口腔扁平苔藓病损组织自噬相关基因微管相关蛋白1轻链3B、p62和Beclin1的表达及意义

Expression and significance of microtubule associated protein 1 light chain 3B, p62 and Beclin1 in lesion tissues of oral lichen planus patients

目的了解自噬相关基因微管相关蛋白1轻链3B(microtubule associated protein 1 light chain 3B, LC3B)、p62及自噬关键分子Beclin1在口腔扁平苔藓(oral lichen planus, OLP)病损组织中的表达变化及其与OLP临床病理特征的关系, 探讨细胞自噬在OLP病因机制中的作用及意义。方法于贵州医科大学口腔医学院·附属口腔医院牙周黏膜科2017年10月至2019年12月的就诊患者中, 选取41例OLP患者的病损组织(OLP组, 21例糜烂型OLP, 20例非糜烂型OLP)及15例来自贵州医科大学附属口腔医院口腔颌面外科手术中收集的正常口腔黏膜组织(对照组), 采用免疫组织化学技术(immunohistochemistry, IHC)与实时荧光定量PCR(real-time quantitative PCR, RT-qPCR)检测OLP组织中LC3B、p62、Beclin1的蛋白及mRNA水平, 采用蛋白质印迹法检测上述41例OLP病损的16例组织(8例糜烂型OLP, 8例非糜烂型OLP)中LC3B、p62、Beclin1的蛋白表达量及LC3B-Ⅱ/LC3B-Ⅰ比值。分析LC3B、p62、Beclin1及LC3B-Ⅱ/LC3B-Ⅰ比值与OLP临床病理特征的关系。结果 IHC结果显示, OLP病损组织中LC3B、p62蛋白阳性表达率[分别为68%(28/41)、59%(24/41)]均显著高于对照组(分别为5/15、3/15)(χ^(2)=5.55, P=0.019;χ^(2)=5.55, P=0.015);糜烂型OLP组织中LC3B、p62蛋白阳性表达率[分别为86%(18/21)、76%(16/21)]均显著高于非糜烂型OLP[分别为50%(10/20)、40%(8/20)](χ^(2)=4.50, P=0.034;χ^(2)=5.53, P=0.019);OLP病损组织中Beclin1蛋白的阳性表达率[20%(8/41)]较对照组(7/15)显著降低(χ^(2)=4.13, P=0.042), 但Beclin1蛋白阳性表达率在OLP两型间差异无统计学意义(P>0.05)。RT-qPCR结果显示, OLP组织中LC3B、p62 mRNA表达水平[分别为2.78(1.59, 6.15)、4.30(2.34, 6.29)]均显著高于对照组[分别为1.05(0.88, 1.21)、1.12(0.89, 1.36)](Z=-4.56, P<0.001;Z=-4.78, P<0.001), 糜烂型OLP组LC3B、p62 mRNA表达水平均显著高于非糜烂型OLP(Z=-2.87, P=0.004;Z=-2.95, P=0.003), 而Beclin1在OLP组织中的mRNA表达显著低于对照组(Z=-2.43, P=0.015), 但在OLP两型间差异无统计学意义(P>0.05)。蛋白质印迹法结果显示, OLP组织中LC3B-Ⅱ/LC3B-Ⅰ比值显著高于对照组(t=-2.45, P=0.021), 非糜烂型OLP组织中LC3B-Ⅱ/LC3B-Ⅰ比值显著高于糜烂型OLP(t=-2.38, P=0.032)。Spearman相关性分析显示, LC3B-Ⅱ/LC3B-Ⅰ比值与OLP临床分型、基底细胞液化变性程度均呈显著负相关关系(r=-0.57, P=0.021;r=-0.54, P=0.032), 与病程及淋巴细胞浸润程度无显著相关性(P>0.05)。结论自噬相关因子LC3B、p62与Beclin1在OLP病损形成及发展中可能发挥了一定作用;与非糜烂型OLP相比, 糜烂型OLP的自噬水平相对不足, 这可能导致糜烂型OLP局部病损破坏程度加重;细胞自噬异常可能在OLP病损形成中起重要作用。

Objective To explore the expression of autophagy related factors microtubule associated protein 1 light chain 3B(LC3B),p62,autophagy key factor Beclin1 in oral lichen planus(OLP)tissues and their relationships with the clinicopathological characteristics of OLP,investigating the function and significance of autophagy in pathogenesis of OLP.Methods Forty-one lesion tissues(OLP group,twenty-one cases of erosive OLP and twenty cases of non-erosive OLP)were selected from OLP patients visiting the Department of Periodontal and Oral Medicine,School and Hospital of Stomatology,Guizhou Medical University from October 2017 to December 2019.Fifteen cases of normal oral mucosal tissues(control group)were collected from oral and maxillofacial surgery at The Affiliated Stomatology Hospital of Guizhou Medical University during the same period.Protein and mRNA expression levels of LC3B,p62 and Beclin1 were detected by immunohistochemistry(IHC)and real-time quantitative PCR(RT-qPCR)in OLP lesions respectively.The protein expression levels of LC3B,p62,Beclin1 and ratio of LC3B-Ⅱ/LC3B-Ⅰin sixteen cases(eight cases of erosive OLP and eight cases of non-erosive OLP)from the OLP group were detected by Western blotting(WB).The potential relationship between LC3B,p62,Beclin1,LC3B-Ⅱ/LC3B-Ⅰratio and clinical features of OLP were analyzed.Results IHC results showed that the positive expression rates of LC3B and p62 proteins in OLP lesion tissues[LC3B:68%(28/41);p62:59%(24/41)]were higher than those in the control group[LC3B:5/15;p62:3/15](LC3B:χ^(2)=5.55,P=0.019;p62:χ^(2)=5.55,P=0.015).The positive expression rates of LC3B and p62 proteins in the erosive OLP group[LC3B:86%(18/21);p62:76%(16/21)]were higher than those in the non-erosive OLP group[LC3B:50%(10/20);p62:40%(8/20)](LC3B:χ^(2)=4.50,P=0.034;p62:χ^(2)=5.53,P=0.019).The positive expression rate of Beclin1 protein in the OLP lesions[20%(8/41)]was lower than that in the control group(7/15)(χ^(2)=4.13,P=0.042),but was not statistically different between the two types of OLP(P>0.05).The RT-qPCR results showed that the mRNA expression levels of LC3B and p62 in OLP lesions[LC3B:2.78(1.59,6.15);p62:4.30(2.34,6.29)]were higher than those in the control group[LC3B:1.05(0.88,1.21);p62:1.12(0.89,1.36)](LC3B:Z=-4.56,P<0.001;p62:Z=-4.78,P<0.001),and the mRNA expression levels of LC3B and p62 in the erosive OLP group were higher than those in the non-erosive OLP group(LC3B:Z=-2.87,P=0.004;p62:Z=-2.95,P=0.003).The mRNA expression level of Beclin1 in OLP tissues was lower than that in the control group(Z=-2.43,P=0.015),but the difference was not statistically significant between the two types of OLP(P>0.05).WB results showed that the LC3B-Ⅱ/LC3B-Ⅰratio was higher in the OLP lesions than that in the control group(t=-2.45,P=0.021),and the LC3B-Ⅱ/LC3B-Ⅰratio was higher in the non-erosive OLP group than in the erosive OLP group(t=-2.38,P=0.032).Spearman′s correlation analysis showed that the ratio was negatively correlated with the clinical staging and the degree of basal cell liquefaction in OLP(clinical staging:r=-0.57,P=0.021;basal cell liquefaction:r=-0.54,P=0.032),but not with the disease duration and the degree of lymphocytic infiltration(P>0.05).Conclusions Autophagy related factors LC3B,p62 and Beclin1 may play a role in the formation and progression of OLP lesions.The autophagy level was relatively lack in erosive OLP compared to non-erosive OLP,contributing to the increased local lesion destruction in erosive OLP.Abnormal cellular autophagy may play an important role in the formation of OLP lesions.