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Study the Function of THAP11: A Candidate Gene for Polyglutamine Disorders and Tumor Suppressor

Author: LiYang
Tutor: WangSiYing；YangXiaoMing
School: Anhui Medical University,
Course: Pathology and Pathophysiology
Keywords: THAP11 CAG repeats Polyglutamine Disease genes Tumor suppressor gene
CLC: R741
Type: Master's thesis
Year: 2008
Downloads: 22
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Abstract

Background: polyglutamine (polyglutamin, polyQ) diseases are a class of neurodegenerative diseases, the disease is due to the presence of the gene coding region appear superfluous CAG trinucleotide repeat, resulting translation products increase in the number of glutamine repeat triggered, Now, there are a large number of disease genes has not been revealed, THAP11 contains a glutamine encoded by the CAG repeat arranged in tandem fragment, then whether it is polyQs candidate disease genes that cause it? Therefore, needs through the normal population and neurodegenerative disease were THAP11 gene CAG repeat screening, and may in patients with abnormal screening to gene amplification of CAG THAP11 functional studies to further prove that it is a candidate disease genes polyQ diseases . On the other hand, because the other members of THAP family of proteins involved in transcriptional repression is a class, the process of apoptosis and proliferation proteins, and some have been clear as tumor suppressor, it is also possible for THAP11 tumor suppressor it? To this end, it still needs to study its function in order to verify this hypothesis. Objective: 1) by examining normal Chinese Han population THAP11 gene CAG repeat number in order to learn its normal range of variation, to understand Chinese Han population neurodegenerative lesions THAP11 gene CAG repeats exceeds this normal range of variation, to explore it as a polyglutamine disease causative gene possibilities. 2) by the number of CAG repeats exceeds the normal range THAP11 gene function studies to further validate its polyglutamine diseases as the possibility of disease genes. 3) By THAP11 gene function studies, exploring its possibilities as a tumor suppressor gene. Method: 1) extraction of blood and tissue genomic DNA, PCR amplification and sequencing of the Chinese Han nationality and neurodegeneration in patients THAP11 gene CAG repeat number of the investigation, and its genotype distribution and other characteristics, clear Patients number of CAG repeats in the gene is more than the normal range. 2) containing the abnormal amplification of CAG THAP11 built in pEGFP-N1 vector and the expression of green fluorescent ecdysone inducible system of pIND vector, was observed by fluorescence microscopy and the presence of the cell localization of the protein polymer characteristic lesion formation. 3) by flow cytometry with abnormal amplification of CAG THAP11 whether the impact on the cell cycle and further detected by Western blot of cell cycle proteins. 4) through the use of chromatin structure detection technology, observe THAP11 chromatin morphology to indicate the impact on other aspects of transcription. Using RT-PCR on liver cancer and adjacent tissues THAP11mRNA expression levels were detected, compare the difference, and further using Real time-PCR in liver cancer cells detected THAP11 right proto-oncogene c-myc mRNA expression levels of impact, Also the protein levels Western blot using validated reveal its relationship with cancer. Results: 1) through the normal Chinese Han population THAP11 gene CAG repeat sequence of investigation, we found that the gene CAG repeat polymorphism obvious, not only in the number of CAG repeats, and is also reflected in the existence of CAG repeats CAA different positions and numbers of the insertion. Among them, the normal Han population CAG / CAA repeat number from 22 to 34 range, in the interval, the most common number of repeats is 29, 69.6% of the total number of chromosomes, the most common genotype (CAG) ₃ CAA (CAG) ₅ CAA (CAG) ₂ CAA (CAG) ₅ CAA (CAG) ₁₀, the total number of chromosomes of 62.35%; followed by the most common CAG / CAA repeat count is 28, 14.5% of the total number of chromosomes. CAA CAG trinucleotide insertion in the number of 2-6 range, which two CAA insertion occurred in 23 and 25 CAG repeats, the six CAA insertion occurred in 29 repetitions of CAG The most common number of CAA insertion is four, which is not interrupted by the successive insertion longest CAG repeat number of 15. 2) By neurodegeneration in patients THAP11 gene CAG repeat screening, we found that, compared with normal control patients THAP11 varying degrees of gene CAG repeat expansion, not only the number of CAG repeats found 35 mutations THAP11 (there are two type of patients were genotype heterologous chromosomes, accounting for 1.66%), but also detect the number of CAG repeat mutation 38 THAP11 (present in a patient heterologous chromosomes, 0.83%), the specific genotype (CAG) ₃ CAA (CAG) ₅ CAA (CAG) ₂ CAA (CAG) ₅ CAA (CAG) ₈ CAA (CAG) ₁₀. 3) Through containing abnormal amplification of CAG THAP11 intracellular fluorescence localization observation, we found THAP11 (35Q) protein with normal controls THAP11 (29Q) compared to the presence of nuclear protein expression was significantly increased. And THAP11 (38Q) not only has a nuclear protein expression increased, but also the emergence polymeric protein precipitation. By ecdysone inducible expression system, we examined the THAP11 (38Q) whole cell protein was initially distributed, as the expression time, gradually to nuclear accumulation and the formation of protein aggregates and deposits. 4) by detecting the THAP11 different polyQs effect on the cell cycle and found no normal population THAP11 (29Q) influence on the cell cycle; while THAP11 (38Q) but they can cause significant G0/G1 arrest, cells accounted for the period 90.24%, 58.52% was significantly higher than normal. Further through the cell cycle protein detection, we found THAP11 (29Q) on cell cycle related protein had no effect, while THAP11 (38Q) allows cells to increased expression of P21 protein, CyclinD1 protein expression was decreased, and the expression of these two proteins We detected the same cell G0/G1 arrest. 5) chromatin structure through the use of detection techniques, we can observe THAP11 condensed chromatin, suggesting that it may have a tumor suppressor function and the inhibition of transcription factors. This leads, by detecting THAP11 in cancer and adjacent normal tissue expression changes found THAP11 expression in cancer tissues compared with normal tissues significantly decreased, thereby further suggest THAP11 may act as tumor suppressor. In addition, by detecting THAP11 oncogene c-myc effects, we find that its c-myc mRNA levels in either the protein level or can inhibit its expression, which further confirms the THAP11 as a candidate tumor suppressor gene. Conclusions: 1) detection of a normal Chinese Han population THAP11 gene CAG repeats in the normal range of variation of 22-34, the number of CAG repeats its clear form of various genotypes and distribution characteristics. 2) By having neurodegeneration in patients THAP11 gene CAG repeat screening and found one case of neurodegeneration in patients whose THAP11 gene containing 38 CAG repeats, which initially prompted its incidence may be related to more diseases associated with polyglutamine; 3) containing abnormal amplification CAG (38) of the THAP11 gene functional studies, found that the mutant gene expression in cells that can produce nuclear inclusions polyglutamine disease is characterized by lesions, but also the existence of distinct cell cycle arrest in G0/G1 phase, further suggesting that it is a candidate for polyglutamine disease causative genes. 4) may have found THAP11 transcriptional repressor function, while the gene expression levels in cancer tissues compared to adjacent tissues is low, and can inhibit the proto-oncogene c-myc expression, suggesting THAP11 as a candidate tumor suppressor gene.

Study the Function of THAP11: A Candidate Gene for Polyglutamine Disorders and Tumor Suppressor

Abstract

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