HOXC8通过调控PDX1的表达促进非小细胞肺癌的生长与EMT过程

摘要 目的：探索HOXC8与PDX1在非小细胞肺癌（non-small lung cancer, NSCLC）细胞生长及上皮间质转化（Epithelial-mesenchymal transition, EMT）的作用机制。方法：通过转录组测序、荧光定量PCR及染色质免疫沉淀等方法筛选并鉴定HOXC8调控的靶基因;通过Western blot、CCK-8、克隆集落生成及生物信息学等手段分析靶基因PDX1在非小细胞肺癌中的作用。结果：实验证明HOXC8可结合到PDX1基因的启动子上,并作为转录因子激活PDX1的表达。PDX1的表达促进NSCLC细胞的生长与EMT过程,而沉默PDX1能显著地抑制NSCLC细胞的生长与EMT过程,并诱导细胞的凋亡。通过分析已知的肿瘤数据库, 我们发现在NSCLC中PDX1的表达显著高于正常组织,且PDX1的高表达与肺癌患者的预后不良呈显著的相关性。结论：本研究发现HOXC8-PDX1轴在非小细胞肺癌中起着重要的调节作用, 可有望成为非小细胞肺癌治疗的新靶点。     

Inhibition of apoptosis in human laryngeal cancer cells by E6 and E7 oncoproteins of human papillomavirus 16

The carcinogenesis of human papillomaviruses type 16 (HPV-16) is mainly due to its two oncoproteins, E6 and E7. Their carcinogenic features in term of their relationship with Bcl-2 family are still unclear. We thus aimed to analyze the expression of Bcl-2 family members, Bcl-2, Bax, and Bak in laryngeal cancer cells transfected with the E6 or E7 and to determine the sensitivity of these cells to apoptotic stimuli. We employed two human laryngeal cancer cell lines, UMSCC12 and UMSCC11A in this study. These two cell lines were stably transfected with HPV16 E6, E7 or empty vector, pcDNA3.1. We found that E6 and E7 inhibited apoptosis induced by TNF-alpha/CHX in both UMSCC11A and UMSCC12 cells, enhanced the stability of Bcl-2 protein and increased the degradation of Bak protein. Furthermore, it was found that HPV-16 E7 statistically enhanced the expression of Bcl-2 in laryngeal cancer. The alteration of Bak by E6 and E7 was not through the influence on the Bak promoter, as the luciferase assay showed that neither E6 nor E7 changed the Bak promoter activity. We conclude that the evasion of apoptosis mediated by HPV-16 E6 and E7 is associated with increased Bcl-2 and decreased Bak in laryngeal carcinogenesis and that the decreased level of Bak by E6 and E7 is not caused by the regulation of the Bak promoter but by reducing its protein stability.     

Evidence of a direct role for Bcl-2 in the regulation of articular chondrocyte apoptosis under the conditions of serum withdrawal and retinoic acid treatment

The regulation of chondrocyte apoptosis in articular cartilage may underlay age-associated changes in cartilage and the development of osteoarthritis. Here we demonstrate the importance of Bcl-2 in regulating articular chondrocyte apoptosis in response to both serum withdrawal and retinoic acid treatment. Both stimuli induced apoptosis of primary human articular chondrocytes and a rat chondrocyte cell line as evidenced by the formation of DNA ladders. Apoptosis was accompanied by decreased expression of aggrecan, a chondrocyte specific matrix protein. The expression of Bcl-2 was downregulated by both agents based on Northern and Western analysis, while the level of Bax expression remained unchanged compared to control cells. The importance of Bcl-2 in regulating chondrocyte apoptosis was confirmed by creating cell lines overexpressing sense and antisense Bcl-2 mRNA. Multiple cell lines expressing antisense Bcl-2 displayed increased apoptosis even in the presence of 10% serum as compared to wild-type cells. In contrast, chondrocytes overexpressing Bcl-2 were resistant to apoptosis induced by both serum withdrawal and retinoic acid treatment. Finally, the expression of Bcl-2 did not block the decreased aggrecan expression in IRC cells treated with retinoic acid. We conclude that Bcl-2 plays an important role in the maintenance of articular chondrocyte survival and that retinoic acid inhibits aggrecan expression independent of the apoptotic process. J. Cell. Biochem. 71:302–309, 1998. © 1998 Wiley-Liss, Inc.     

Neural Epidermal Growth Factor-Like Like Protein 2 (NELL2) Promotes Aggregation of Embryonic Carcinoma P19 Cells by Inducing N-Cadherin Expression

NELL2 was first identified as a mammalian homolog of chick NEL (Neural EGF-like) protein. It is almost exclusively expressed in neurons of the rat brain and has been suggested to play a role in neural differentiation. However, there is still no clear evidence for the detailed function of NELL2 in the differentiation of neurons. In this study, we identified NELL2 function during neural differentiation of mouse embryonic carcinoma P19 cells. Endogenous expression of NELL2 in the P19 cells increased in parallel with the neuronal differentiation induced by retinoic acid (RA). We found that the mouse NELL2 promoter contains RA response elements (RAREs) and that treatment with RA increased NELL2 promoter activity. Transfection of P19 cells with NELL2 expression vectors induced a dramatic increase in cell aggregation, resulting in the facilitation of neural differentiation. Moreover, NELL2 significantly increased N-cadherin expression in the P19 cell. These data suggest that NELL2 plays an important role in the regulation of neuronal differentiation via control of N-cadherin expression and cell aggregation.     

急性力竭运动后小鼠肾细胞凋亡水平的变化及牛磺酸的保护作用

为探讨急性力竭运动后小鼠(Mus musculus)肾细胞凋亡水平的时相性变化及牛磺酸对肾的保护作用,将56只雄性小鼠随机分为对照组、力竭运动组(分为运动后即刻组、12h组、24 h组和48 h组)及牛磺酸运动组(分为12h组和24 h组),每小组8只,一次性力竭游泳运动后检测肾细胞凋亡水平、Bcl-2和Bax蛋白表达、一氧化氮(NO)含量及结构型一氧化氮合酶(cNOS)、诱导型一氧化氮合酶(iNOS)活性的变化.结果显示,力竭运动后各组小鼠肾细胞凋亡水平呈先升高后下降的趋势,其中运动后24 h组的凋亡水平达峰值(P＜0.05).与对照组相比,运动各组Bax表达均显著增强(P＜0.05).除运动后即刻组外,运动各组Bcl-2表达显著减弱(P＜0.05).各组Bax/Bcl-2比值显著升高,并在运动后24 h达峰值(P＜0.01),后出现下降趋势.小鼠力竭游泳后24 h和48 h肾组织NO含量显著高于对照组(P＜0.05),同时iNOS活性升高(P＜0.01),cNOS活性无显著性变化.相比同时刻运动组,牛磺酸运动组小鼠肾细胞凋亡水平、Bax表达及Bax/Bcl-2比值、iNOS活性显著降低(P＜0.05),Bcl-2表达显著升高(P＜0.05).以上结果表明,急性力竭运动可导致肾细胞凋亡的发生,iNOS、Bax、Bcl-2水平及Bax/Bcl-2比值可能在肾细胞凋亡的发生过程中发挥重要的介导作用.牛磺酸可通过调控iNOS活性及Bax/Bcl-2比值,抑制急性力竭运动后小鼠肾细胞凋亡的发生.     

Homeobox Proteins as Signal Transduction Intermediates in Regulation of NCAM Expression by Recombinant Human Bone Morphogenetic Protein-2 in Osteoblast-like Cells

The role of homeobox genes in signaling of recombinant human bone morphogenetic protein-2 (rhBMP-2) was studied in osteoblast-like cells. Expression of several homeobox genes was decreased by rhBMP-2. The finding that this regulation of homeobox gene expression by rhBMP-2 was not dependent on protein synthesis suggests that homeobox proteins can act as direct intermediates in signal transduction of BMPs. Therefore, we studied the regulation of neural cell adhesion molecule (NCAM), which has previously been described as a target gene of both rhBMP-2 and homeobox proteins. We now show that in osteoblast-like cells, rhBMP-2 inhibits NCAM expression, while HOXC6 increases its expression, both acting via the same region of the promoter. As overexpression of HOXC6 could abolish effects of rhBMP-2 on NCAM promoter activity, these data show for the first time that members of the homeobox gene family may form direct functional intermediates in the signaling mechanism of the TGF-β superfamily.     

Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors

Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2''deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC.     

Upregulation of microRNA-141 suppresses epithelial-mesenchymal transition and lymph node metastasis in laryngeal cancer through HOXC6-dependent TGF-β signaling pathway

Laryngeal cancer is one of the most malignant cancers among the head and neck malignant tumors. Abnormal expression of microRNAs (miRNAs) contributes to cancer development through regulating proliferation and apoptosis of cancer cells. In this study, we aim to explore the roles of microRNA-141 (miR-141), Homeobox C6 (HOXC6) and TGF-β signaling pathway in epithelial-mesenchymal transition (EMT) and lymph node metastasis in laryngeal cancer. Initially, we identified differentially expressed genes in laryngeal cancer, among which HOXC6 was identified. Then the target miRNA of HOXC6 was predicted and verified. Next, expression of miR-141, HOXC6, TGF-β1, Smad3, Vimentin and Snail in cancer tissues was detected. Then, AMC-HN-8 cells were transfected with miR-141 mimic, miR-141 inhibitor and HOXC6-siRNA to investigate specific role of miR-141, HOXC6 and TGF-β signaling pathway in laryngeal cancer in vivo and in vitro. Our results showed that HOXC6 was a target gene of miR-141, which was downregulated in laryngeal cancer. Besides, overexpression of miR-141 could downregulate HOXC6 and inhibit the TGF-β signaling pathway. Upregulation of miR-141 or silencing of HOXC6 can repress EMT, viability, migration and invasion abilities of laryngeal cancer cells. In addition, upregulation of miR-141 inhibited the tumor growth and lymph node metastasis in vivo. In summary, our findings demonstrated that upregulated miR-141 decreased HOXC6 expression, and inhibited the TGF-β signaling pathway, EMT and lymph node metastasis in laryngeal cancer, which is of clinical significance in the treatment of laryngeal cancer.     

MAGEB2 is Activated by Promoter Demethylation in Head and Neck Squamous Cell Carcinoma

Purpose

Although promoter hypermethylation has been an accepted means of tumor suppressor gene inactivation, activation of otherwise normally repressed proto-oncogenes by promoter demethylation has been infrequently documented.

Experimental Design

In this study we performed an integrative, whole-genome analysis for discovery of epigenetically activated proto-oncogenes in head and neck cancer tumors. We used the 47K GeneChip U133 Plus 2.0 Affymetrix expression microarray platform to obtain re-expression data from 5-aza treated normal cell line and expression data from primary head and neck squamous cell carcinoma (HNSCC) tumor tissues and normal mucosa tissues. We then investigated candidate genes by screening promoter regions for CpG islands and bisulfite sequencing followed by QUMSP and RT PCR for the best candidate genes. Finally, functional studies were performed on the top candidate gene.

Results

From the top 178 screened candidates 96 had CpG islands in their promoter region. Seven candidate genes showed promoter region methylation in normal mucosa samples and promoter demethylation in a small cohort of primary HNSCC tissues. We then studied the demethylation of the top 3 candidate genes in an expanded cohort of 76 HNSCC tissue samples and 17 normal mucosa samples. We identified MAGEB2 as having significant promoter demethylation in primary head and neck squamous cell carcinoma tissues. We then found significantly higher expression of MAGEB2 in tumors in a separate cohort of 73 primary HNSCC tissues and 31 normal tissues. Finally, we found that MAGEB2 has growth promoting effects on minimally transformed oral keratinocyte cell lines but not a definite effect on HNSCC cell lines.

Conclusion

In conclusion, we identified MAGEB2 as activated by promoter demethylation in HNSCCand demonstrates growth promoting effects in a minimally transformed oral keratinocyte cell line. More studies are needed to evaluate MAGBE2''s exact role in HNSCC.