第一部分 原发性甲状旁腺功能亢进症的外周血白细胞microRNA表达谱

背景

原发性甲状旁腺功能亢进症（primary hyperparathyroidism，PHPT）是因甲状旁腺本身病变导致分泌过量甲状旁腺素的全身性疾病，病理类型多为良性的腺瘤（parathyroid adenoma，PA），少数为增生及恶性的腺癌（parathyroid carcinoma，PC）。PC患者术后复发及死亡率高，然而术前鉴别PA及PC仍十分困难。多发性内分泌腺瘤病1型（multiple endocrine neoplasia type 1，MEN1）是最常见的遗传性PHPT。microRNA（miRNA）是一类单链非编码小分子RNA，对基因进行转录后调控。外周血白细胞miRNA可作为分子标志物指导病理分型、预后等。

目的

1. 寻找与散发性PA、PC以及MEN1相关的PHPT（MEN1 related PHPT，MHPT）发病相关的外周血白细胞miRNAs。

2. 寻找有助于术前鉴别PC与PA的外周血白细胞miRNAs分子标志物。

对象与方法

1. 研究对象：北京协和医院内分泌科确诊的22例散发性PA、14例散发性PC及16例MHPT患者。年龄、性别与PA组匹配的23例正常对照（normal control，NC）。

2. 研究方法：（1）遗传分析：提取研究对象的外周血细胞总RNA和总DNA。应用Sanger测序对散发性PHPT患者进行MEN1基因、CDC73基因、CaSR基因、CDKN1B基因以及RET基因突变分析，排除遗传性PHPT。筛查MHPT患者的MEN1基因突变。（2）miRNA表达谱分析：应用Affymetrix miRNA 4.0芯片（含2578个人成熟miRNA）分析各组中性别、年龄相匹配的6例对象的外周血白细胞miRNA表达谱，得到组间差异miRNAs（P<0.05，差异倍数>1.5或<-1.5）。使用GCBI平台进行生物信息学分析。（3）结果验证：应用实时定量PCR（quantitative real time PCR，qRT-PCR）在22例PA、14例PC、16例MHPT患者以及23例NC中验证候选miRNAs的表达水平。

结果

1. PA组与NC组相比：①芯片：共有45个差异表达的miRNAs（同时满足Q<0.05），差异倍数在-3.259~2.818之间。②qRT-PCR验证：挑选14个miRNAs进行验证，PA组let-7e-5p、let-7f-5p及has-miR-1260b的表达水平显著升高，分别为NC组的3.01倍（P=0.028）、2.60倍（P=0.031）及1.98倍（P= 0.043）；miR-98-5p的表达在PA组呈上调趋势，为NC组的2.49倍（P=0.068）。

2. PC组与NC组相比：①芯片：共有37个差异表达的miRNAs，差异倍数在-3.309~2.447之间。②qRT-PCR验证：挑选10个miRNAs进行验证，PC组的miR-4646-5p、miR-5196-5p及miR-6757-5p的表达分别是NC组的1.73（P=0.035）倍、1.84倍（P=0.033）及1.58倍（P= 0.017）。

3. PC组与PA组相比：①芯片：共有64个差异表达的miRNAs，差异倍数在-2.421~2.498之间。②qRT-PCR验证：挑选15个miRNAs进行验证，校正性别后，miR-3136-3p及miR-5088-5p的表达在PC组呈上调趋势（P=0.070及0.076）。通过二元Logistic回归将性别分别与上述两个miRNAs进行建模，得到的新变量PRE_1及PRE_2有助于术前鉴别PC与PA（曲线下面积：0.776，0.805）。

4. MHPT组与NC组相比：①遗传分析：MHPT组中有10例携带MEN1基因突变。②芯片：共有30个差异表达的miRNAs，差异倍数在-2.614~2.244之间。③qRT-PCR验证：挑选7个miRNAs进行验证，MHPT组miR-1268b及miR-5194的表达水平显著低于NC组，分别为NC组的38.85%（P=0.001）及56.63%（P=0.004）。

结论

本研究首次探讨了PHPT的外周血白细胞的miRNA表达谱。与正常对照相比，PA组的let-7e-5p、let-7f-5p及has-miR-1260b的表达显著升高，PC组的miR-4646-5p、miR-5196-5p及miR-6757-5p的表达显著升高，MHPT组的miR-1268b及miR-5194的表达显著降低。这些异常表达的miRNAs可能有助于未来深入研究PHPT的发生、发展机制。miR-3136-3p及miR-5088-5p有望成为术前鉴别良恶性甲状旁腺肿瘤的分子标志物之一，但需要更大的样本量研究进行验证。

第二部分 多发性内分泌腺瘤病1型相关的甲状旁腺肿瘤p27^Kip1及β-连环蛋白的表达

目的

分析p27^Kip1及β-连环蛋白（β-catenin）在多发性内分泌腺瘤病1型（multiple endocrine neoplasia type 1，MEN1）相关的原发性甲状旁腺功能亢进症（primary hyperparathyroidism，PHPT）肿瘤组织中的表达，以探索其在MEN1相关的甲状旁腺肿瘤发病机制中的作用。

方法

收集2002年至2013年北京协和医院收治的31例MEN1相关的PHPT（MEN1 related PHPT，MHPT）患者的31份甲状旁腺肿瘤组织以及5份正常甲状旁腺（normal parathyroid，NP）组织的石蜡标本，应用免疫组织化学染色法检测p27^Kip1及β-catenin蛋白的表达情况。

结果

MHPT肿瘤组织中，p27^Kip1在细胞核表达为缺失、弱阳性或中等阳性（比例分别为12.9%、32.3%、54.8%）；细胞膜β-catenin表达为强阳性或中等阳性、细胞质β-catenin表达为中等阳性或弱阳性，未见有β-catenin表达于细胞核。MHPT组织p27^Kip1表达水平显著低于NP组织（P=0.001），但细胞膜及细胞质β-catenin的表达在MHPT组无显著性统计学意义（P值分别为0.087，0.357）。在MHPT组中，腺瘤亚组与增生亚组间p27^Kip1与β-catenin的表达差异均无统计学意义。

结论

MEN1相关甲状旁腺肿瘤组织p27^Kip1蛋白表达显著减少，而未发现β-catenin异位聚集于细胞核现象。

Part 1 MicroRNA expression profile in peripheral blood leukocytes from primary hyperparathyroidism

Background

Primary hyperparathyroidism (PHPT) is due to increased activity of parathyroid glands, secreting excess parathyroid hormone. Parathyroid adenoma (PA) is the most common cause, parathyroid hyperplasia (PH) and parathyroid carcinoma (PC) is rare. PC is associated with high rate of recurrence and fatality, while no tools are available to distinguish PC from PA preoperatively. The most common form of hereditary PHPT is multiple endocrine neoplasia type 1 (MEN1). MicroRNA (miRNA) is abundant small noncoding RNA that regulate gene expression post-transcriptionally. MiRNAs in peripheral blood leukocytes are helpful for pathological classification and prognosis evaluation.

Aims

To investigate miRNA expression profile in leukocytes from sporadic PA, PC and MEN1 related PHPT (MHPT).

To investigate miRNA expression profile in leukocytes from PC compared with PA, which may serve as preoperative diagnostic markers.

Subjects and Methods

Twenty-two PA, 14 PC and 16 MHPT patients were studied. Twenty-three subjects were used as normal controls (NC). Total DNA and RNA of peripheral blood leukocytes were extracted. Direct sequence of the MEN1, CDC73, CaSR, CDKN1B and RET gene were conducted in sporadic PHPT cases to excluded hereditary PHPT, while MEN1 gene was screened in MHPT patients. Affymetrix GeneChip miRNA 4.0 Array containing 2578 human mature miRNAs were used to profile miRNA expression in leukocytes. Bioinformatics analysis was performed in GCBI platform (GMINIX Informatics Ltd. Co, China) to find candidate miRNAs (P<0.05, and fold change [FC]>1.5or<-1.5). Differentially expressed miRNAs were validated by quantitative real-time PCR (qRT-PCR).

Resµlts

Compared with NC, 45 miRNAs were significantly dysregulted in leukocytes from PA group (Q<0.05). FC differed from -3.259 to 2.818. Forteen miRNAs were validated by qRT-PCR. PA showed significantly higher expression of let-7e-5p, let-7f-5p and has-miR-1260b than NC (P=0.028, 0.031 and 0.043, respectively). PA showed trend of higher expression of miR-98-5p than NC group (FC: 2.49, P=0.068).

Compared with NC, 37 miRNAs were significantly dysregulted in leukocytes from PC group. FC differed from -3.309 to 2.447. Ten miRNAs were validated by qRT-PCR. PC demonstrated significantly higher expression of miR-4646-5p, miR-5196-5p and miR-6757-5p than NC (P=0.035, 0.033 and 0.017, respectively).

Compared with PA, 64 miRNAs were significantly dysregulted in leukocytes from PC group. FC differed from -2.421 to 2.498. Fifteen miRNAs were validated by qRT-PCR. After adjusted for sex, PC exhibited trend of higher expression of miR-3136-3p and miR-5088-5p than PA (P=0.070 and 0.076, respectively). Using binary Logistic regression, new variates (PRE_1 and PRE_2) were calculated by sex and the relative quantity of miR-3136-3p and miR-5088-5p respectively. Receiver operative characteristic curve analysis showed PRE_1 and PRE_2 could be diagnostic markers of PC and PA, with an area under the curve of 0.776 and 0.805.

Ten MHPT patients had gemline MEN1 gene mutation. Compared with NC, 30 miRNAs were significantly dysregulted in leukocytes from MHPT group. FC differed from -2.614 to 2.244. Seven miRNAs were validated by qRT-PCR. MHPT demonstrated significantly lower expression of miR-1268b and miR-5194 than NC (P=0.001 and 0.004, respectively).

Conclusion

This is the first study detecting miRNAs expression profile in peripheral blood leukocytes from PHPT patients. Compared with normal controls, PA patients showed higher expression of let-7e-5p, let-7f-5p and has-miR-1260b, PC patients showed higher expression of miR-4646-5p, miR-5196-5p and miR-6757-5p, while MHPT patients showed lower expression of miR-1268b and miR-5194. These dysregulated miRNAs may be helpful to understand the pathogenesis of PHPT. Furthermore, miR-3136-3p and miR-5088-5p could be used as preoperative markers to differentiate PC from PA.

Part 2 Expression of p27^Kip1 and β-catenin in multiple endocrine neoplasia type 1 related parathyroid tumors

Objective

To explore the expression of cyclin-dependent kinase inhibitor p27^Kip1 and β-catenin in multiple endocrine neoplasia type 1 (MEN1) related parathyroid tumor.

Methods

Immunohistochemistry was used to analysis the expression of p27^Kip1 and β-catenin in 31 parathyroid tumors of MEN1 related primary hyperparathyroidism (MHPT) which were removed at Peking Union Medical College Hospital from 2002 to 2013. Five normal parathyroid glands were used as controls.

Results

In MHPT group, the nuclear expression of p27^Kip1 was absent in 4 specimens (12.9%), while 10 tumors showed weak nuclear staining and 17 tumors showed moderated nuclear staining (32.3%, 54.8% respectively). All normal parathyroid glands showed marked nuclear expression of p27^Kip1 which were significant stronger than MEN1 related parathyroid tumors (P=0.001). Concerning the status of β-catenin, normal parathyroid showed a distinct to moderate membrane staining, a moderate to weak cytoplasmic staining and negative nuclear staining. Nuclear staining of β-catenin was not observed in all tumors. Moreover, MEN1 related parathyroid tumors showed a marked to moderate membrane and a moderated to weak cytoplasmic staining of β-catenin which were similar to those of normal parathyroid (P=0.087, 0.357 respectively). Only one tumor demonstrated weak cytoplasmic and membrane staining of β-catenin. There were no significant difference in the expression of p27^Kip1 and β-catenin between parathyroid adenoma and parathyroid hyperplasia in MEN1 patients.

Conclusion

Our results suggest that the expression of p27^Kip1 is reduced or absent in MEN1 related parathyroid tumors, while nuclear accumulation of β-catenin is not involved in the development of the tumors.