干细胞因子及其受体c-KIT对羊驼毛囊黑色素细胞增殖与分化的影响及其机制

目的 探讨SCF/c-KIT信号通路对羊驼毛囊黑色素细胞分化、增殖和定位的作用及羊驼丰富毛色性状形成的细胞学机制。 方法 选取8只成年雄性羊驼（4只有色被毛,4只白色被毛）,采用免疫组织化学方法研究SCF和c-KIT受体在羊驼皮肤组织中的表达定位;采用实时定量PCR方法分析SCF和c-KIT基因在羊驼皮肤组织中的表达水平。 结果 SCF和c-KIT受体在不同毛色皮肤组织中均有表达,但在不同被毛颜色羊驼皮肤组织中的表达量和表达部位存在差异;ΔΔCt法统计分析SCF和c-KIT基因在不同颜色被毛羊驼皮肤组织中的表达情况显示,SCF基因在有色被毛皮肤组织中的相对表达量是白色被毛皮肤组织的2.41倍,而c-KIT基因在有色被毛皮肤组织中的相对表达量是白色被毛组织的1.20倍。 结论 SCF/c-KIT信号通路参与调节黑色素细胞在皮肤组织中的增殖、分化;成熟黑色素细胞的数量及其在毛囊组织中的分布位置决定羊驼被毛颜色的形成;SCF信号调节不同分化程度黑色素细胞在毛囊组织中的分布。     

Low expression of KLF17 is associated with tumor invasion in esophageal carcinoma

Purpose: KLF17 belongs to the Sp/KLF zinc-finger protein family as a regulator in tumor development. However, its expression and biologic function has remained unclear in EC. Methods: The esophageal carcinoma tissue samples and adjacent normal tissues were obtained from the Second Hospital of Hebei Medical University. Immunohistochemistry, Western blot, and transfection were applied to evaluate the expression and clinical significance of KLF17 in esophageal cancer. Results: In this study, we showed that KLF17 was overexpressed in esophageal normal samples compared to the cancer. Moreover, KLF17 was upregulated at lymph node non-metastatic cancer tissues when compared to metastatic cancer tissues. KLF17 overexpression decreased EC cell proliferation, migration and invasion ability. In contrast, the knockdown of KLF17 increased EC cell proliferation, migration and invasion ability. Conclusion: These results suggest that KLF17 inhibits tumor development and may serve as a potential therapeutic target.     

TNF-α通过调控KLF4抑制口腔鳞癌细胞CAL27增殖

目的：探讨肿瘤坏死因子-α（TNF-α）通过上调口腔鳞癌细胞CAL27中Krüppel样因子4（KLF4）表达对细胞增殖的影响。方法：用不同浓度的TNF-α（0、25、5、10、20 ng/ml）处理CAL27细胞24 h,免疫印迹试验（Western blot）检测KLF4表达变化;用20 ng/ml TNF-α处理细胞,分别于24 h、48 h、72 h用噻唑蓝（MTT）法检测细胞增殖变化;构建过表达KLF4的CAL27细胞,MTT检测其对细胞增殖的影响;MTT检测沉默KLF4（siKLF4）及siKLF4联合TNF-α 处理对细胞增殖能力的影响。结果：MTT检测发现TNF-α可抑制细胞增殖;TNF-α可呈剂量依赖性地诱导CAL27细胞中KLF4表达;Western blot和qPCR检测显示成功构建过表达KLF4的CAL27细胞;过表达KLF4可抑制细胞增殖;沉默KLF4可部分恢复TNF-α对CAL27细胞增殖的抑制作用。结论：TNF-α可诱导口腔鳞癌细胞CAL27中KLF4表达增加,KLF4可能参与了TNF-α对CAL27细胞增殖的抑制作用。     

KLF4过表达抑制人白血病K562细胞系的增殖及迁移

目的探索人锌指转录因子Krüppel样因子4(KLF4)过表达对人慢性髓系白血病细胞K562增殖及迁移能力的影响。方法设稳定过表达KLF4的白血病K562细胞系作为实验组(命名为K562-KLF4细胞),空白K562细胞及空质粒转染的K562细胞(命名为K562-C1细胞)作为对照组。实时荧光定量PCR检测各组细胞KLF4mRNA的表达含量;Western blot检测各组细胞KLF4的蛋白相对表达含量;MTS法检测各组细胞的增殖水平;Transwell检测各组细胞的迁移能力。结果 K562-KLF4细胞KLF4 mRNA的相对表达量比2个对照组明显增高(P0.05);与对照组相比,K562-KLF4细胞KLF4蛋白相对表达含量明显增加(P0.05),其过表达率为77.78%;K562-KLF4细胞增殖和迁移能力明显被抑制(P0.05)。结论 KLF4过表达能抑制K562细胞的增殖及迁移能力。     

过表达miR-10b促进肺癌细胞系A549增殖

目的探讨非小细胞肺癌(NSCLC)患者肺癌及癌旁组织中miR-10b(miR-10b)表达水平及miR-10b是否通过调控锌指转录蛋白基因(KLF4)对肺癌细胞系A549恶性化的影响。方法 40例NSCLC患者病理切片,原位杂交检测肺癌及癌旁组织中miR-10b的表达量;对肺癌细胞系A549转染miR-10b mimics后,CCK-8法检测肺癌细胞增殖;real-time PCR及Western blot检测KLF4 mRNA及蛋白水平;软琼脂克隆形成实验检测过表达miR-10b对A549细胞的肿瘤恶性化程度的影响。结果肺癌细胞A549及肺癌组织中miR-10b的表达量分别高于正常肺上皮细胞16HBE及癌旁组织;过表达miR-10b模拟物的A549细胞中,KLF4蛋白水平显著下降(P0.05);过表达的miR-10b可显著增加A549细胞的增殖速度及在软琼脂内的成瘤性。结论 miR-10b在不同类型细胞及组织中具有分布差异性、可能是通过抑制KLF4的表达促进肺癌细胞增殖及恶性化。     

Altered expression of the KLF4 in colorectal cancers

KLF4 is an important regulator of cell proliferation and is maximally expressed in epithelial cells of the gastrointestinal tract. Inactivation of the KLF4 gene by genetic and epigenetic alterations has been reported in colorectal cancers, but the expression pattern of the KLF4 protein has not been studied. Here, to investigate the roles of KLF4 in colorectal carcinogenesis, we examined the expression pattern of the KLF4 protein in 123 colorectal cancers by immunohistochemistry using tissue microarray. Moderate to strong nuclear staining for KLF4 was found in normal colonic mucosa. Interestingly, loss of KLF4 expression was observed in 30 (24.4%) of 123 colorectal cancers. Statistically, loss of KLF4 protein expression was not associated with clinocopathologic parameters, including tumor stage (Bartholomew test, P>0.05), lymph node metastasis, differentiation, tumor location, and tumor size (chi2 test, P>0.05). These results indicate that loss of the KLF4 expression might play a role in tumor development as an early event for a subset of colorectal cancers.     

PDGF-BB对血管平滑肌细胞表型标志物表达的影响

目的:观察血小板源性生长因子BB(PDGF-BB)对血管平滑肌细胞(VSMCs)增殖及分化相关基因表达的影响,探讨其可能的机制。方法:分离体外培养的SD大鼠胸腹主动脉VSMCs,分为空白对照组和不同浓度PDGF-BB处理组。分别采用MTT法、流式细胞术和伤口愈合实验检测PDGF-BB对VSMCs增殖、细胞周期和迁移活性的影响;用W estern b lotting分析检测VSMCs表型标志物的表达;用免疫沉淀和免疫共沉淀分析检测Krüppel样因子4(KLF4)磷酸化及与其它转录因子的相互作用。结果:PDGF-BB促进VSMCs增殖和迁移;上调增殖相关蛋白PCNA的表达,下调增殖抑制蛋白p27、分化相关蛋白SM22α的表达。PDGF-BB诱导KLF4的表达和磷酸化,促进KLF4与NF-κB的相互作用,抑制KLF4与Sm ad3、HDAC2的结合。结论:PDGF-BB可能通过影响KLF4磷酸化及其与不同转录调节因子的相互作用而诱导VSMCs表型转化。     

HISTOGENESIS OF THE INTRADERMAL MELANOCYTIC TUMOR IN BDF1 MICE INDUCED BY TOPICAL APPLICATION OF 9, 10-DIMETHYL-1, 2-BENZANTHRACENE (DMBA) AND 12–0-TETRADECANOYLPHORBOL-13-ACETATE (TPA)

The histogenesis of the intradermal melanocytic tumor induced in the skin of 88 female BDF₁ mice by DMBA and TPA is studied light-and electron-micro-scopically. Four types of melanocytes were found in the mouse skin. [1] The epidermal melanocytes transiently appeared several days after topical application of DMBA. [2] Hair follicles turned into anagen phase and follicular melanocytes became apparent by week 3. [3] After week 3, intradermal melanocytes of the perifollicular melanocytic networks (PFM), which were scattered in untreated mouse skin, proliferated to form the intradermal melanocytic tumors, and 267 tumors, composed of oval melanocytes in medullary growth, were induced in all of the 19 treated mice by week 32. [4] Large dendritic melanocytes located deep in the reticular dermis, which did not participate in the formation of the tumor. Only 3 papillomas were induced in 2 of the 19. Light-and electron-microscopically, there was no evidence of migration of epidermal and hair follicular melanocytes into the dermis. Additionally, melanin-producing activity in the cytoplasm of Schwann cell and perineural epithelium was observed. The histogenesis of the intradermal melanocytic tumor was closely related to the melanocytes of the perifollicular melanocytic network (PFM).     

Silencing KLF16 inhibits oral squamous cell carcinoma cell proliferation by arresting the cell cycle and inducing apoptosis

Krüppel-like factor 16 (KLF16), a member of the Krüppel-like factor (KLF) family, has been extensively investigated in multiple cancer types. However, the role of KLF16 in oral squamous cell carcinoma (OSCC) remains unknown. Thus, we conducted this study to investigate its related mechanism. KLF16 expression in OSCC cell lines was quantified by western blotting. Then, OECM1 and OC3 cells were divided into Blank, siCtrl, siKLF16#1 and siKLF16#2 groups. Subsequently, cell proliferation was detected using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assays, cell migration and invasion were detected with wound healing and Transwell assays, and cell cycle distribution and cell apoptosis were detected via flow cytometry. KLF16, p21, CDK4, Cyclin D1 and p-Rb expression was detected by western blotting. Finally, xenograft models were established in nude mice to observe the in vivo effects of KLF16 on OSCC. KLF16 protein expression was upregulated in OSCC cells. Compared to the cells in the Blank group, the OECM1 and OC3 cells in the siKLF16#1 group and siKLF16#2 group exhibited a sharp decrease in proliferation but a remarkable increase in apoptosis. Moreover, the proportion of cells in the G0/G1 phase notably increased and that in the S phase decreased, with evident decreases in cell invasion and migration. Moreover, KLF16, cyclin-dependent kinase 4 (CDK4), Cyclin D1 and p-Rb protein expression was upregulated, but p21 expression was downregulated. The mice in the siKLF16#1 and siKLF16#2 xenograft model groups exhibited slower tumour growth and smaller tumours with evident downregulation of Ki67 expression compared to the mice in the Blank group. KLF16 expression was upregulated in OSCC cells, and interfering with KLF16 led to cell cycle arrest, inhibited OSCC cell growth and promoted cell apoptosis.     

Histogenesis of the intradermal melanocytic tumor in BDF1 mice induced by topical application of 9,10-dimethyl-1,2-benzanthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA)

The histogenesis of the intradermal melanocytic tumor induced in the skin of 88 female BDF1 mice by DMBA and TPA is studied light-and electron-microscopically. Four types of melanocytes were found in the mouse skin. The epidermal melanocytes transiently appeared several days after topical application of DMBA. Hair follicles turned into anagen phase and follicular melanocytes became apparent by week 3. After week 3, intradermal melanocytes of the perifollicular melanocytic networks (PFM), which were scattered in untreated mouse skin, proliferated to form the intradermal melanocytic tumors, and 267 tumors, composed of oval melanocytes in medullary growth, were induced in all of the 19 treated mice by week 32. Large dendritic melanocytes located deep in the reticular dermis, which did not participate in the formation of the tumor. Only 3 papillomas were induced in 2 of the 19. Light-and electron-microscopically, there was no evidence of migration of epidermal and hair follicular melanocytes into the dermis. Additionally, melanin-producing activity in the cytoplasm of Schwann cell and perineural epithelium was observed. The histogenesis of the intradermal melanocytic tumor was closely related to the melanocytes of the perifollicular melanocytic network (PFM).     

microRNA-204 inhibits cell proliferation in T-cell acute lymphoblastic leukemia by down-regulating SOX4

Background: MicroRNAs (miRNAs) are a group of small non-coding RNAs that play important roles in the pathogenesis of human diseases by negatively regulating gene expression. The aim of this study was to explore the effect of miR-204 on cell proliferation migration and invasion in T-cell acute lymphoblastic leukaemia (T-ALL). Method: miR-204 expression was determined in bone marrow samples from 32 leukemia patients and 32 healthy controls by quantitative real-time PCR (qRT-PCR). The effect of miR-204 on cell proliferation was evaluated by CCK8 assay, cell migration and invasion were evaluated by transwell migration and invasion assays, In addition, the regulation of SOX4 by miR-204 was evaluated by luciferase reporter assay and western blot. Results: our results revealed that miR-204 was low expressed in T-ALL. Cell proliferation assay showed that the cell proliferation ability was inhibited by miR-204 mimics. Moreover, migration and invasion assay suggested that overexpression of miR-204 could significantly suppressed the migration and invasion ability of T-ALL cells. Luciferase reporter assay confirmed that miR-204 directly bound to the 3’ untranslated region of SOX4, and western blot suggested that miR-204 inhibited the expression of SOX4 at the protein levels. Conclusions: Our findings indicated that miR-204 negatively regulates SOX4 and inhibited proliferation, migration and invasion of T-ALL cell lines. Thus, miR-204 might represent a potential therapeutic target for T-ALL intervention.     

KLF4 is a novel candidate tumor suppressor gene in pancreatic ductal carcinoma

Ductal pancreatic carcinoma (DPC) is a deadly disease with an incidence of 9 cases in 100,000 people per year and a mortality rate close to 100%. Allelic losses in the long arm of chromosome 9 are commonly encountered in many human malignancies but no data are yet available about DPC. We screened 40 laser-microdissected DPC samples and 6 pre-invasive lesions for 9 microsatellite mapping markers of region 9q21.3 through 9q34.2. A small overlapping region of deletion, spanning 8 million base pairs, was identified between D9S127 and D9S105. Two genes, RSG3 and KLF4, mapped to 9q31.1 through 9q32, were further investigated. A highly significant association was found between KLF4 gene expression levels and genomic status. Similarly, absence of immunohistochemical expression of KLF4 protein was found in 86.8% cases of DPC (33/38). Overexpression of KLF4 in a human pancreatic carcinoma cell line induced a significant decrease in the proliferation associated with up-regulation of p21 and the down-regulation of cyclin D1. In conclusion, we identified a novel oncosuppressor region located at the 9q 31.1-3 locus that is lost in DPC at high frequency. Loss of KLF4 expression is closely related to the genomic loss, and its restoration inhibits cancer cell proliferation, suggesting a key suppressor role in pancreatic tumorigenesis.     

Notch1 and Notch2 receptors influence progressive hair graying in a dose-dependent manner.

The Notch signaling pathway is involved in diverse biological processes such as cell fate decisions or stem cell maintenance. In this study, we assessed the role of this pathway for melanocyte development and hair pigmentation using RBP-Jkappa, Notch1, and Notch2 conditional knockout mice. Disruption of the Notch pathway by inactivating RBP-Jkappa in the melanocyte lineage using Tyr::Cre mice led to a severe coat color dilution. Similarly, hair graying was observed when Notch1 and/or Notch2 receptors were ablated in melanocytes. This phenotype was proportional to the number of floxed Notch alleles, with the most pronounced effect seen in Tyr::Cre/degrees; Notch1(flox/flox); Notch2(flox/flox) mice. Deletion of Notch1 and/or Notch2 in melanoblasts did not induce a congenital defect. The number of Dct-expressing cells at embryonic stages was not affected, but melanocytes located within the hair matrix progressively disappeared during the first regeneration of the hair follicle. In contrast, non-follicular melanocytes and pigmentation in the dermis and in the choroid were not affected. We suggest that both Notch1 and Notch2 receptors contribute to the maintenance of melanoblasts and melanocyte stem cells, and are essential for proper hair pigmentation.     

lncRNA COLCA1/miR-423-5p/FAM172A分子轴对皮肤鳞状细胞癌A431细胞的增殖和迁移的影响

目的探讨长链非编码RNA(long-chain non-coding RNA, lncRNA)COLCA1在皮肤鳞状细胞癌(cutaneous squamous cell carcinoma, CSCC)组织中的表达及其对细胞增殖和迁移的影响、分子机制。方法采用qRT-PCR法检测COLCA1在正常皮肤组织和CSCC组织、细胞系及人角质形成细胞系中的表达;选择表达最低的细胞系,分别转染空白质粒(对照组)或COLCA1过表达质粒(实验组);采用MTT法和细胞划痕实验分别检测过表达COLCA1对CSCC细胞系增殖和迁移的影响;通过生物信息学法预测COLCA1潜在的分子机制。采用qRT-PCR和Western blot法检测过表达COLCA1对靶基因表达的影响。结果 CSCC组织中COLCA1的表达显著低于正常皮肤组织(1.01±0.20 vs 5.71±0.78,P<0.01)。CSCC细胞系中COLCA1的表达显著低于人角质形成细胞系(P<0.01),A431细胞系中的表达最低(P<0.01)。与对照组相比,过表达质粒可有效促进COLCA1的表达(1.00±0.04 vs 9.31±0.99,P<0.01)。过表达COLCA1可抑制A431细胞的增殖能力和迁移能力(P均<0.01)。COLCA1可能与miR-423-5p互补结合,miR-423-5p可能与FAM172A mRNA互补结合。与对照组相比,过表达COLCA1可抑制A431细胞中miR-423-5p的表达(1.00±0.05 vs0.20±0.05,P<0.01),并促进FAM172A在mRNA和蛋白的表达(P<0.01)。结论 COLCA1在CSCC组织和细胞系中呈低表达,过表达COLCA1可能通过miR-423-5p/FAM172A分子轴抑制CSCC细胞系A431的增殖和迁移能力。     

The role of hyaluronan-binding protein in assembly of pericellular matrices.

Hyaluronan-dependent pericellular matrices or "coats" are expressed by a variety of cell types in culture and modulation of their expression may be important in regulation of cell interactions in vivo during development. Monoclonal antibody IVd4, which recognizes hyaluronan-binding protein with the properties of a hyaluronan receptor, was shown to block formation of these coats by a variety of cells. Using rat fibrosarcoma cells, it was found that the antibody not only blocked initial formation of the coats but also caused their loss when added subsequent to formation. The loss of preformed coats in the presence of antibody occurred at 4 degrees and 37 degrees, implying that the function of hyaluronan-binding protein in coat formation is not in mediating metabolic processes. The antibody also had no significant effect on hyaluronan production by the fibrosarcoma cells. In addition, hyaluronan hexasaccharide, a competitive inhibitor of the interaction between polymeric hyaluronan and its cell surface receptor, was found to inhibit coat formation. Thus it is concluded that a hyaluronan-binding protein with the properties of a hyaluronan receptor is required for pericellular matrix formation.     

MicroRNA-145 suppresses cell migration and invasion by targeting paxillin in human colorectal cancer cells

A number of cancers show increased expression of paxillin which plays a central role in tumor progression, including colorectal cancer. However, the mechanisms causing paxillin upregulation remains unclear. In our study, bioinformatics analyses suggested that paxillin is predicted to be a direct target of miR-145. We firstly identified paxillin as a new target of miR-145 and demonstrated that miR-145 inhibits paxillin expression by binding to the paxillin mRNA 3’UTR. Therefore, we assume overexpression of paxillin induced by suppression of miR-145 may promote cell migration and invasion. We detected the expression of paxillin and miR-145 in human colorectal cancer tissues by real-time quantitative PCR. Higher expression of paxillin and lower expression of miR-145 was observed in colorectal cancer tissues than corresponding paracancerous tissue. Moreover, the expression of paxillin was negatively correlated with miR-145 expression. A dual-luciferase reporter assay was used to confirm that paxillin was a direct target of miR-145. In CRC cell lines, overexpression of miR-145 could downregulate paxillin protein expression levels, and ectopic overexpression of miR-145 mimics or inhibitor could inhibit or promote cell migration, invasion, proliferation and clone formation in vitro. Taken together, these data suggested that miR-145 plays a pivotal role in colon cancer through inhibiting cell proliferation migration and invasion, and miR-145 may serve as a tumor suppressor by targeting paxillin gene.