Depletion of intermediate filament protein Nestin,a target of microRNA-940, suppresses tumorigenesis by inducing spontaneous DNA damage accumulation in human nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a major malignant tumor of the head and neck region in southern China. The understanding of its underlying etiology is essential for the development of novel effective therapies. We report for the first time that microRNA-940 (miR-940) significantly suppresses the proliferation of a variety of cancer cell lines, arrests cells cycle, induces caspase-3/7-dependent apoptosis and inhibits the formation of NPC xenograft tumors in mice. We further show that miR-940 directly binds to the 3′-untranslated regions of Nestin mRNA and promotes its degradation. Likewise, depletion of Nestin inhibits tumor cell proliferation, arrest cells at G2/M, induces apoptosis and suppresses xenograft tumor formation in vivo. These functions of miR-940 can be reversed by ectopic expression of Nestin, suggesting that miR-940 regulates cell proliferation and survival through Nestin. Notably, we observed reduced miR-940 and increased Nestin levels in NPC patient samples. Protein microarray revealed that knockdown of Nestin in 5-8F NPC cells alters the phosphorylation of proteins involved in the DNA damage response, suggesting a mechanism for the miR-940/Nestin axis. Consistently, depletion of Nestin induced spontaneous DNA damage accumulation, delayed the DNA damage repair process and increased the sensitivity to irradiation and the chemotherapeutic agent doxorubicin. Collectively, our findings indicate that Nestin, which is downregulated by miR-940, can promote tumorigenesis in NPC cells through involvement in the DNA damage response. The levels of microRNA-940 and Nestin may serve as indicators of cancer status and prognosis.Nasopharyngeal carcinoma (NPC), a major malignant tumor of the head and neck region, is endemic to Southeast Asia, southern China, the Arctic, the Middle East and North Africa.¹ Low differentiation and high metastatic potential and recurrence rates are major pathologic features of NPC. The incidence of NPC in southern China has remained very high, with a 5-year overall survival rate of approximately 70%.² Within 4 years after radiation therapy, about 30–40% of NPC patients develop distant metastasis, which is associated with poor prognosis.³ Therefore, an understanding of the underlying etiology is essential for the development of novel effective therapies for NPC.MicroRNAs (miRNAs), a class of small (∼22 nucleotides) noncoding RNAs, reduce mRNA stability and/or suppress translation by binding to the 3′-untranslated regions (3′-UTRs) or coding sequences of target mRNAs.⁴ As such, miRNAs are involved in the majority of basic biologic processes, including cell proliferation, apoptosis, differentiation and development.⁵ Cumulative evidence also suggests that miRNAs can function as potential oncogenes or tumor suppressor genes.^{6, 7} Abnormal expression of miRNAs and mutations of their genes have been documented in various types of tumors.⁸ Recently, a growing number of miRNAs have been implicated in the development of NPC. For instance, the decreased expression of miR-100 has been reported to cause Plk1 overexpression, which in turn contributes to NPC progression.⁹ MiR-200a upregulation in the undifferentiated cell line C666-1 inhibits cell growth, migration and invasion by targeting ZEB2 and CTNNB1.¹⁰ Inhibition of miR-141, which is upregulated in NPC specimens, may affect cell cycle, apoptosis, cell growth, migration and invasion through targeting of BRD3, UBAP1 and PTEN.¹¹ In addition, reduced levels of let-7 in NPC might have a role in the proliferation through DNA methylation.¹² In view of the roles of miRNAs in tumorigenesis, identification of key miRNAs and their targets that contribute to NPC progression may provide novel targets for NPC diagnosis and treatment.Nestin, a member of the type VI intermediate filament protein family, is widely expressed in mammalian nervous tissue, some immortalized mammalian stem cell lines¹³ and precursor cells of some tissues, for which expression is decreased with differentiation.^{14, 15, 16} As a stem cell/progenitor cell marker,¹⁷ Nestin is essential for mitogen-stimulated proliferation of neural progenitor cells,¹⁸ and loss of Nestin leads to apoptosis of neural progenitor cells in zebrafish.¹⁹ Recently, Nestin has been detected in various cell lines established from human solid tumors²⁰ and has been associated with aggressive nervous system tumors.²¹ All of these findings suggest that Nestin is associated with tumorigenesis; however, the precise role of Nestin and the relationship between Nestin and NPC progression are still unknown.In this study, we screen 350 different miRNAs and determined that miR-940 inhibits the proliferation of the NPC cell lines 5-8F and CNE2. Furthermore, miR-940 expression induces G2/M arrest, promotes apoptosis and suppresses xenograft tumor growth. Bioinformatic and luciferase reporter assays revealed that miR-940 targets two putative binding sites in the Nestin 3′-UTR region. A physiologic role for miR-940 was suggested by its common downregulation in NPC tissues, whereas Nestin showed a converse pattern of upregulation. Knockdown of Nestin in 5-8F and CNE2 cells induces G2/M arrest and apoptosis and inhibits cell proliferation and xenograft tumor growth; conversely, ectopic expression of Nestin partially reverses the effects of miR-940 on cell proliferation, cell cycle and apoptosis. Interestingly, knockdown of Nestin induces spontaneous DNA damage accumulation, delays DNA damage repair and enhances sensitivity to ionizing radiation (IR) of 5-8F cells both in vitro and in vivo. These results elucidate a pathway by which miR-940 regulates tumor progression in NPC by targeting Nestin.     

The LEF1/CYLD axis and cIAPs regulate RIP1 deubiquitination and trigger apoptosis in selenite-treated colorectal cancer cells

Inhibitor-of-apoptosis protein (IAP) inhibitors have been reported to synergistically reduce cell viability in combination with a variety of chemotherapeutic drugs via targeted cellular IAP (cIAP) depletion. Here, we found that cIAP silencing sensitised colorectal cancer (CRC) cells to selenite-induced apoptosis. Upon selenite treatment, the K63-linked ubiquitin chains on receptor-interacting protein 1 (RIP1) were removed, leading to the formation of the death-inducing complex and subsequent caspase-8 activation. Although the ubiquitinases cIAP1 and cIAP2 were significantly downregulated after a 24-h selenite treatment, cylindromatosis (CYLD) deubiquitinase protein levels were marginally upregulated. Chromatin immunoprecipitation assays revealed that lymphoid enhancer factor-1 (LEF1) dissociated from the CYLD promoter upon selenite treatment, thus abolishing suppression of CYLD gene expression. We corroborated these findings in a CRC xenograft animal model using immunohistochemistry. Collectively, our findings demonstrate that selenite caused CYLD upregulation via LEF1 and cIAP downregulation, both of which contribute to the degradation of ubiquitin chains on RIP1 and subsequent caspase-8 activation and apoptosis. Importantly, our results identify a LEF1-binding site in the CYLD promoter as a potential target for combinational therapy as an alternative to cIAPs.     

Spatio-temporal incidence of malaria patients in Incheon Metropolitan City

Malaria, a globally significant mosquito-borne infectious disease, re-emerged in the Republic of Korea, and manifested annually in regions close to the demilitarized zone. Notably, Incheon Metropolitan City has witnessed an alarming upswing in malaria infections in recent years, drawing attention to this public health issue. This research was conducted to catch spatio-temporal and ecological landscape encompassing malaria patients and mosquito vectors in Incheon over the past decade. The top two incidences of malaria cases were found in Ganghwa-gun and Seo-gu, an occurrence potentially attributed to their geographic proximity to North Korea. Furthermore, the incidence of malaria infections displayed a seasonal pattern commencing in March, reaching its peak between June and August, and decreasing to a minimum in November. A correlation was noted between prevalence of malaria cases and number of mosquito breeding sites, such as ponds and rice fields within the region. Collectively, these research outcomes underlined the importance of systematically and holistically advocating mosquito elimination measures to enhance the efficacy of malaria eradication policies. These measures encompass the establishment of a robust mosquito outbreak surveillance system, targeted control of vector mosquitoes, residual pesticide spray, management of mosquito breeding sites, and adoption of repellents during outdoor activities.     

Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish

During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1^-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of spo11 does not suppress the sex reversal phenotype. Compared to tp53 single mutant controls, rad21l1^-/- tp53^-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1^-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis.     

Cancer-associated fibroblasts-derived exosomal miR-3656 promotes the development and progression of esophageal squamous cell carcinoma via the ACAP2/PI3K-AKT signaling pathway

Esophageal squamous cell carcinoma (ESCC) is one of the most common gastrointestinal tumors, accounting for almost half a million deaths per year. Cancer-associated fibroblasts (CAFs) are the major constituent of the tumor microenvironment (TME) and dramatically impact ESCC progression. Recent evidence suggests that exosomes derived from CAFs are able to transmit regulating signals and promote ESCC development. In this study, we compared different the component ratios of miRNAs in exosomes secreted by CAFs in tumors and with those from normal fibroblasts (NFs) in precancerous tissues. The mRNA level of hsa-miR-3656 was significantly upregulated in the former exosomes. Subsequently, by comparing tumor cell development in vitro and in vivo, we found that the proliferation, migration and invasion capabilities of ESCC cells were significantly improved when miR-3656 was present. Further target gene analysis confirmed ACAP2 was a target gene regulated by miR-3656 and exhibited a negative regulatory effect on tumor proliferation. Additionally, the downregulation of ACAP2 triggered by exosomal-derived miR-3656 further promotes the activation of the PI3K/AKT and β-catenin signaling pathways and ultimately improves the growth of ESCC cells both in vitro and in xenograft models. These results may represent a potential therapeutic target for ESCC and provide a new basis for clinical treatment plans.     

两种类型人碱性成纤维生长因子的体外稳定性研究

目的:通过野生型bFGF和改构型bFGF蛋白溶液中的聚集过程的比较,初步探讨bFGF在水溶液中发生聚集的机理。方法:在选择合适溶液体系后,采用蛋白溶解度和促有丝分裂活性能力为指标,表征野生型bFGF和突变型bFGF聚合程度,分析共价聚合和非共价聚合所起的作用。结果:在相同的溶液体系中,野生型bFGF的聚集程度高于突变型bFGF。bFGF聚合程度与浓度有依赖性。沉淀结果分析非共价聚合占主要作用。结论:野生型bFGF在溶液中共价聚和和非共价聚合同时发生,两种bFGF聚合过程中非共价聚合占较大比例。突变半胱氨酸可以减少聚合发生。     

中国鄂温克、鄂伦春、达斡尔族永生细胞系的建立与保存

本采用ＥＢＶ（Ｅｐｓｔｅｉｎ－Ｂａｒｒ Ｖｉｒｕｓ）上清液转化Ｂ淋巴细胞,并加入环胞霉素Ａ（Ｃｙｃｌｏｓｐｏｒｉｎｅ Ａ）抑制Ｔ淋巴细胞,成功地对中国东北地区鄂温克族、鄂伦春族及达斡尔族的部分个体建立了永生细胞系,其中鄂温克族４９株,鄂伦春族４０株,达斡尔族５１例,总计１４０株。永久保存我国特有民族的基因组,为分析其遗传学差异奠定了基础。     

叶绿康球肥在果树上的应用研究

用叶绿康球肥在苹果、梨、桃和李树上进行大田对比试验,通过田间调查和室内分析,研究了球肥对果树叶片的复绿效果、对叶内矿质营养元素及叶绿素含量的影响以及对果树的增产效果和改善苹果品质的作用等。研究结果表明：施用叶绿康球肥可使失绿果树叶片的活性铁提高８．７％￣２０．２％,苹果叶片的叶绿素含量提高１８％;果树失绿程度降低２￣３级,果实单产提高５６％￣８９％。可降低叶片中Ｐ、Ｋ、Ｚｎ、Ｍｎ、Ｃｕ的含量,使铁     

Differential expression of the potato proteinase inhibitor II promoter in transgenic tobacco

We studied temporal and spatial expression patterns of the potato proteinase inhibitor II (PI-II) promoter, using transgenic tobacco (Nkotiana tabacum L cv. Xanthi) plants that carried a fusion between the PI-II promoter and the chloramphenicol acetyltransferase (cat) gene. Pl-ll promoter activity was low when plants were young, but increased as plants grew. In 8-week-old plants, old leaves showed higher activity than young leaves. At flowering stage (ca. 15 weeks), the overall promoter activity was reduced to a lower level except in the petals. Compared with stems or petioles at the flowering stage, the roots and floral organs showed minimal activity for the Pl-ll promoter. We used several environmental stimuli to examine the induction of the Pl-ll promoter in different organs. Promoter induction was effected by wounding or methyl jasmonate in stems, petioles, sepals, and leaves. The induction was highest in leaves, as was sucrose-enhanced wound induction. These results suggest that the Pl-ll gene is temporally and spatially regulated. We also established a transient assay system in tobacco BY2 suspension cells to elucidate the upstream regulatory region of the Pl-ll promoter. A field strength of 0.75 kV/cm and 400 μF capacitance were optimal electroporation conditions for our transient assay.     

Antibiotics resistance of a red alga,Griffithsia japonica

To identify antibiotics suitable for stable transformation, we tested the resistance of a red alga,Griffithsia japonica Okamura, to four commonly used antibiotics. Very young germlings, with 1;3 cells, that germinated from the tetraspores were cultured for 40 d in a half PES medium containing kanamycin, streptomycin, hygromycin B, or phleomycin.G. japonica was highly sensitive to 1 μg mL^-1of phleomycin and g mL^-1of hygromycin B. However, it was resistant to kanamycin and low levels of streptomycin and hygromycin B. These results suggest that resistance genes for phleomycin or hygromycin can be used as selectable markers for transformation of G.japonica.