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.     

MARCH5 RNA promotes autophagy,migration, and invasion of ovarian cancer cells

MARCH5 is a crucial regulator of mitochondrial fission. However, the expression and function of MARCH5 in ovarian cancer have not been determined. This study investigated the expression and function of MARCH5 in ovarian cancer with respect to its potential role in the tumorigenesis of the disease as well as its usefulness as an early diagnostic marker. We found that the expression of MARCH5 was substantially upregulated in ovarian cancer tissue in comparison with the normal control. Silencing MARCH5 in SKOV3 cells decreased TGFB1-induced cell macroautophagy/autophagy, migration, and invasion in vitro and in vivo, whereas the ectopic expression of MARCH5 in A2780 cells had the opposite effect. Mechanistic investigations revealed that MARCH5 RNA may function as a competing endogenous RNA (ceRNA) to regulate the expression of SMAD2 and ATG5 by competing for MIR30A. Knocking down SMAD2 or ATG5 can block the effect of MARCH5 in A2780 cells. Also, silencing the expression of MARCH5 in SKOV3 cells can inhibit the TGFB1-SMAD2/3 pathway. In contrast, the ectopic expression of MARCH5 in A2780 cells can activate the TGFB1-SMAD2/3 pathway. In turn, the TGFB1-SMAD2/3 pathway can regulate MARCH5 and ATG5 through MIR30A. Overall, the results of this study identified MARCH5 as a candidate oncogene in ovarian cancer and a potential target for ovarian cancer therapy.     

Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease

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甲状腺乳头状癌中TLR4对Foxp3表达的调控作用分析

目的:探讨人甲状腺乳头状癌(PTC)细胞中TLR4信号通路对Foxp3表达的调控作用。方法:以人甲状腺乳头状癌K1细胞株为实验材料,选用脂多糖(LPS)作为配体来激活TLR4信号通路,采用RT-PCR方法检测LPS在不同浓度(0,5,10,20,40μg/mL)和不同时间点(12,24,36,48 h)Foxp3的mRNA表达量,流式细胞术检测相应浓度和时间点的Foxp3蛋白和TLR4蛋白表达量的变化;加入LPS抑制剂多粘菌素B(PMB)后Foxp3和mRNA和蛋白的表达量分别利用RT-PCR和流式细胞术检测。结果:10μg/mL的LPS可以显著上调PTC细胞中Foxp3 mRNA和蛋白的表达量(P0.05),在第24小时达到最佳;LPS作用下,TLR4表达量上调;PMB阻断TLR4信号通路后,Foxp3蛋白表达量较未阻断组显著降低(P0.05)。结论:在甲状腺乳头状癌K1细胞株中,TLR4作为上游信号调控因子,通过自身表达量改变,进而参与调控Foxp3的表达。     

甲状腺乳头状癌组织SMYD2、GRK6表达与临床病理特征、增殖基因和预后的关系分析

摘要 目的：探讨甲状腺乳头状癌组织中SET和MYND域蛋白2（SMYD2）、G蛋白偶联受体激酶6（GRK6）表达与临床病理特征、增殖基因和预后的关系。方法：选择2015年1月至2017年12月期间我院收治的83例甲状腺乳头状癌患者,取甲状腺乳头状癌患者手术切除的癌组织及癌旁组织（距肿瘤组织边缘≥5 cm）标本,采用qRT-PCR检测SMYD2、GRK6以及增殖基因[叉头盒蛋白A1（FOXA1）、程序性细胞死亡基因4（PDCD4）、Xklp2靶蛋白(TPX2)]表达。分析SMYD2、GRK6表达与临床病理特征、增殖基因和预后的关系。分析甲状腺乳头状癌患者预后的影响因素。结果：癌组织SMYD2、GRK6、FOXA1、PDCD4、TPX2表达高于癌旁组织（P＜0.05）。Pearson相关性分析显示：SMYD2、GRK6表达与FOXA1、PDCD4、TPX2表达呈正相关（P＜0.05）。肿瘤直径＞1 cm、低中度分化、T3～T4a期、N1a～N1b期患者癌组织中SMYD2、GRK6表达高于肿瘤直径≤1 cm、高度分化、T1～T2期、N0期患者（P＜0.05）。SMYD2、GRK6高表达组患者3年无进展生存期（PFS）、总生存期（OS）生存率低于SMYD2、GRK6低表达组（P＜0.05）。Cox比例风险模型分析结果显示N1a～N1b期、SMYD2≥2.95、GRK6≥3.02是甲状腺乳头状癌患者不良预后的危险因素（P＜0.05）。结论：甲状腺乳头状癌组织中SMYD2、GRK6表达增高,SMYD2、GRK6过表达与癌细胞增殖、侵袭和术后预后不良有关。     

PI3K/AKT/mTOR信号通路及其在卵巢癌治疗中的应用

卵巢癌是女性生殖系统常见的恶性肿瘤,发病率居于妇科恶性肿瘤第三位,死亡率居于妇科恶性肿瘤之首。目前对卵巢癌的标准治疗包括肿瘤细胞减灭术及卡铂和紫杉醇的联合化疗。PI3K/AKT/mTOR信号通路在卵巢癌的细胞增殖、侵袭、细胞周期进展、血管生成及耐药中发挥重要作用,是卵巢癌中最常发生改变的细胞内途径。本文对PI3K/AKT/mTOR信号通路及其在卵巢癌增殖和进展中的影响、PI3K/AKT/mTOR信号通路抑制剂在卵巢癌中的治疗应用做简要综述。     

The ubiquitin E3 ligase MARCH7 is differentially regulated by the deubiquitylating enzymes USP7 and USP9X

Protein modification by one or more ubiquitin chains serves a critical signalling function across a wide range of cellular processes. Specificity within this system is conferred by ubiquitin E3 ligases, which target the substrates. Their activity is balanced by deubiquitylating enzymes (DUBs), which remove ubiquitin from both substrates and ligases. The RING-CH ligases were initially identified as viral immunoevasins involved in the downregulation of immunoreceptors. Their cellular orthologues, the Membrane-Associated RING-CH (MARCH) family represent a subgroup of the classical RING genes. Unlike their viral counterparts, the cellular RING-CH proteins appear highly regulated, and one of these in particular, MARCH7, was of interest because of a potential role in neuronal development and lymphocyte proliferation. Difficulties in detection and expression of this orphan ligase lead us to search for cellular cofactors involved in MARCH7 stability. In this study, we show that MARCH7 readily undergoes autoubiquitylation and associates with two deubiquitylating enzymes – ubiquitin-specific protease (USP)9X in the cytosol and USP7 in the nucleus. Exogenous expression and short interfering RNA depletion experiments demonstrate that MARCH7 can be stabilized by both USP9X and USP7, which deubiquitylate MARCH7 in the cytosol and nucleus, respectively. We therefore demonstrate compartment-specific regulation of this E3 ligase through recruitment of site-specific DUBs.     

USF1-ATRAP-PBX3 Axis Promote Breast Cancer Glycolysis and Malignant Phenotype by Activating AKT/mTOR Signaling

Angiotensin II type 1 receptor-associated protein (ATRAP) is widely expressed in different tissues and organs, although its mechanistic role in breast cancer remains unclear. Here, we show that ATRAP is highly expressed in breast cancer tissues. Its aberrant upregulation promotes breast cancer aggressiveness and is positively correlated with poor prognosis. Functional assays revealed that ATRAP participates in promoting cell growth, metastasis, and aerobic glycolysis, while microarray analysis showed that ATRAP can activate the AKT/mTOR signaling pathway in cancer progression. In addition, ATRAP was revealed to direct Ubiquitin-specific protease 14 (USP14)-mediated deubiquitination and stabilization of Pre-B cell leukemia homeobox 3 (PBX3). Importantly, ATRAP is a direct target of Upstream stimulatory factor 1 (USF1), and that ATRAP overexpression reverses the inhibitory effects of USF1 knockdown. Our study demonstrates the broad contribution of the USF1/ATRAP/PBX3 axis to breast cancer progression and provides a strong potential therapeutic target.     

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。     

microRNA-10b confers cisplatin resistance by activating AKT/mTOR/P70S6K signaling via targeting PPARγ in esophageal cancer

It is well known that the acquisition of chemoresistance is a major obstacle for the effective treatment of human cancers. It is reported that microRNAs (miRNAs) are implicated in chemotherapy resistance of various malignancies. miR-10b was previously proved as an oncogene in multiple malignancies, including esophageal cancer. However, its biological significance in regulating cisplatin (DDP) resistance in esophageal cancer is still elusive. Here, we observed that miR-10b expression was upregulated and peroxisome proliferator-activated receptor-γ (PPARγ) expression was downregulated in esophageal cancer tumor tissues and cells. PPARγ was proved as a functional target of miR-10b. Moreover, suppression of miR-10b enhanced the chemosensitivity of esophageal cancer cells to DDP in vitro and in vivo. In addition, PPARγ-mediated DDP sensitivity was weakened by miR-10b overexpression. Furthermore, miR-10b-activated AKT/mTOR/p70S6K signaling pathway through targeting PPARγ. Inactivation of AKT/mTOR/p70S6K by AKT inhibitor (GSK690693) attenuated miR-10b-induced DDP resistance in esophageal cancer cells. Taken together these observation, miRNA-10b-mediated PPARγ inhibition enhanced DDP resistance by activating the AKT/mTOR/P70S6K signaling in esophageal cancer, suggesting a potential target to improve therapeutic response of patients with esophageal cancer to DDP.     

Propensity Score-Matched Analysis to Identify Pathways Associated with Loss of Sodium Iodide Symporter in Papillary Thyroid Cancer

Sodium iodide symporter (NIS) expression in thyroid follicular cells plays an important role in normal physiology and radioactive iodine therapy for thyroid cancer. Loss of NIS expression is often seen in thyroid cancers and may lead to radioiodine refractoriness. To explore novel mechanisms of NIS repression beyond oncogenic drivers, clinical and RNA-seq data from the thyroid cancer dataset of The Cancer Genome Atlas were analyzed. Propensity score matching was used to control for various genetic background factors. We found that tumoral NIS expression was negatively correlated with tumor size. Additionally, low NIS expression was the only factor associated with recurrence-free survival in a Cox multivariate regression analysis. After matching for clinicopathologic profiles and driver mutations, the principal component analysis revealed distinct gene expressions between the high and low NIS groups. Gene set enrichment analysis suggested the downregulation of hedgehog signaling, immune networks, and cell adhesions. Positively enriched pathways included DNA replication, nucleotide excision repair, MYC, and Wnt/β-catenin pathways. In summary, we identified several potential targets which could be exploited to rescue the loss of NIS expression and develop redifferentiation strategies to facilitate radioactive iodine therapy for thyroid cancer.     

Application of Molecular Diagnostics to the Evaluation of the Surgical Approach to Thyroid Cancer

Recent important studies that include long-term follow-up have shown that BRAF and RAS mutations can have negative implications for disease recurrence and survival. BRAF positivity has been shown to be associated with decreased survival and is an independent predictor of poor prognosis. Reliable pre-operative identification of high-risk papillary thyroid cancer (PTC) patients may productively guide initial surgical management since reoperative neck surgery is associated with increased morbidity. However, it is probably too early to conclude that at present it is possible to tailor surgical therapy patient by patient only on the basis of their mutational status. Other important parameters, not including molecular testing, represented by some specific morphological aspects, still play an important role, probably still more significant than molecular diagnostics, such as neck ultrasonography. Pre-operative knowledge of BRAF-positive PTC could alter the initial surgical treatment for at least 20% of patients and can potentially prevent the increased morbidity associated with reoperative neck exploration. However, additional multi-institutional and randomized studies will be needed to further define the role of the pre-operative identification of BRAF positivity to guide not only the initial extent of total thyroidectomy (TT) but also the need for and extent of lymphadenectomy.     

KDM6A promotes chondrogenic differentiation of periodontal ligament stem cells by demethylation of SOX9

Objectives

KDM6A has been demonstrated critical in the regulation of cell fates. However, whether KDM6A is involved in cartilage formation remains unclear. In this study, we investigated the role of KDM6A in chondrogenic differentiation of PDLSCs, as well as the underlying epigenetic mechanisms.

Methods

KDM6A shRNA was transfected into PDLSCs by lentivirus. The chondrogenic differentiation potential of PDLSCs was assessed by Alcian blue staining. Immunofluorescence was performed to demonstrate H3K27me3 and H3K4me3 levels during chondrogenesis. SOX9, Col2a1, ACAN and miRNAs (miR‐29a, miR‐204, miR‐211) were detected by real‐time RT‐PCR. Western blot was performed to evaluate SOX9, H3K27me3 and H3K4me3.

Results

The production of proteoglycans in PDLSCs was decreased after knockdown of KDM6A. Depletion of KDM6A inhibited the expression of SOX9, Col2a1, ACAN and resulted in increased H3K27me3 and decreased H3K4me3 levels. EZH2 inhibitor rescued the chondrogenic potential of PDLSCs after knockdown of KDM6A by regulating H3K27me3. Additionally, miR‐29a, miR‐204 and miR‐211 were also involved in the process of PDLSCs chondrogenesis.

Conclusions

KDM6A is required in chondrogenic differentiation of PDLSCs by demethylation of H3K27me3, and EZH2 inhibitor could rescue chondrogenesis of PDLSCs after knockdown of KDM6A. It could be inferred that upregulation of KDM6A or application of EZH2 inhibitor might improve mesenchymal stem cell mediated cartilage regeneration in inflammatory tissue destruction such as osteoarthritis.

     

结肠癌组织中p-p70S6K的表达及临床意义

目的:检测p-p70S6K在结肠癌组织中的表达并探讨其临床意义。方法:选取40例结肠癌组织蜡块以及40例同一患者的正常结肠组织蜡块进行免疫组化实验,其中又随机选取3组新鲜结肠癌组织和正常结肠组织,通过免疫印迹(Western blot)技术检测p-p70S6K在各组织中的表达情况。结果:在免疫组化实验中,癌组织阳性28例,阴性12例,阳性率为70%,正常结肠组织阳性14例,阴性26例,阳性率为35%,采用Pearson卡方检验,得出x2=9.825,P=0.0020.05,说明癌组织与正常结肠组织中p-p70S6K的表达差异有统计学意义;在免疫印迹实验中,以甘油醛-3-磷酸脱氢酶(GAPDH)为内参,重复试验三次,均显示目标蛋白(p-p70S6K)分子量约70 KD,癌组织中p-p70S6K表达较正常结肠组织明显增加,两组表达水平的比较采用t检验,得出P=0.0250.05,说明差异具有统计学意义。结论:p-p70S6K在结肠癌组织中异常表达,提示该分子在结肠癌的发生、发展过程中具有重要的调控作用,进一步的研究可为结肠癌的靶向治疗提供分子生物学基础。     

O‐GlcNAcylation promotes colorectal cancer progression by regulating protein stability and potential catcinogenic function of DDX5

The RNA helicase p68 (DDX5), a key player in RNA metabolism, belongs to the DEAD box family and is involved in the development of colorectal cancer. Here, we found both DDX5 and O‐GlcNAcylation are up‐regulated in colorectal cancer. In addition, DDX5 protein level is significantly positively correlated with the expression of O‐GlcNAcylation. Although it was known DDX5 protein could be regulated by post‐translational modification (PTM), how O‐GlcNAcylation modification regulated of DDX5 remains unclear. Here we show that DDX5 interacts directly with OGT in the SW480 cell line, which is the only known enzyme that catalyses O‐GlcNAcylation in humans. Meanwhile, O‐GlcNAcylation could promote DDX5 protein stability. The OGT‐DDX5 axis affects colorectal cancer progression mainly by regulating activation of the AKT/mTOR signalling pathway. Taken together, these results indicated that OGT‐mediated O‐GlcNAcylation stabilizes DDX5, promoting activation of the AKT/mTOR signalling pathway, thus accelerating colorectal cancer progression. This study not only reveals the novel functional of O‐GlcNAcylation in regulating DDX5, but also reveals the carcinogenic effect of the OGT‐DDX5 axis in colorectal cancer.     

CHRDL2 promotes osteosarcoma cell proliferation and metastasis through the BMP-9/PI3K/AKT pathway

Various studies demonstrated that bone morphogenetic proteins (BMPs) and their antagonists contribute to the development of cancers. Chordin-like 2 (CHRDL2) is a member of BMP antagonists. However, the role and its relative mechanism of CHRDL2 in osteosarcoma remains unclear. In the present study, we demonstrated that the expression of CHRDL2 was significantly upregulated in osteosarcoma tissues and cell lines compared with adjacent tissues and human normal osteoblast. Inhibition of CHRDL2 decreased the proliferation and colony formation of osteosarcoma cells in vitro, as well as the migration and invasion. CHRDL2 overexpression induced the opposite effects. CHRDL2 can bind with BMP-9, thus decreasing BMP-9 expression and the combination to its receptor protein kinase ALK1. It was predicted that BMP-9 regulates PI3K/AKT pathways using gene set enrichment analysis. Inhibition of CHRDL2 decreased the activation of PI3K/AKT pathway, while overexpression of CHRDL2 upregulated the activation. Increasing the expression of BMP-9 reversed the effects of CHRDL2 overexpression on the activation of PI3K/AKT pathway, as well as the proliferation and metastasis of osteosarcoma cells. Take together, our present study revealed that CHRDL2 upregulated in osteosarcoma tissues and cell lines, and promoted osteosarcoma cell proliferation and metastasis through the BMP-9/PI3K/AKT pathway. CHRDL2 maybe an oncogene in osteosarcoma, as well as novel biomarker for the diagnosis of osteosarcoma.