【作者】 杨晶；

【导师】 刘谦虚；

【作者基本信息】 暨南大学 ， 临床医学硕士（耳鼻咽喉头颈外科学）（专业学位）， 2021， 硕士

【摘要】 目的:研究微小RNA-10a-5p(microRNA-10a-5p,miR-10a-5p)及靶基因磷脂酰肌醇-4,5-双磷酸3-激酶催化亚基α(phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha,PIK3CA)在中耳胆脂瘤中的表达,并分析其相关性,探讨它们在中耳胆脂瘤的发病机制中发挥的作用,为中耳胆脂瘤的非手术治疗提供新的研究思路和方法。方法:1.收集共计27例中耳胆脂瘤和耳后正常皮肤组织标本。其中实验组为中耳胆脂瘤组织,对照组为耳后正常皮肤组织。2.通过Target Scan、miRBase等生物信息学软件预测PIK3CA是miR-10a-5p的下游靶基因。3.实时荧光定量PCR(quantitative Real-time PCR,qRT-PCR)分别检测miR-10a-5p及PIK3CA的mRNA在中耳胆脂瘤及耳后正常皮肤组织中的表达情况。4.分别用免疫组织化学法、蛋白免疫印迹(Western Blot)检测PIK3CA在中耳胆脂瘤及耳后正常皮肤组织中的表达。5.分析miR-10a-5p及PIK3CA mRNA在中耳胆脂瘤中表达的相关性,探讨miR-10a-5p是否通过调控PIK3CA的功能进而参与中耳胆脂瘤组织的生长和增殖等生物学过程。结果:1.qRT-PCR结果提示:miR-10a-5p在中耳胆脂瘤和耳后正常皮肤组织中均有表达,在中耳胆脂瘤中的表达量(0.99±0.03)显著低于耳后正常皮肤组织(1.61±0.32),差异具有统计学意义(P<0.05)。2.qRT-PCR结果提示:PIK3CA mRNA在中耳胆脂瘤和耳后正常皮肤组织中均有表达,在中耳胆脂瘤中的表达量(2.06±0.23)显著高于耳后正常皮肤组织(1.02±0.05),差异具有统计学意义(P<0.05)。3.免疫组织化学结果提示:PIK3CA在中耳胆脂瘤中从基底层到基底上层均有表达,主要位于细胞质及细胞膜,染色程度多呈中至强阳性;在耳后正常皮肤组织的基底层和少部分基底上层细胞染色,主要位于细胞核及细胞膜,染色程度多为弱阳性。4.Western Blot结果提示:PIK3CA在中耳胆脂瘤中的表达量显著高于耳后正常皮肤组织,差异具有统计学意义(P<0.05)。5.Pearson相关分析结果显示:中耳胆脂瘤组织中miR-10a-5p与PIK3CA呈显著负相关关系(r=﹣0.926,P<0.001)。结论:1.在中耳胆脂瘤组织中低表达的miR-10a-5p可能对胆脂瘤的生长和增殖起抑制作用。2.在中耳胆脂瘤组织中高表达的PIK3CA可能对胆脂瘤的生长和增殖起促进作用。3.中耳胆脂瘤组织中miR-10a-5p与PIK3CA的表达呈负相关,提示下调的miR-10a-5p可能通过负性调控其靶基因PIK3CA的功能,来影响胆脂瘤角质形成细胞的增殖和分化过程。更多还原

【Abstract】 Objective: To study the expression differences,distribution and the correlation of micro-RNA-10a-5p(miR-10a-5p)and it’s target gene phosphatidy-linositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PIK3CA)protein in cholesteatoma tissues.This study aims to provide new research ideas and directions for the non-surgical treatment of middle ear cholesteatoma by exploring the role of miR-10a-5p and PIK3CA in the molecular biological mechanism of the occurrence and development of middle ear cholesteatoma.Methods: 1.A total of 27 cases of cholesteatoma and normal skin tissues behind the ear were collected.The experimental group was cholesteatoma tissue of the middle ear,and the control group was normal skin tissue behind the ear.2.PIK3CA was predicted to be the downstream target gene of miR-10a-5p by Target Scan,miRBase and other bioinformatics software.3.Quantitative Real-time PCR(qRT-PCR)was used to detect the mRNA expression of miR-10a-5p and PIK3CA in the cholesteatoma and the normal skin tissues behind the ear.4.Immunohistochemistry and Western Blot were used to detect the expression of PIK3CA in middle ear cholesteatoma and normal skin tissue behind ear.5.The correlation between miR-10a-5p and PIK3CA mRNA expression in cholesteatoma tissues was analyzed by statistical methods to further explore whether miR-10a-5p participates in the growth and proliferation of middle ear cholesteatoma epithelial tissues by regulating the function of PIK3CA.Results: 1.QRT-PCR results indicated that the expression of miR-10a-5p could be detected in cholesteatoma and normal skin tissues behind the ear,and the expression level of miR-10a-5p in cholesteatoma(0.99±0.03)was significantly lower than that in normal skin tissues(1.61±0.32),with statistical significance(P < 0.05).2.QRT-PCR results showed that PIK3CA mRNA expression could be detected in cholesteatoma group and normal skin tissues behind the ear,and the expression level of PIK3CA mRNA in cholesteatoma(2.06±0.23)was significantly higher than that in normal skin tissues(1.02±0.05),with statistical significance(P < 0.05).3.Immunohistochemical results indicated that the expression of PIK3CA in cholesteatoma tissues was mainly localized in the cytoplasm and cell membrane,with positive expression from basal layer to basal layer,and the staining degree was mostly moderate to strong positive.PIK3CA was mainly localized in the nucleus and cell membrane in the normal skin tissues behind the ear,showing staining in the basal layer and a few basal superlayer cells,and the degree of staining was mostly weak positive.4.Western Blot results showed that the expression level of PIK3CA in cholesteatoma tissues was significantly higher than that in the control group of normal skin tissues,with statistical significance(P < 0.05).5.Pearson correlation analysis showed that miR-10a-5p was significantly negatively correlated with PIK3CA in cholesteatoma tissues of middle ear(r=-0.926,P <0.001).Conclusions: 1.The lower expression of miR-10a-5p in middle ear cholesteatoma tissues may inhibit the growth and proliferation of cholesteatoma.2.The higher expression of PIK3CA in middle ear cholesteatoma tissues may promote the growth and proliferation of cholesteatoma.3.MiR-10a-5p was negatively correlated with PIK3CA expression in middle ear cholesteatoma tissues,suggesting that down-regulated miR-10a-5p may affect the proliferation and differentiation of cholesteatoma keratinocytes by negatively regulating the function of its target gene PIK3CA.更多还原