敲低EDAG抑制乳头状甲状腺癌细胞的增殖

为研究EDAG在人乳头状甲状腺癌病人组织中的表达及在乳头状甲状腺癌细胞中的作用,利用免疫组化检测31例乳头状甲状腺癌癌组织及癌旁组织中EDAG蛋白的表达,并进行数据分析.包装EDAG敲低慢病毒颗粒,感染乳头状甲状腺癌细胞系K1,建立EDAG敲低稳定细胞株,检测EDAG敲低对细胞增殖、克隆形成、周期和凋亡的影响. 结果显示,EDAG蛋白在乳头状甲状腺癌癌组织中异常高表达,而在对应癌旁组织极低表达或不表达.建立稳定敲低EDAG的K1细胞株,敲低效果达到约96%,敲低EDAG后细胞增殖变缓,倍增时间由18.49±0.19 h变为19.47±0.11 h,且克隆形成能力下降,G0/G1期比例升高,无血清培养时凋亡增多.本文报道了EDAG在乳头状甲状腺癌病人中高表达,且敲低甲状腺癌细胞系K1中内源EDAG抑制细胞增殖,降低细胞克隆形成能力,G0/G1期增多,凋亡升高,提示EDAG异常高表达可能在甲状腺癌发生发展中具有重要作用.     

Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants

Single base substitutions in DNA mismatch repair genes which are predicted to lead either to missense or silent mutations, or to intronic variants outside the highly conserved splicing region are often found in hereditary nonpolyposis colorectal cancer (HNPCC) families. In order to use the variants for predictive testing in persons at risk, their pathogenicity has to be evaluated. There is growing evidence that some substitutions have a detrimental influence on splicing. We examined 19 unclassified variants (UVs) detected in MSH2 or MLH1 genes in patients suspected of HNPCC for expression at RNA level. We demonstrate that 10 of the 19 UVs analyzed affect splicing. For example, the substitution MLH1,c.2103G>C in the last position of exon 18 does not result in a missense mutation as theoretically predicted (p.Gln701His), but leads to a complete loss of exon 18. The substitution MLH1,c.1038G>C (predicted effect p.Gln346His) leads to complete inactivation of the mutant allele by skipping of exons 10 and 11, and by activation of a cryptic intronic splice site. Similarly, the intronic variant MLH1,c.306+2dupT results in loss of exon 3 and a frameshift mutation due to a new splice donor site 5 bp upstream. Furthermore, we confirmed complete exon skipping for the mutations MLH1,c.1731G>A and MLH1,c.677G>A. Partial exon skipping was demonstrated for the mutations MSH2,c.1275A>G, MLH1,c.588+5G>A, MLH1,c.790+4A>G and MLH1,c.1984A>C. In contrast, five missense mutations (MSH2,c.4G>A, MSH2,c.2123T>A, MLH1,c.464T>G, MLH1,c.875T>C and MLH1,c.2210A>T) were found in similar proportions in the mRNA as in the genomic DNA. We conclude that the mRNA examination should precede functional tests at protein level. Databases: HNPCC – OMIM 114500, MSH2 – OMIM: 120435; GenBank: NM_000251.1, MLH1 – OMIM: 120436; GenBank: NM_000249.2, InSiGHT mutation database: , Programs: BDGP: , ESEfinder program:     

Molecular pathology and thyroid FNA

This review summarises molecular pathological techniques applicable to thyroid FNA. The molecular pathology of thyroid tumours is now fairly well understood. Molecular methods may be used as a rule‐in test for diagnosis of malignancy in thyroid nodules, eg BRAF V600E point mutation, use of a seven‐gene mutational panel (BRAF V600E, RAS genes, RET/PTC or PAX8/PPARG rearrangement), or as a comprehensive multigene next‐generation sequencing panel, eg ThyroSeq v2. Molecular methods can also be applied as rule‐out tests for malignancy in thyroid nodules, eg Afirma or ThyroSeq v2 or as markers of prognosis, eg TERT promoter mutation or other gene mutations including BRAF V600E, TP53 and AKT1, and as tests for newly defined tumour entities such as non‐invasive follicular thyroid neoplasm with papillary like nuclei, or as a molecular marker(s) for targeted therapies. This review describes practical examples of molecular techniques as applied to thyroid FNA in routine clinical practice and the value of molecular diagnostics in thyroid FNA. It describes the range of molecular abnormalities identified in thyroid nodules and thyroid cancers with some practical applications of molecular methods to diagnosis and prognosis of thyroid nodules and thyroid cancer.     

The rare intracellular RET mutation p.S891A in a Chinese Han family with familial medullary thyroid carcinoma

We report intracellular RET mutation in a Han Chinese pedigree with familial medullary thyroid carcinoma (FMTC). Direct sequencing of RET proto-oncogene identified a missense c.2671T>G (p.S891A) mutation in 6 of 14 family members. The single nucleotide polymorphisms c. 135A>G (p.A45A), IVS4+48A>G, c. 1296A>G (p.A432A), c. 2071G>A (p.G691S), c. 2307T>G (p.L769L) and a variant c. 833C>A (p.T278N) were also found in 6 carriers. Among 5 of the 6 carriers presented medullary thyroid carcinoma (MTC) as an isolated clinical phenotype, with elevated basal serum calcitonin (Ct). Two underwent non-normative thyroidectomy either two or four times without physician awareness or diagnosis of this disease at initial treatment, but with elevated Ct. One with elevated pre-Ct accepted total thyroidectomy (TT) with modified bilateral neck dissection (MBiND), and whose seventh posterior rib MTC metastases was confirmed 5 months after surgery. Moreover, results of two affected individuals with elevated Ct were reduced to normal after TT with MBiND or prophylactic VI compartmental dissection. However, only another carrier with the variant p.T278N had slightly elevated Ct rejected surgery and was strictly monitored. Given these case results, we suggest that screening of RET and pre-surgical Ct levels in the management of MTC patients is essential for earlier diagnosis and more normative initial treatment, that FMTC patients with cervical lymph nodes metastases may be cured by TT with MBiND, and that prophylactic VI compartmental dissection should be avoided when Ct levels are low.     

SNHG15 functions as a tumor suppressor in thyroid cancer

Small nucleolar RNA host gene 15 (SNHG15) has been suggested to be overexpressed, and function as an oncogenic long noncoding RNA (lncRNA) in various types of human malignancies. However, the expression status and function of SNHG15 were still unknown in thyroid cancer. In our study, we assessed the expression status and clinical value in thyroid cancer samples, and explored the effect of SNHG15 on thyroid cancer cell proliferation, migration, and invasion. In results, SNHG15 expression was downregulated in thyroid cancer tissues and cells, and correlated with age, pathology classification, clinical stage, tumor size, distant metastasis, and disease-free survival. The in vitro studies suggested SNHG15 overexpression suppressed cell proliferation, migration, and invasion in thyroid cancer. In summary, SNHG15 serves as tumor suppressive role in thyroid cancer.     

Association of single nucleotide polymorphisms in the microRNA miR‐1596 locus with residual feed intake in chickens

MicroRNAs are an abundant class of small non‐coding RNAs that regulate gene expression. Genetic variations in microRNA sequences may be associated with phenotype differences by influencing the expression of microRNAs and/or their targets. This study identified two single nucleotide polymorphisms (SNPs) in the genomic region of the microRNA miR‐1596 locus of chicken. Of the two SNPs, one was 95 bp upstream of miR‐1596 (g.5678784A>T) and the other was in the middle of the sequence producing the mature microRNA gga‐miR‐1596‐3p (g.5678944A>G). Genotypic distribution of the two SNPs had large differences among 12 chicken breeds (lines), especially between the fast‐growing commercial lines and the slow‐growing Chinese indigenous breeds for the g.5678784A>T SNP. Only the g.5678784A>T SNP was significantly associated with residual feed intake (RFI) in the F₂ population derived from a fast‐growing and a slow‐growing broiler as well as in the pure Huiyang bearded chicken. The birds with the AA genotype of the g.5678784A>T SNP had lower RFI and higher expression of the mature gga‐miR‐1596‐3p microRNA of miR‐1596 than did those with the other genotypes of the same SNP. We also found that the expression of the mature gga‐miR‐1596‐3p microRNA of miR‐1596 was significantly associated with RFI. These findings suggest that miR‐1596 can become a candidate gene related to RFI, and its genetic variation may contribute to changes in RFI by altering expression levels of the mature gga‐miR‐1596‐3p microRNA in chicken.     

microRNAs regulate human embryonic stem cell division

RNA interference-mediated suppression of DICER and DROSHA in human embryonic stem cells (hESCs) attenuates cell proliferation, supporting a role for an intact microRNA (miRNA) pathway in the control of hESC cell division. Normal cell growth can be partially restored by introduction of the mature miRNAs miR-195 and miR-372. These miRNAs regulate two tumor suppressor genes, respectively: WEE1, which encodes a negative G2/M kinase modulator of the cycB/CDK complex and CDKN1A, which encodes p21, a cycE/CDK cyclin dependent kinase inhibitor that regulates the G1/S transition. We show that in wild-type hESCs, WEE1 levels control the rate of hESC division, whereas p21 levels must be maintained at a low level for hESC division to proceed. These data support a model for hESC cell cycle control in which miRNAs regulate negative cell cycle modulators at two phases of the cell cycle to ensure proper replenishment of the stem cell population.

Supplemental information can be found here.     

Association between MT-CO3 haplotypes and high-altitude adaptation in Tibetan chicken

Genetic mutation in cytochrome c oxidase subunit III gene (MT-CO3) could influence the kinetics of cytochrome c oxidase (COX), which catalyzes oxygen transport capacity in oxidative phosphorylation. However, the potential relationship between MT-CO3 variants and high-altitude adaptation remains poorly understood in Tibetan chicken. Here, we sequenced MT-CO3 gene of 125 Tibetan chickens and 144 Chinese domestic chickens in areas at a low elevation (below 1000 m). Eight single nucleotide polymorphisms (SNPs) were detected; and five of them (m.10081A>G, m.10115G>A, m.10270G>A, m.10336A>G and m.10447C>T) shared by Tibetan chicken and lowland chicken with the significant difference in their respective allele frequencies. Nine haplotypes (H1–H9) were finally defined. Among them, haplotype H4 was positively associated with high-altitude adaptation whereas haplotypes H6, H7 and H8 had negative association with high-altitude adaptation. The Median-joining profile suggested that haplotype H5 had the ancestral position to the other haplotypes but had no significant relationship with high-altitude adaptation. However, there was only m.10081A>G mutation differed from haplotype H4 and H5. Results also suggested that chickens with A allele at m.10081A>G, had over 2.6 times than those with G allele in the probability of the ability to adapt hypoxia. It suggests that the synonymous mutation m.10081A>G may be a prerequisite for shaping high-altitude adaptation-specific haplotypes.     

Role of MDM2 309T>G (rs2279744) and I/D (rs3730485) polymorphisms and haplotypes in risk of papillary thyroid carcinoma,tumor stage,tumor size,and early onset of tumor: A case control study

Murine double minute clone 2 (MDM2) protein plays an important role in the regulation of p53 tumor suppressor. Genetic polymorphisms of the MDM2 gene are the candidate variants in susceptibility to various cancers. In the present study, we aimed to investigate the possible effects of MDM2 309T>G (rs2279744) and I/D (rs3730485) polymorphisms on papillary thyroid carcinoma (PTC) susceptibility and clinical or pathological features of the disease. A case control study was carried out involving in a total of 131 patients with PTC and 144 healthy controls. Both cases and controls were genotyped for MDM2 309T>G and I/D polymorphisms. There was no significant difference regarding MDM2 309T>G and I/D genotypes between patients with PTC and controls in neither dominant nor recessive and allelic models. The frequency of G-D haplotype was higher in patients with PTC and this haplotype was associated with a 1.7-fold increased risk of PTC. The MDM2 309T>G polymorphism was associated with a higher risk of III–IV stages in patients with PTC. The MDM2 ID genotype was significantly higher in patients with PTC less than 40 years and associated with larger tumor size (≥1 cm). In conclusion, the G-D haplotype but not MDM2 309T>G and I/D polymorphisms were associated with higher risk of PTC. MDM2 309T>G polymorphism was associated with a higher incidence of III–IV stages, however, I/D polymorphism was associated with larger tumor size and a lower age of disease occurrence.     

Genetic polymorphism c.1438A>G of the 5-HT<Subscript>2A</Subscript> receptor is associated with abdominal obesity in Chinese Northern Han population

Objective We examined the potential impact of the 5-hydroxytryptamine 2A receptor (5-HT_2AR) c.1438A>G promoter polymorphism on obesity and estimates of insulin, glucose as well as lipid metabolism. Methods The genotypes and allelic frequencies of the 5-HT_2AR c.1438A>G were examined with polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) in 210 patients with overweight/obesity and 216 unrelated healthy subjects. Results The genotype (AA, AG, and GG) distribution of c.1438A>G polymorphism of the 5-HT_2AR gene promoter was 35%, 46%, and 19% in patients, and 32%, 56%, 12% in controls, respectively, no significant difference was found between two groups. Association of genetic diversity of 5-HT_2AR c.1438A>G with the total body fat, fat distribution and clinical characteristics revealed that overweight/obese men carrying G allele were associated with increased body mass index (P = 0.043), waist circumference (P = 0.038), waist-to-hip ratio (P = 0.045), in comparison with patients who carrying A allele, but there were no significant difference between the c.1438A>G genotype groups in overweight/obese women. Conclusion No significant associations were detected. However, the present study suggests the possibility that an abnormal production rate of the 5-HT_2AR c.1438A>G gene product might lead to the development of abdominal obesity in men but not in women. Su Ying and Yan-Ming Sun equally contributed to this study.     

促甲状腺激素并非甲状腺乳头状癌发生发展的关键因素

目的:血清促甲状腺激素(TSH)在甲状腺乳头状癌(PTC)中的作用及机制尚不明确,本研究主要探讨TSH对甲状腺细胞系及乳头状癌细胞系的作用。方法:体外培养人甲状腺细胞系和乳头状癌细胞系,分别给予不同剂量(0 mU/L、5 mU/L及20 mU/L)的TSH干预。通过MTS及流式细胞术,观察TSH对甲状腺及乳头状癌细胞系增殖和细胞周期的作用;通过RNA-seq、ELISA检测TSH对细胞因子的影响;通过实时荧光定量PCR及Western blot寻找潜在的作用靶点。结果:MTS及流式细胞术结果显示,TPC-1和Nthy-ori-3-1细胞经TSH干预后增殖指数下降,20 mU/L浓度的TSH干预组细胞周期缩短。ELISA结果显示TPC-1中TSH下调CXCL8,上调CXCL10,而CXCL12的表达无明显变化。在Nthy-ori-3-1细胞中CXCL8和CXCL10的表达也观察到类似的结果,但CXCL12表达受到TSH的抑制。TSH可使Nthy-ori-3-1和Bcpap细胞中细胞命运决定因子(DACH1)的表达呈剂量依赖性上调,且TSH可抑制Bcpap中BRAF(V600E)以及Nthy-ori-3-1和TPC-1中BRAF的表达。结论:综上所述,我们并未发现TSH对甲状腺癌细胞有明显的促肿瘤作用。相反,本研究提示TSH可能呈部分抗癌作用。因此,TSH对甲状腺的致癌作用仍有待进一步研究。     

A novel de novo PDE4D gene mutation identified in a Chinese patient with acrodysostosis

Acrodysostosis is an extremely rare disorder at birth, that is, characterized by skeletal dysplasia with short stature and midfacial hypoplasia, which has been reported to be caused by PDE4D and PRKAR1A gene mutations. Here, a Chinese boy with acrodysostosis, ventricular septal defect, and pulmonary hypertension was recruited for our study, and his clinical and biochemical characteristics were analyzed. A novel de novo heterozygous missense mutation (NM_001104631: c.2030A>C, p.Tyr677Ser) of the PDE4D gene was detected by whole exome sequencing and confirmed by Sanger sequencing. The c.2030A>C (p.Tyr677Ser) variant was located in exon 15 of the PDE4D gene, predicted to be damaging by a functional prediction program and shown to be highly conserved among many species. Further functional analysis showed that the p.Tyr677Ser substitution changes the function of the PDE4D protein, affects its subcellular localization in transfected cells, increases PDE4 activity in the regulation of cAMP signaling and affects cell proliferation. Our study identified a novel de novo PDE4D mutation in acrodysostosis of Chinese origin that not only contributes a deeper appreciation of the phenotypic characteristics of patients with PDE4D mutations but also expands the spectrum of PDE4D mutations.     

Yak OXGR1 promotes fibroblast proliferation via the PI3K/AKT pathways

Oxoglutarate receptor 1 (OXGR1), as one of the intermediates in G protein-coupled receptors (GPCRs), plays a crucial role in the citric acid cycle receptor of α-ketoglutarate and metabolism. GPCR can control the cell proliferation by regulating the downstream signaling of G protein signaling pathways. The PI3K/AKT pathway transmits the downstream signals of GPCRs and receptor tyrosine kinases. However, the specific role of OXGR1 promoting cell proliferation and differentiation are still unknown. In current study, the over-expression vector and knockdown sequence of yak OXGR1 were transfected into yak fibroblasts, and the effects were detected by a series of assays. The results revealed that OXGR1 expression in yak lung parenchyma tissue was significantly higher than that of other tissues. In yak fibroblasts, the upregulated expression of OXGR1 resulted in activating the PIK3CG (downstream signal) of the PI3K/AKT1 pathway that can upregulated the expression of proliferation genes ( CDK1, PCNA, and CyclinD1) and promote cell proliferation. Conversely, the downregulated expression of OXGR1 inhibited cell proliferation via PI3K/AKT1 pathway. Cell cycle and cell proliferation assays demonstrated that over-expression of OXGR1 can enhanced the DNA synthesis and promoted yak fibroblasts proliferation. While the conversely, knockdown of OXGR1 can decreased DNA synthesis and inhibited cell proliferation. These results illustrated that changes of OXGR1 expression can trigger the fibroblasts proliferation via PI3K/AKT signaling pathway, which indicating that OXGR1 is a novel regulator for cell proliferation and differentiation. Furthermore, these results provide evidence supporting the functional role of GPCRs-PI3K-AKT1 and OXGR1 in cell proliferation.     

Hypermethylated RASSF1 and SLC5A8 promoters alongside BRAFV600E mutation as biomarkers for papillary thyroid carcinoma

Circulating cell-free DNA (cfDNA) has been considered as a diagnostic source to track genetic and epigenetic alterations in cancer. We aimed to study mutation in addition to the methylation status in the promoter regions of RASSF1 and SLC5A8 genes in tissues and circulating free DNA samples of patients affected with papillary thyroid carcinoma (PTC) and thyroid nodules as controls. BRAF^V600E mutation was studied by ARMS-scorpion real-time polymerase chain reaction method in 57 PTC and 45 thyroid nodule cases. Methylation status of RASSF1 and SLC5A8 promoter regions was analyzed by methylation-specific high-resolution melting curve analysis. BRAF^V600E mutation was found in 39 (68.4%) out of 57 PTC tissue samples, while in 33 (49.1%) cases of cfDNA, this mutation was detected. The frequency of BRAF^V600E mutation in cfDNA was significantly different between metastatic and nonmetastatic PTC cases (22 of 33 PTC cases vs. 5 of 34 thyroid nodule samples). Methylation levels of three promoter regions of SLC5A8 and proximal promoter region of RASSF1 was significantly different between PTC and thyroid nodule cases in both cfDNA and tissue DNA. In addition, the methylation status of these two genes in tissue DNA was reflected in methylation status observed in cfDNA. This study confirmed that BRAF^V600E mutation is better for discrimination between papillary thyroid carcinoma and thyroid nodules. On the other hand, hypermethylation in the more proximal promoter regions to RASSF1 and SLC5A8 genes showed higher sensitivity and more acceptable specificity for this discrimination.