.00) 17(68.00) 0.53(1.15~1.93) 0.34 AHRR 565 C/G (rs2292596) CC 5(20.00) 10(40.00) Referent CG 18(72.00) 15(60.00) 0.37(1.00~1.45) 0.37 GG 2(8.00) 0 — — CG, GG 20(80.00) 15(60.00) 0.44(0.02~9.03) 0.598 CYP1A1 6235 T/C (rs4646903) TT 8(32.00) 10(40.00) Referent TC 5(20.00) 13(52.00) 0.48(0.12~1.93) 0.30 CC 12(48.00) 2(8.00) 7.50(1.29~43.69) 0.03 TC, CC 17(68.00) 15(60.00) 1.41(0.44~4.52) 0.56 GSTP1 313 A/G (rs1695) AA 15(60.00) 15(60.00) Referent AG 8(32.00) 10(40.00) 1.25(0.39~4.04) 0.71 GG 2(8.00) 0 — — AG, GG 10(40.00) 10(40.00) — — IL-1β 3953 C/T (rs1143634) CC 25(100.00) 25(100.00) — —
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表 4 基因-基因在EMT发病中的交互作用
基因型 基因位点 OR(95%CI) TT TC+CC CYP1A1 6235 T/C ARNT 522 G/C CG+CCGSTP1 313 A/G AG+GG 1010 1515 9.33(1.47~59.48)13.50(1.34~135.98) AHR 1661 G/A AHRR 565 C/G CG+GG GG5 GA+AA20 13.5(1.34~159.98)
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