Cellular stress induced alterations in microRNA let-7a and let-7b expression are dependent on p53

Genotoxic stressors, such as radiation, induce cellular damage that activates pre-programmed repair pathways, some of which involve microRNAs (miRNA) that alter gene expression. The let-7 family of miRNA regulates multiple cellular processes including cell division and DNA repair pathways. However, the role and mechanism underlying regulation of let-7 genes in response to stress have yet to be elucidated. In this study we demonstrate that let-7a and let-7b expression decreases significantly following exposure to agents that induce stress including ionizing radiation. This decrease in expression is dependent on p53 and ATM in vitro and is not observed in a p53(-/-) colon cancer cell line (HCT116) or ATM(-/-) human fibroblasts. Chromatin Immunoprecipitation (ChIP) analysis showed p53 binding to a region upstream of the let-7 gene following radiation exposure. Luciferase transient transfections demonstrated that this p53 binding site is necessary for radiation-induced decreases in let-7 expression. A radiation-induced decrease in let-7a and let-7b expression is also observed in radiation-sensitive tissues in vivo and correlates with altered expression of proteins in p53-regulated pro-apoptotic signaling pathways. In contrast, this decreased expression is not observed in p53 knock-out mice suggesting that p53 directly repress let-7 expression. Exogenous expression of let-7a and let-7b increased radiation-induced cytotoxicity in HCT116 p53(+/+) cells but not HCT116 p53(-/-) cells. These results are the first demonstration of a mechanistic connection between the radiation-induced stress response and the regulation of miRNA and radiation-induced cytotoxicity and suggest that this process may be a molecular target for anticancer agents.     

RAS is regulated by the let-7 microRNA family

MicroRNAs (miRNAs) are regulatory RNAs found in multicellular eukaryotes, including humans, where they are implicated in cancer. The let-7 miRNA times seam cell terminal differentiation in C. elegans. Here we show that the let-7 family negatively regulates let-60/RAS. Loss of let-60/RAS suppresses let-7, and the let-60/RAS 3'UTR contains multiple let-7 complementary sites (LCSs), restricting reporter gene expression in a let-7-dependent manner. mir-84, a let-7 family member, is largely absent in vulval precursor cell P6.p at the time that let-60/RAS specifies the 1 degrees vulval fate in that cell, and mir-84 overexpression suppresses the multivulva phenotype of activating let-60/RAS mutations. The 3'UTRs of the human RAS genes contain multiple LCSs, allowing let-7 to regulate RAS expression. let-7 expression is lower in lung tumors than in normal lung tissue, while RAS protein is significantly higher in lung tumors, providing a possible mechanism for let-7 in cancer.     

Down-regulation of circulating microRNA let-7a in Egyptian smokers

Altered miRNAs were associated with cigarette smoking. The study aimed to examine the gene expression level of plasma let-7a among healthy smokers and compared it with the non-smokers. Forty subjects were recruited for the present study and classified into 21 smokers and 19 non-smokers, age, and sex were matched. The software that used to design functional primers was MIRprimer. Quantitative real-time PCR was employed to compare the relative expression of plasma let-7a. Results showed that the level of let-7a was down-regulated in smokers to 0.34fold (p?=?0.006) that of the non-smokers. Plasma let-7a showed an area under curve (AUC) of 0.749 with sensitivity 43% and specificity 100%. In conclusion, plasma let-7a was significantly down-regulated in the smokers, and it might be considered a candidate biomarker to discriminate between smokers and non-smokers.     

let-7 microRNA调控动物器官发育的研究进展

微小RNA（microRNA,miRNA）是一类在进化上高度保守、长度约20～24 nt的小分子非编码RNA,能通过与靶基因3′非翻译区相结合从而抑制靶基因的翻译或降解靶基因。let-7 microRNA是发现较早的一类miRNA,最早在线虫中发现能调控细胞分裂的时序。此后大量证据表明,let-7参与动物多个器官发育的调控过程,并与人类疾病发生密切相关。该文综述了近年来let-7调控动物脑、神经及心肺系统等器官发育的研究成果,初步阐述了let-7调控动物器官发育可能的作用机制,以期为深入研究let-7的功能奠定基础。     

The Effects of Chronic Lymphocytic Thyroiditis on Clinicopathologic Factors in Papillary Thyroid Cancer

ObjectiveThis study evaluated the impact of chronic lymphocytic thyroiditis (CLT) on clinicopathologic parameters, prognostic outcome, and initial treatment responses in patients with papillary thyroid cancer (PTC).MethodsA retrospective review was conducted of 1409 patients with PTC, comprising 443 patients with pathology-proven PTC with CLT and 447 patients with PTC without CLT.ResultsThe median follow-up time was 58 months (range, 8-380 months), and the median age at the time of diagnosis was 43 years. The age at diagnosis was significantly lower in patients with CLT than in those without CLT (42 years vs 45 years, respectively; P = .001). The preoperative thyroid-stimulating hormone level was found to be significantly higher in patients with CLT than in those without CLT (1.71 mIU/L vs 1.28 mIU/L, respectively; P < .001). Multifocality and capsular, lymphovascular, and perineural invasion were detected at a higher rate in the group with CLT than in the group without CLT (P = .015, P = .024, P = .004, and P = .039, respectively). No difference was found between the 2 groups in terms of tumor size, bilaterality, extrathyroidal invasion, lymph node metastasis, disease stage, or response to treatment (P > .05).ConclusionThe results of the present study demonstrated that the coexistence of PTC and CLT is very frequent. Patients with the coexistence of PTC and CLT were diagnosed at a younger age, and the thyroid-stimulating hormone level was higher in these patients. Contrary to previous studies, no positive effect of the CLT and PTC combination was detected on any clinicopathologic factor. In addition, lymphovascular and perineural invasions, which had negative effects on prognosis, were more common in the group with CLT.     

Identification of specific let-7 microRNA binding complexes in Caenorhabditis elegans

Little is known about the protein complexes required for microRNA formation and function. Here we used native gel electrophoresis to identify miRNA ribonucleoprotein complexes (miRNPs) in Caenorhabditis elegans. Our data reveal multiple distinct miRNPs that assemble on the let-7 miRNA in vitro. The formation of these complexes is affected but not abolished by alg-1 or alg-2 null mutations. The largest complex (M*) with an estimated molecular mass of >669 kDa cofractionates with the known RISC factors ALG-1, VIG-1, and TSN-1. The M* complex and two complexes, M3 and M4, with similar molecular weights of ~500 kDa, also assemble on all other miRNAs used in our experiments. Two smaller complexes, M1 (~160 kDa) and M2 (~250 kDa), assemble on the members of the let-7 miRNAs family but not lin-4 or mir-234, and their formation is highly dependent on specific sequences in the 5′ seed region of let-7. Moreover, an unidentified protein, p40, which only appears in the M1 and M2 complexes, was detected by UV triggered cross-linking to let-7 but not to lin-4. The cross-linking of p40 to let-7 is also dependent on the let-7 sequence. Another unidentified protein, p13, is detected in all let-7 binding complexes and lin-4 cross-linked products. Our data suggest that besides being present in certain large miRNPs with sizes similar to reported RISC, the let-7 miRNA also assembles with specific binding proteins and forms distinct small complexes.     

Increased Pleiotrophin Concentrations in Papillary Thyroid Cancer

Background

Thyroid nodules are common, and approximately 5% of these nodules are malignant. Pleiotrophin (PTN) is a heparin-binding growth factor which is overexpressed in many cancers. The expression of PTN in papillary thyroid cancer (PTC) is unknown.

Method and Findings

74 subjects (age 47 ± 12 y, 15 males) who had thyroidectomy with a histological diagnosis: 79 benign nodules and 23 PTCs (10 classic, 6 tall cell, 6 follicular variant and 1 undetermined). Fine-needle aspiration (FNA) samples were obtained ex vivo from surgically excised tissue and assayed for PTN and thyroglobulin (Tg). Immunohistochemistry (IHC) was performed on tissue sections. In FNA samples, PTN concentration normalized to Tg was significantly higher in PTC than in benign nodules (16 ± 6 vs 0.3 ± 0.1 ng/mg, p < 0.001). In follicular variant of PTC (n = 6), the PTN/Tg ratio was also higher than in benign nodules (1.3 ± 0.6 vs 0.3 ± 0.1 ng/mg, P < 0.001, respectively). IHC showed cytoplasmic localization of PTN in PTC cells.

Conclusion

In ex vivo FNA samples, the PTN to thyroglobulin ratio was higher in PTCs, including follicular variant PTC, than in benign thyroid nodules. The findings raise the possibility that measurement of the PTN to Tg ratio may provide useful diagnostic and/or prognostic information in the evaluation of thyroid nodules.     

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.     

P68 RNA helicase unwinds the human let-7 microRNA precursor duplex and is required for let-7-directed silencing of gene expression

MicroRNAs are short, single-stranded RNAs that arise from a transient precursor duplex. We have identified a novel activity in HeLa cell extracts that can unwind the let-7 microRNA duplex. Using partially purified material, we have shown that microRNA helicase activity requires ATP and has a native molecular mass of approximately 68 kDa. Affinity purification of the unwinding activity revealed co-purification of P68 RNA helicase. Importantly, recombinant P68 RNA helicase was sufficient to unwind the let-7 duplex. Moreover, like its native homolog, P68 RNA helicase did not unwind an analogous small interfering RNA duplex. We further showed that knockdown of P68 inhibited let-7 microRNA function. From our data, we conclude that P68 RNA helicase is an essential component of the let-7 microRNA pathway, and in conjunction with other factors, it may play a role in the loading of let-7 microRNA into the silencing complex.     

An Unusual Case of Recurrent Hyperparathyroidism and Papillary Thyroid Cancer

ObjectiveTo report an unusual occurrence of recurrent hyperparathyroidism due to papillary thyroid carcinoma.MethodsWe describe the clinical history, physical examination findings, laboratory values, imaging findings, and pathologic findings of a woman who developed recurrent hyperparathyroidism 13 years after successful parathyroidectomy.ResultsA 59-year-old woman presented to our clinic with recurrent primary hyperparathyroidism. In 1994, she presented with nephrolithiasis and underwent resection of a right superior parathyroid adenoma that resulted in clinical and biochemical cure. Her clinical course had been followed at periodic intervals, and she had been symptom-free and normocalcemic. In 2007, she again developed nephrolithiasis and was documented to have recurrent hyperparathyroidism. Imaging studies suggested a parathyroid adenoma near the right inferior pole of the thyroid. The patient had reoperative neck exploration. No obvious parathyroid adenoma was found and a right thyroid lobectomy was performed, which resulted in normalization of intraoperative intact parathyroid hormone levels, and the incision was closed. Final pathology demonstrated no parathyroid adenoma, but instead, a 1-cm papillary thyroid carcinoma that stained positive for parathyroid hormone. More than 6 months after surgery, she remains clinically and biochemically cured.ConclusionsRecurrent hyperparathyroidism occurs secondary to multiple causes. This case demonstrates the challenge a surgeon faces in managing recurrent disease and highlights a rare phenomenon of papillary thyroid cancer causing recurrent hyperparathyroidism. (Endocr Pract. 2009;15:349-352)     

miR-26a and its Target CKS2 Modulate Cell Growth and Tumorigenesis of Papillary Thyroid Carcinoma

Background

While many studies have shown that levels of miR-26a are lower in papillary thyroid carcinoma (PTC), the role and mechanism of miR-26a in PTC are unclear.

Method

We used database searches to select potential mRNA targets of miR-26a. Anti-miR-26a, miR-26a mimic, siRNA for CKS2 and their effects on cell growth, cell-cycle distribution and colony formation were evaluated. We also evaluate the over-expressed miR-26a in TPC-1 cells in severe combined immune-deficient mice. We used luciferase reporter assays, real-time PCR and western blot analysis to measure the expression and activity of miR-26a, CKS2, and related factors such as cyclin B1, cyclin A, cdk1, bcl-xl and Akt. Finally, we measured the relationship between the levels of miR-26a and CKS2 in PTC and normal thyroid tissues.

Results

Relative to normal thyroid tissues, miR-26a is consistently down-regulated in TPC specimens, and CKS2 was identified as a potential target. Up-regulated miR-26a expression or down-regulated CKS2 expression in TPC-1 and CGTH W3 cells lines caused G2 phase-arrest. Decreased miR-26a expression or increased CKS2 expression could have inverse function on PTC cell lines. CyclinB1, cyclinA, bcl-xl and AKt are indirectly regulated by miR-26a in a CKS2-dependent manner. Finally, CKS2 is overexpressed in PTC specimens relative to normal thyroid tissue, and a significant inverse correlation exists between miR-26a and CKS2 expression in clinical PTC specimens.

Conclusion

Our data indicate that miR-26a functions as a growth-suppressive miRNA in PTC, and that its suppressive effects are mediated mainly by repressing CKS2 expression.     

The Significance of Thyroglobulin Antibodies in Papillary Thyroid Cancer

Abbreviations:CLT = chronic lymphocytic thyroiditisPTC = papillary thyroid cancerTgAb = thyroglobulin antibodies     

Molecular Pathways Associated with Aggressiveness of Papillary Thyroid Cancer

The most common thyroid malignancy is papillary thyroid cancer (PTC). Mortality rates from PTC mainly depend on its aggressiveness. Geno- and phenotyping of aggressive PTC has advanced our understanding of treatment failures and of potential future therapies. Unraveling molecular signaling pathways of PTC including its aggressive forms will hopefully pave the road to reduce mortality but also morbidity from this cancer. The mitogen-activated protein kinase and the phosphatidylinositol 3-kinase signaling pathway as well as the family of RAS oncogenes and BRAF as a member of the RAF protein family and the aberrant expression of microRNAs miR-221, miR-222, and miR-146b all play major roles in tumor initiation and progression of aggressive PTC. Small molecule tyrosine kinase inhibitors targeting BRAF-mediated events, vascular endothelial growth factor receptors, RET/PTC rearrangements, and other molecular targets, show promising results to improve treatment of radioiodine resistant, recurrent, and aggressive PTC.     

miRNA let-7e Modulates the Wnt Pathway and Early Nephrogenic Markers in Mouse Embryonic Stem Cell Differentiation

This study indicates that embryonic stem cells [ESCs] cultured with retinoic acid and activin A significantly upregulate the miRNA let-7e. This specific miRNA modulates the Wnt pathway and the expression of early nephrogenic markers under these differentiation conditions. The differentiation markers WT1, Pax2 and Wnt4 were downregulated when miRNA let-7e was silenced, thus indicating the role of miRNA let-7e in the differentiation process. PKCβ, GSK3β phosphorylation (GSK3β^P) and β-catenin expression was reduced in differentiated cells and reversed by miRNA let-7e silencing. Addition of a PKCβ inhibitor to the miRNA let-7e silenced cells abolished let-7e-derived effects in differentiation markers, and reversed the increase in GSK3β^P and β-catenin, thus indicating that miRNA let-7e is involved in differentiation via the modulation of GSK3β phosphorylation and β-catenin production.     

Hormonal regulation of Drosophila microRNA let-7 and miR-125 that target innate immunity

The steroid 20-hydroxy-ecdysone (20-HE) and the sesquiterpenoid Juvenile Hormone (JH) coordinate insect life stage transitions. 20-HE exerts these effects by the sequential induction of response genes. In the nematode Caenorhabditis elegans hormones also play a role in such transitions, but notably, microRNA such as let-7 and lin-4 have likewise been found to help order developmental steps. Little is known about the corresponding function of homologous microRNA in Drosophila melanogaster, and the way microRNA might be regulated by 20-HE in the fly is ambiguous. Here we used Drosophila S2 cells to analyze the effects of 20-HE on D. melanogaster microRNA let-7 and miR-125, the homolog of lin-4. The induction by 20-HE of let-7 and miR-125 in S2 cells is inhibited by RNai knockdown of the ecdysone receptor and, as previously shown, by knockdown of its cofactor broad-complex C. To help resolve the currently ambiguous role of 20-HE in the control of microRNa, we show that nanomolar concentrations of 20-HE primes cells to subsequently express microRNa when exposed to micromolar levels of 20-HE. We then explore the role microRNa plays in the established relationship between 20-HE and the induction of innate immunity. We show that the 3′UTR of the antimicrobial peptide diptericin has a let-7 binding site and that let-7 represses translation from this site. We conclude that 20-HE facilitates the initial expression of innate immunity while it simultaneously induces negative regulation via microRNa control of antimicrobial peptide translation.Key words: microRNA, ecdysone, innate immunity, let-7, diptericin, inflammation     

Hormonal regulation of Drosophila microRNA let-7 and miR-125 that target innate immunity

The steroid 20-hydroxy-ecdysone (20-HE) and the sesquiterpenoid Juvenile Hormone (JH) coordinate insect life stage transitions. 20-HE exerts these effects by the sequential induction of response genes. In the nematode Caenorhabditis elegans hormones also play a role in such transitions, but notably, microRNA such as let-7 and lin-4 have likewise been found to help order developmental steps. Little is known about the corresponding function of homologous microRNA in Drosophila melanogaster, and the way microRNA might be regulated by 20-HE in the fly is ambiguous. Here we used Drosophila S2 cells to analyze the effects of 20-HE on D. melanogaster microRNA let-7 and miR-125, the homolog of lin-4. The induction by 20-HE of let-7 and miR-125 in S2 cells is inhibited by RNAi knockdown of the ecdysone receptor and, as previously shown, by knockdown of its cofactor broad-complex C. To help resolve the currently ambiguous role of 20-HE in the control of microRNA, we show that nanomolar concentrations of 20-HE primes cells to subsequently express microRNA when exposed to micromolar levels of 20-HE. We then explore the role microRNA plays in the established relationship between 20-HE and the induction of innate immunity. We show that the 3'UTR of the antimicrobial peptide diptericin has a let-7 binding site and that let-7 represses translation from this site. We conclude that 20-HE facilitates the initial expression of innate immunity while it simultaneously induces negative regulation via microRNA control of antimicrobial peptide translation.     

Rhabdomyolysis after Withdrawal of Thyroid Hormone in a Patient with Papillary Thyroid Cancer

ObjectiveTo report a case of rhabdomyolysis presenting with severe hyperkalemia after withdrawal of thyroid hormone in a patient with differentiated thyroid cancer.MethodsWe describe the clinical and laboratory findings of the study patient and review the relevant literature.ResultsA 54-year-old man with progressive generalized weakness and myalgias presented with acute renal failure and hyperkalemia. He had undergone total thyroidectomy for papillary thyroid cancer 6 weeks earlier and had discontinued thyroid hormone 2 weeks before his current presentation in preparation for thyroid remnant ablation. He had a history of multiple colon and small-bowel resections for familial adenomatous polyposis and desmoid tumor. He was severely dehydrated on examination. Laboratory tests results included the following values: creatine phosphokinase, 5265 U/L (reference range, 52-336 U/L); creatinine, 2.1 mg/dL; potassium, > 8.0 mEq/L; and thyrotropin, 92.2 mIU/L. His condition was diagnosed as rhabdomyolysis, and his fluid deficit and hyperkalemia were treated aggressively. Cardiac status remained stable, and both acute renal failure and hyperkalemia improved. He then received remnant ablation, and thyroid hormone was restarted. His muscle complaints resolved over the following 3 months.ConclusionsHypothyroidism-induced rhabdomyolysis can occur during thyroid hormone withdrawal and can present with life-threatening hyperkalemia. Patients undergoing thyroid hormone withdrawal should be assessed for risk of rhabdomyolysis, and preventive strategies should be implemented, including prevention of dehydration.The use of recombinant thyrotropin, rather than thyroid hormone withdrawal, should be considered in those who are at high risk for such complications. (Endocr Pract. 2008;14:1023-1026)