1.Microrna Expression Profile In Peripheral Blood Leukocytes From Primary Hyperparathyroidism 2.Expression Of P27^kip1and ?-catenin In Multiple Endocrine Neoplasia Type 1 Related Parathyroid Tumors

Part 1MicroRNA expression profile in peripheral blood leukocytes from primary hyperparathyroidismBackgroundPrimary 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.Aims1.To investigate miRNA expression profile in leukocytes from sporadic PA,PC and MEN1 related PHPT?MHPT?.2.To investigate miRNA expression profile in leukocytes from PC compared with PA,which may serve as preoperative diagnostic markers.Subjects and MethodsTwenty-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?.Res?lts1.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?.2.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 thanNC?P=0.035,0.033 and 0.017,respectively?.3.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?PRE₁ and PRE₂?were calculated by sex and the relative quantity of miR-3136-3p and miR-5088-5p respectively.Receiver operative characteristic curve analysis showed PRE₁ and PRE₂ could be diagnostic markers of PC and PA,with an area under the curve of 0.776 and 0.805.4.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?.ConclusionThis 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 2Expression of p27Kip1 and ?-catenin in multiple endocrine neoplasia type 1 related parathyroid tumorsObjectiveTo explore the expression of cyclin-dependent kinase inhibitor p27Kip1 and ?-catenin in multiple endocrine neoplasia type 1?MEN1?related parathyroid tumor.MethodsImmunohistochemistry was used to analysis the expression of p27Kip1 and ?-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.ResultsIn MHPT group,the nuclear expression of p27Kip1 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 p27Kip1 which were significant stronger than MEN1 related parathyroid tumors?P=0.001?.Concerning the status of(3-catenin,normal parathyroid showed a distinct to moderate membrane staining,a moderate to weak cytoplasmic staining and negative nuclear staining.Nuclear staining of ?-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 ?-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 ?-catenin.There were no significant difference in the expression of p27Kip1 and ?-catenin between parathyroid adenoma and parathyroid hyperplasia in MEN1 patients.ConclusionOur results suggest that the expression of p27Kip1 is reduced or absent in MEN1 related parathyroid tumors,while nuclear accumulation of ?-catenin is not involved in the development of the tumors.