miR-452-5p promotes the proliferation, invasion and EMT of esophageal cancer KYSE-150 cells via targeting SOX7 / 中国肿瘤生物治疗杂志

@# [摘 要] 目的：检测miR-452-5p在食管鳞状细胞癌（ESCC）中的表达，并探讨其异常表达对食管癌KYSE-150细胞增殖、侵袭能力和EMT进程的影响及其分子机制。方法：收集2012年3月至2015年12月在河北医科大学第四医院就诊的86名ESCC患者的癌组织样本和对应的癌旁组织，用qPCR法检测miR-452-5p及其他相关基因在ESCC组织和细胞中的表达；向KYSE-150细胞中分别转染miR-452-5p mimic或pcDNA3.1-SOX7构建过表达的细胞株。分析miR-452-5p表达与ESCC病理特征和患者5年OS的关系。用MTS、Tanswell法检测miR-452-5p过表达对食管癌KYSE-150细胞增殖、侵袭能力和EMT进程的影响；用双荧光素酶报告基因实验及TOP/FOP报告基因系统检测miR-452-5p与SRY盒转录因子（SOX7）3'UTR区的结合作用及对Wnt/β-catenin通路活化水平的影响。结果：miR-452-5p在ESCC组织中呈明显高表达（P<0.01），并与ESCC患者的淋巴结转移、TNM分期及5年OS密切相关（均P<0.01）。miR-452-5p过表达明显促进食管癌KYSE-150细胞的增殖、侵袭能力及EMT进程（P<0.05或P<0.01）。SOX7是miR-452-5p的直接靶基因，miR-452-5p通过对SOX7的负向调控影响了Wnt通路活化水平（P<0.05或P<0.01），同时，miR-452-5p表达也受Wnt通路活化水平的影响（P<0.05或P<0.01），其可能为Wnt通路下游靶基因。结论：miR-452-5p通过miR-452-5p/SOX7/Wnt/miR-452-5p正反馈环路提高Wnt/β-catenin通路活化水平，进而促进ESCC KYSE-150细胞的增殖、侵袭能力及EMT进程，miR-452-5p有望成为ESCC患者靶向治疗的潜在靶点及预后评估的新型分子标志物。

Clinical significance of LINC00997 expression in gastric cardia adenocarcinoma and its effect on migration, invasion and epithelial-mesenchymal transition of SGC7901 cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：检测LINC00997在胃贲门腺癌（GCA）组织及胃癌细胞中的表达，分析其表达与患者临床病理特征和预后的关系，探讨敲减LINC00997对胃癌SGC7901细胞迁移、侵袭及上皮间质转化（EMT）的影响。方法：基于TCGA和GTEx数据库分析LINC00997在胃癌组织中的表达及其与患者预后的关系。应用qPCR法检测68例GCA组织和相应癌旁组织以及胃癌细胞中LINC00997的表达水平，分析其表达与患者临床病理特征及预后的关系。通过划痕愈合、Transwell侵袭实验分别检测敲减LINC00997对SGC7901细胞迁移和侵袭的影响，qPCR法和WB法检测敲减LINC00997对EMT相关标志物E-cadherin、N-cadherin及vimentin表达的影响。结果：LINC00997在胃癌组织中的表达水平显著高于癌旁组织（P<0.05），且LINC00997高表达组患者的OS及DFS显著低于LINC00997低表达组患者（P<0.01或P<0.05）。在68例在GCA组织中，LINC00997的表达水平显著高于癌旁组织（P<0.01），其表达与患者淋巴结转移、TNM分期及OS相关联（P<0.05或P<0.01）。敲减LINC00997的SGC7901细胞的迁移和侵袭能力均显著降低（均P<0.01），细胞中E-cadherin的表达显著升高，N-cadherin、vimentin的表达均显著降低（P<0.05或P<0.01）。结论：LINC00997在GCA组织和胃癌细胞中高表达，其高表达可能促进了胃癌细胞的迁移、侵袭及EMT进程，有望成为GCA患者预后评估的分子标志物。

Mechanisms of Coreopsis tinctoria Nutt in the treatment of diabetic nephropathy based on network pharmacyology analysis of its active ingredients / Mecanismos de Coreopsis tinctoria Nutt. En el tratamiento de la nefropatía diabética basado en el análisis farmacológico de red de sus principios activos

SUMMARY: Coreopsis tinctoria Nutt. (C. tinctoria Nutt.) can protect diabetic kidneys, but the mechanisms are unclear. This work is to investigate the potential mechanisms of C. tinctoria Nutt. in the treatment of diabetic nephropathy based on network pharmacology analysis of its active ingredients. Twelve small molecular compounds of C. tinctoria Nutt. and targets related to diabetic nephropathy were docked by Discovery Studio 3.0. DAVID database was used for GO enrichment and KEGG pathway analysis. Cytoscape 3.6.1 was used to construct active ingredient-target network. Cell viability was detected with MTT. Glucose consumption was analyzed with glucose oxidase method. Protein expression was measured with Western blot and immunofluorescence. Electron microscopy observed autophagosomes. The core active ingredients of C. tinctoria Nutt. included heriguard, flavanomarein, maritimein, and marein. Twenty-one core targets of the 43 potential targets were PYGM, TLR2, RAF1, PRKAA2, GPR119, INS, CSF2, TNF, IAPP, AKR1B1, GSK3B, SYK, NFKB2, ESR2, CDK2, FGFR1, HTRA1, AMY2A, CAMK4, GCK, and ABL2. These 21 core targets were significantly enriched in 50 signaling pathways. Thirty- four signaling pathways were closely related to diabetic nephropathy, of which the top pathways were PI3K/AKT, insulin, and mTOR, and insulin resistance. The enriched GO terms included biological processes of protein phosphorylation, and the positive regulation of PI3K signaling and cytokine secretion; cellular components of cytosol, extracellular region, and extracellular space; and molecular function of protein kinase activity, ATP binding, and non-membrane spanning protein tyrosine kinase activity. In vitro experiments found that marein increased the expression of phosphorylated AKT/AKT in human renal glomerular endothelial cells of an insulin resistance model induced by high glucose, as well as increased and decreased, respectively, the levels of the microtubule-associated proteins, LC3 and P62. C. tinctoria Nutt. has many active ingredients, with main ingredients of heriguard, flavanomarein, maritimein, and marein, and may exert anti-diabetic nephropathy effect through various signaling pathways and targets.

RESUMEN: Coreopsis tinctoria Nutt. (C. tinctoria Nutt.) puede proteger riñones diabéticos, sin embargo los mecanismos son desconocidos. Este trabajo se realizó para investigar los potenciales mecanismos de C. tinctoria Nutt. en el tratamiento de la nefropatía diabética basado en el análisis de farmacología en red de sus principios activos. Doce compuestos moleculares pequeños de C. tinctoria Nutt. y los objetivos relacionados con la nefropatía diabética fueron acoplados por Discovery Studio 3.0. La base de datos DAVID se utilizó para el enriquecimiento GO y el análisis de la vía KEGG. Se usó Cytoscape 3.6.1 para construir una red de ingrediente-objetivo activa. La viabili- dad celular se detectó mediante MTT. El consumo de glucosa se analizó con el método de glucosa oxidasa. La expresión proteica fue determinada mediante Western blot e inmunofluorescencia. En la microscopía electrónica se observó autofagosomas. Los principales ingredientes activos de C. tinctoria Nutt. incluyeron heriguard, flavanomarein, maritimin y marein. Veintiún de los 43 objetivos potenciales fueron PYGM, TLR2, RAF1, PRKAA2, GPR119, INS, CSF2, TNF, IAPP, AKR1B1, GSK3B, SYK, NFKB2, ESR2, CDK2, FGFR1, HTRA1, AMY2A, CAMK4, GCK y ABL2. Estos 21 objetivos principales se enriquecieron significativamente en 50 vías de señalización. Treinta y cuatro vías de señalización estuvieron estrechamente relacionadas con la nefropatía diabética, de las cuales las principales vías fueron PI3K/ AKT, insulina y mTOR, y resistencia a la insulina. Los términos GO enriquecidos incluyeron procesos biológicos de fosforilación proteica, la regulación positiva de la señalización de PI3K y la secreción de citoquinas; componentes celulares del citosol, región extracelular y espacio extracelular; y la función molecular de la actividad de la proteína quinasa, la unión de ATP y la actividad de la proteína tirosina quinasa que no se extiende por la membrana. Los experimentos in vitro encontraron que la mareína aumentaba la expresión de AKT/AKT fosforilada en células endoteliales glomerulares renales humanas en un modelo de resistencia a la insulina inducida por niveles elevados de glucosa, así como aumentaron y disminuyeron respectivamente, los niveles de las proteínas asociadas a los microtúbulos, LC3 y P62. C. tinctoria Nutt. tiene muchos principios activos, con ingredientes principales de heriguard, flavanomarein, maritimain y marein, y puede ejercer un efecto de nefropatía antidiabética a través de distintass vías de señalización y objetivos.

Marein regulates insulin resistance in diabetic nephropathy mice by inducing autophagy / Marein regula la resistencia a la insulina en ratones con nefropatía diabética induciendo la autofagia

SUMMARY: Marein is the main active substance of Coreopsis tinctoria nutt. It not only has anti-oxidation and anti-tumor effects, but also can lower blood lipid, prevent high blood glucose, improve insulin resistance, inhibit gluconeogenesis and promote glycogen synthesis. However, the exact mechanism of its action is still unclear. Here, we explored the effect and mechanism of Marein on insulin resistance. The mice were divided into db/m, db/db, metformin+db/db, and marein+db/db groups. The body weight and kidney weight were recorded. Serum biochemical and renal function tests were measured after 8 weeks of continuous administration. Kidney tissues were subjected to HE staining, PAS staining, and Masson staining. The effect of marein on PI3K/Akt signal and autophagy pathway was detected by Western blot. After 8 weeks of Marein intervention, the body weight and kidney weight of mice did not change significantly, but the fasting blood glucose and blood lipid levels were significantly reduced than db/db group. Marein significantly improved the insulin resistance index, increased serum adiponectin and improved glucose and lipid metabolism disorders of db/db mice. Moreover, marein improved the basement membrane thickness of glomeruli and tubules, improved glomerular sclerosis and tubular fibrosis, as well as renal insufficiency, thereby protecting kidney function and delaying the pathological damage. Furthermore, marein increased the expression of PI3K and the phosphorylation of Akt/Akt (Ser473), and promoted the expression of LC3II/I, Beclin1 and ATG5. Additionally, it promoted the expression of FGFR1 in the kidney of db/db mice, and promoted the increase of serum FGF21 and FGF23. Marein has a protective effect on the kidneys of diabetic mice. It protects diabetic nephropathy by regulating the IRS1/PI3K/Akt signaling pathway to improve insulin resistance. Therefore, marein may be an insulin sensitizer.

RESUMEN: Marein es la principal sustancia activa de Coreopsis tinctoria nutt. No solo tiene efectos antioxidantes y antitumorales, sino que también puede reducir los lípidos en sangre, prevenir la glucemia alta, mejorar la resistencia a la insulina, inhibir la gluconeogénesis y promover la síntesis de glucógeno. Sin embargo, el mecanismo exacto de su acción aún no está claro. Se analizó el efecto y el mecanismo de Marein sobre la resistencia a la insulina. Los ratones se dividieron en grupos db / m, db / db, metformina + db / db y mareína + db / db. Se registró el peso corporal y el peso de los riñones. Se midieron las pruebas de función renal y bioquímica sérica después de 8 semanas de administración continua. Los tejidos renales se sometieron a tinción HE, tinción PAS y tinción Masson. El efecto de la mareína sobre la señal de PI3K / Akt y la vía de autofagia se detectó mediante Western blot. Al término de 8 semanas de tratamiento con mareína, el peso corporal y el peso de los riñones de los ratones no cambiaron significativamente, pero los niveles de glucosa en sangre y lípidos en sangre en ayunas se redujeron significativamente en relación a los del grupo db / db. Marein mejoró significativamente el índice de resistencia a la insulina, aumentó la adiponectina sérica y mejoró los trastornos del metabolismo de la glucosa y los lípidos de los ratones db / db. Además, la mareína mejoró el grosor de la membrana basal de los glomérulos y túbulos, mejoró la esclerosis glomerular y la fibrosis tubular, así como la insuficiencia renal, protegiendo la función renal y retrasando el daño patológico. Además, la mareína aumentó la expresión de PI3K y la fosforilación de Akt / Akt (Ser473), y promovió la expresión de LC3II / I, Beclin1 y ATG5. Además, promovió la expresión de FGFR1 en el riñón de ratones db / db y el aumento de FGF21 y FGF23 en suero. Marein tiene un efecto protector sobre los riñones de ratones diabéticos. Protege la nefropatía diabética regulando la vía de señalización IRS1 / PI3K / Akt para mejorar la resistencia a la insulina. Por tanto, la mareína puede ser un sensibilizador a la insulina.

LINC01140 regulates the proliferation and invasion of esophageal squamous cell carcinoma Eca109 cells via miR-452-5p/Wnt/β-catenin axis / 中国肿瘤生物治疗杂志

@# [摘 要] 目的： 探讨长链非编码RNA（lncRNA）LINC01140在食管鳞状细胞癌（esophageal squamous cell carcinoma，ESCC）组织及细胞中的表达及其对Eca109细胞增殖与侵袭的影响及其分子机制。方法：选取2012年3月至2015年5月河北医科大学第四医院收治的133例ESCC患者的临床资料和GEPIA数据库中收集的182例ESCC组织及286例食管正常黏膜组织的LINC01140表达数据，以及ESCC细胞系Kyse150、Eca109和TE13。用qPCR法检测癌组织和细胞中LINC01140的表达水平，分析其表达水平与患者临床病理特征及预后的关系。分别将pcDNA3.1-LINC01140、阴性对照（pcDNA3.1-NC）或miR-452-5p mimic及阴性对照（miR-NC）转染到Eca109细胞，MTS、Transwell实验分别检测细胞的增殖与侵袭能力。用双荧光报告基因实验及TOP/FOP报告基因系统检测LINC01140与miR-452-5p的靶向结合作用及LINC01140对Wnt/β-catenin通路活化水平的影响。结果：LINC01140在ESCC组织和细胞中表达均显著下调（均P<0.01），LINC01140低表达与ESCC患者年龄、淋巴结转移、TNM分期及OS密切相关（均P<0.05）。LINC01140过表达明显抑制Eca109细胞的增殖及侵袭能力（均P<0.01）。机制研究表明，LINC01140可能通过竞争结合miR-452-5p影响Wnt/β-catenin信号通路的活化水平继而调控Eca109细胞的恶性生物学行为。结论：LINC01140通过靶向miR-452-5p/Wnt/β-catenin轴促进ESCC细胞的增殖与侵袭能力，其有望成为ESCC患者靶向治疗的潜在靶点及预后评估的标志物。

Expression of forkhead box protein D3 in gastric cardia adenocarcinoma tissues and its effect on biological behaviors of SGC-7901 cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨叉头框蛋白D3（forkhead box protein D3，FOXD3）在胃贲门腺癌（gastric cardia adenocarcinoma，GCA）中的表达及其对SGC-7901细胞生物学行为的影响。方法：从河北医科大学第四医院生物标本库中选取2014年6月至2016年12月手术切除的49例GCA组织及相应癌旁组织标本，qRT-PCR检测FOXD3在GCA组织、癌旁组织以及在5种胃癌细胞系中（BGC-823、SGC-7901、HGC-27、MGC-803及NCI-N87）的表达。向SGC-7901细胞转染pc-DNA3.1-FOXD3或pc-DNA3.1，采用细胞增殖实验、克隆形成实验、划痕愈合实验和Transwell小室侵袭实验分别检测FOXD3过表达对SGC-7901细胞增殖、克隆形成、迁移和侵袭的影响，qRT-PCR及WB法检测细胞转染前后上皮-间质转化（epithelial-mesenchymal transition，EMT）相关分子mRNA及蛋白的表达情况，流式细胞术检测转染前后细胞周期改变。结果：GCA组织中FOXD3 mRNA的表达量明显降低，其表达水平与患者临床分期和淋巴结转移密切关联；FOXD3在胃癌细胞系中的表达均低于正常细胞（均P<0.01）。FOXD3过表达能明显抑制SGC-7901细胞的增殖、克隆形成、迁移和侵袭能力（均P<0.01），提高SGC-7901细胞中E-cadherin的表达水平，减少N-cadherin、β-catenin和vimentin的表达水平（均P<0.01），使细胞周期阻滞在G0/G1期（P<0.01）。结论: FOXD3在GCA组织中的表达明显下调，其过表达可以抑制胃癌细胞的生物学行为，FOXD3可作为抑癌基因为肿瘤治疗提供新思路。

TGF- β induced lncRNA linc01503 promotes proliferation, invasion and EMT process of esophageal squamous cell carcinoma cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the expression of lncRNA linc01503 in esophageal squamous cell carcinoma (ESCC) tissues and cell lines, as well as its effect on the proliferation, invasion and epithelial-mesenchymal transition (EMT) of ESCC cells. Methods: A total of 119 pairs of tumor tissues and corresponding para-cancerous tissues of ESCC patients were obtained from the Fourth Affiliated Hospital of Hebei Medical University between Jan. 2012 and Dec. 2014. The expression of linc01503 in ESCC tissues and cell lines (Kyse150, Kyse170, Eca109, TE1 and TE13) was detected by qPCR. The ESCC cells were transfected with pGPU6-shRNA-linc01503 or treated with TGF-β. The expressions of EMT related genes before and after transfection as well as linc01503 expression before and after TGF-β treatment were detected with qPCR. MTS and Transwell assay were performed to assess the effect of linc01503 on proliferation and invasion of ESCC cells. Results: The expression of linc01503 was significantly elevated in ESCC tissues and cell lines (all P<0.05). High expression of linc01503 was correlated with lymph node metastasis, depth of infiltration, TNM stage and the survival of ESCC patients (all P<0.05). Treatment with TGF-β promoted EMT of ESCC cells and induced a significant up-regulation of linc01503 expression. Knockdown of linc01503 significantly inhibited proliferation and invasion ability of ESCC cells; Meanwhile, the low expression of linc01503 increased the expression of E-cadherin but decreased the expressions of N-cadherin and vimentin (all P<0.05). Conclusion: lncRNA linc01503, as one of the downstream effect genes of TGF- β, promotes the proliferation, invasion and EMT process of ESCC cells.

Screening and verification of key Hub genes in esophageal squamous cell carcinoma based on bioinformatics analysis / 中国肿瘤生物治疗杂志

@#Objective: To screen the Hub genes associated with the occurrence and development of esophageal squamous cell carcinoma (ESCC) and to analyze their biological functions by using various bioinformatics analysis tools. Methods: ESCC chip profile GSE100942 from GEO database was used as study subject; GEO2R tool was used to analyze the data and to screen the differentially expressed genes (DEGs), and the bioinformatics tools (DAVID, String, Cytoscape) were further used to construct protein-protein interaction (PPI) network and identify the key Hub genes. GO and KEGG were used for the biological function enrichment analysis. In the meanwhile, MiRDB was applied to identify the miRNAs that might regulate Hub genes and to construct Hub gene–miRNA network. Importantly, the expression of DEGs and the patient survival were verified by the GEPIA analysis tool. Results: By analyzing GSE100942 database, a total of 1229 DEGs with difference of 2 times and 223 DEGs with difference of 4 times were screened out. In addition, 20 Hub genes, which were all up-regulated in ESCC tissues, were also identified. The functional enrichment analysis showed that these DEGs were mainly enriched in cancer related pathways and involved in cell division and mitotic nuclear division. Among those 20 Hub genes, DLGAP5, BUB1B, TPX2, TTK, CDC20, CCNB2, AURKA and DEPDC1 were identified as 8 key Hub genes that related with ESCC, and involved in many important biological processes, such as cell proliferation, cell cycle and signal pathway. Five Hub genes, CEP55, ECT2, NEK2, DEPDC1 and NUSAP1, were identified to be highly regulated by the miRNA regulatory network. Conclusion: Microarray combined with bioinformatics can effectively analyze the DEGs associated with the occurrence and development of ESCC. The identification of the 20 Hub genes and the 8 key Hub genes can provide theoretical guidance for further research on the molecular mechanism and molecular marker screening of ESCC. ·

Expression of lncRNA NUP50-AS1 in esophageal squamous cell carcinoma tissues and its effect on malignant biological behaviors of Eca109 cells / 中国肿瘤生物治疗杂志

@# Objective: To investigate the expression of long non-coding RNA NUP50-AS1 (lncRNA NUP50-AS1) in esophageal squamous cell carcinomas (ESCC) tissues and cell lines, and to explore its effect on proliferation, migration and invasion of human esophageal cancer Eca109 cells. Methods: 49 pairs of ESCC tissues and corresponding para-cancerous tissues obtained from the Biological Specimen Base of the Fourth Hospital of Hebei Medical University during Jan. 2015 to Jan. 2016 were used in this study. qRT-PCR method was applied to detect the expression of NUP50-AS1 in collected tissues samples and five esophageal cancer cell lines (TE1, TE13, Eca109, Kyse150 and Kyse170). ShRNAs were transiently transfected into Eca109 cells to interfere the expression of NUP50AS1 gene, and finally, sh2-NUP50-AS1 was used for the following experiments. The effect of NUP50-AS1 gene knockdown on the proliferation of Eca109 cells was detected by MTS and colony formation assay; the effect of NUP50-AS1 gene knockdown on the migration of Eca109 cells was detected by scratch test, and the effect on cell invasion was detected by Transwell assay. Results: The expression of NUP50-AS1 in ESCC was correlated with the lymphnode metastasis and TNM stage (all P<0.01). The expression of NUP50AS1 in ESCC tissues was significantly higher than that in corresponding normal tissues (2.003±0.870 vs 1.000±0.000, P<0.05). The expression of NUP50-AS1 in five esophageal cancer cell lines was significantly up-regulated (P<0.05), and it had the highest expression in Eca109 cell line. After transfection, sh2-NUP50-AS1 had the highest transfection efficiency, and knocking down NUP50-AS1 gene significantly inhibited the proliferation, invasion and migration of the Eca109 cells. Conclusion: The expression of lncRNA NUP50AS1 in ESCC tissues was significantly higher than that in the para-cancerous tissues, and correlated with the TNM stage and lymphnode metastasis. The down-regulation of NUP50-AS1 inhibited the proliferation, invasion and migration of esophageal cancer cells. The high expression of NUP50-AS1 gene may be closely related to the occurrence and development of ESCC.