[摘 要] 目的：探究包被蛋白复合体β1亚基（COPB1）在食管鳞状细胞癌（ESCC）中的表达，及其对ESCC细胞恶性生物学行为的影响、作用机制及临床意义。方法：采用2014～2018年间在河北医科大学第四医院生物样本库中82例ESCC组织及癌旁组织，常规培养正常食管鳞状上皮细胞HEEC和食管癌细胞KYSE-150、KYSE-170、Eca109、TE1、KYSE-30、KYSE-450，用转染试剂将pcDNA3.1-vector（空载体）、pcDNA3.1-COPB1载体，si-NC和si-COPB1转染至KYSE-150、TE1细胞中，记为NC、COPB1-OE、si-NC和si-COPB1组。用数据库数据分析COPB1 mRNA在泛癌组织中的表达及其表达与免疫细胞浸润的关系，qPCR法检测ESCC组织和细胞中COPB1、PIK3CB、CD68、CD163、CD206、ARG1、IL-10 mRNA水平表达情况，WB法检测ESCC组织和各组细胞中的COPB1、PI3K、CD68、CD163、CD206、p-AKT蛋白表达，克隆形成实验和MTS实验检测各组细胞的增殖能力，划痕愈合实验和Transwell实验检测各组细胞的迁移和侵袭能力，免疫组织化学染色（IHC）法检测ESCC组织中COPB1和CD206蛋白表达。以人单核细胞白血病细胞（THP-1）构建巨噬细胞模型，用佛波酯（PMA）和IL-3和IL-4和ESCC细胞上清液诱导巨噬细胞转型，用qPCR和WB法检测CD68和CD206m RNA和蛋白的表达。结果：COPB1在泛癌组织和ESCC组织中均呈高表达且与淋巴结转移和TNM分期有关联（均P < 0.01），COPB1高表达的ESCC患者总生存期短（P < 0.05），COPB1是潜在的ESCC的诊断标志物。COPB1在KYSE-150和TE1细胞中也呈高表达（均P < 0.05），过表达或敲减COPB1可明显抑制或促进KYSE-150和TE1细胞的增殖能力、迁移和侵袭能力（均P < 0.05）。COPB1表达变化诱导的差异表达基因主要富集于PI3K/AKT通路（均P < 0.001）, COPB1可促进PI3K/AKT通路的活化（P < 0.05），COPB1高表达可导致M2型巨噬细胞浸润增加（P < 0.05），COPB1高表达促进TAM/M2极化（P < 0.05）。结论：COPB1在ESCC组织中呈高表达，其可激活PI3K/AKT通路及调控肿瘤免疫微环境促进 ESCC发生发展，COPB1有望成为ESCC诊断和预后的生物标志物及治疗靶点。

Objective To observe the effect of self-formulated Tongluo Yizhi formula on memory deficits and endoplasmic reticulum stress (ERS) in rats with vascular dementia (VD) and its mechanism. Methods Fifty SPF-grade Wistar rats were randomly divided into sham surgery group,model group,TLYZF low-dose (12.5 mg/kg/d) group,TLYZF medium dose (25 mg/kg/d) group and TLYZF high-dose (50 mg/kg/d) group,with 10 rats in each group. Except for the Sham group,other rats were subjected to modified bilateral carotid artery ligation to construct VD models. After 4 weeks of TLYZF intervention,Morris water maze experiment was used to evaluate the learning and memory abilities of rats.Hematoxylin eosin staining was used to detect the pathological structure of the hippocampus.Nissl staining was used to detect hippocampal neuronal damage.In situ end transfer enzyme labeling staining was used to detect hippocampal neuronal apoptosis.Enzyme-linked immunosorbent assay was used to detect the content of inflammatory factors in hippocampal tissue.Protein immunoblotting was used to detect apoptosis of hippocampal tissue cells and the expression of ERS related proteins.Results Compared with the Sham group,the average escape latency of the model group was prolonged(t=14.059),the number of crossing the platform was reduced(t=8.534),the hippocampal neurons were disorderly arranged,the morphology was fuzzy,the cytoplasmic vacuolization,the nuclear pyknosis,the inflammatory cell infiltration increased,the number of Nissl bodies decreased(t=17.131),and the neuronal apoptosis rate increased(t=17.701). The contents of tumor necrosis factor-α (TNF-α),interleukin-1 β (IL-1β) and IL-6 increased(t=6.541,6.957,10.014),the expression of Bcl-2 associated X protein (Bax),cleaved caspase-3,glucose-regulated protein 78 (GRP78),phosphorylated endoplasmic reticulum kinase (p-PERK)/PERK,phosphorylated eukaryotic initiation factor 2α (p-eIF2α)/eIF2α,activating transcription factor 4 (ATF4) and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) increased(t=13.548～76.468),while the expression of B-cell lymphoma-2 (Bcl-2) proteinwas decreased (t=39.691),and the difference were statistically significant (all P<0.05),respectively. Compared with the model group,the average escape latency of TLYZF-treated rats was shortened(t=2.476,7.266,11.306),the number of platform crossing was increased(t=2.187,4.471,6.932),the hippocampal neurons were relatively regular,the nucleus was clear,the inflammatory cell infiltration was significantly reduced,the content of Nissl bodies was increased(t=4.359,9.477,11.449),the apoptosis rate of neurons was decreased(t=3.631,6.145,7.580),the contents of TNF-α,IL-1β and IL-6 were decreased (t=2.382～8.293),the expression of Bax(t=4.696,16.250,20.250),cleaved caspase-3(t=15.205,27.000,26.833),GRP78(t=5.918,13.139,13.741),p-PERK/PERK(t=13.416,14.230,17.889),p-eIF2α/eIF2α(t=20.152,39.346,50.750),ATF4(t=12.093,24.395,22.946)and CHOP protein((t=21.592,19.207,22.136)decreased,while Bcl-2 protein increased(t=7.474,20.761,35.350),the differences were statistically significant (all P<0.05),respectively,and the high-dose group had the best therapeutic effect.Conclusion Self-formulated Tongluo Yizhi formula could reduce the apoptosis and inflammatory injury of hippocampal neurons in VD rats,and its mechanism may be related to inhibiting ERS mediated by PERK.

Objective To observe the effect of self-formulated Tongluo Yizhi formula on memory deficits and endoplasmic reticulum stress (ERS) in rats with vascular dementia (VD) and its mechanism. Methods Fifty SPF-grade Wistar rats were randomly divided into sham surgery group,model group,TLYZF low-dose (12.5 mg/kg/d) group,TLYZF medium dose (25 mg/kg/d) group and TLYZF high-dose (50 mg/kg/d) group,with 10 rats in each group. Except for the Sham group,other rats were subjected to modified bilateral carotid artery ligation to construct VD models. After 4 weeks of TLYZF intervention,Morris water maze experiment was used to evaluate the learning and memory abilities of rats.Hematoxylin eosin staining was used to detect the pathological structure of the hippocampus.Nissl staining was used to detect hippocampal neuronal damage.In situ end transfer enzyme labeling staining was used to detect hippocampal neuronal apoptosis.Enzyme-linked immunosorbent assay was used to detect the content of inflammatory factors in hippocampal tissue.Protein immunoblotting was used to detect apoptosis of hippocampal tissue cells and the expression of ERS related proteins.Results Compared with the Sham group,the average escape latency of the model group was prolonged(t=14.059),the number of crossing the platform was reduced(t=8.534),the hippocampal neurons were disorderly arranged,the morphology was fuzzy,the cytoplasmic vacuolization,the nuclear pyknosis,the inflammatory cell infiltration increased,the number of Nissl bodies decreased(t=17.131),and the neuronal apoptosis rate increased(t=17.701). The contents of tumor necrosis factor-α (TNF-α),interleukin-1 β (IL-1β) and IL-6 increased(t=6.541,6.957,10.014),the expression of Bcl-2 associated X protein (Bax),cleaved caspase-3,glucose-regulated protein 78 (GRP78),phosphorylated endoplasmic reticulum kinase (p-PERK)/PERK,phosphorylated eukaryotic initiation factor 2α (p-eIF2α)/eIF2α,activating transcription factor 4 (ATF4) and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) increased(t=13.548～76.468),while the expression of B-cell lymphoma-2 (Bcl-2) proteinwas decreased (t=39.691),and the difference were statistically significant (all P<0.05),respectively. Compared with the model group,the average escape latency of TLYZF-treated rats was shortened(t=2.476,7.266,11.306),the number of platform crossing was increased(t=2.187,4.471,6.932),the hippocampal neurons were relatively regular,the nucleus was clear,the inflammatory cell infiltration was significantly reduced,the content of Nissl bodies was increased(t=4.359,9.477,11.449),the apoptosis rate of neurons was decreased(t=3.631,6.145,7.580),the contents of TNF-α,IL-1β and IL-6 were decreased (t=2.382～8.293),the expression of Bax(t=4.696,16.250,20.250),cleaved caspase-3(t=15.205,27.000,26.833),GRP78(t=5.918,13.139,13.741),p-PERK/PERK(t=13.416,14.230,17.889),p-eIF2α/eIF2α(t=20.152,39.346,50.750),ATF4(t=12.093,24.395,22.946)and CHOP protein((t=21.592,19.207,22.136)decreased,while Bcl-2 protein increased(t=7.474,20.761,35.350),the differences were statistically significant (all P<0.05),respectively,and the high-dose group had the best therapeutic effect.Conclusion Self-formulated Tongluo Yizhi formula could reduce the apoptosis and inflammatory injury of hippocampal neurons in VD rats,and its mechanism may be related to inhibiting ERS mediated by PERK.

[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。

[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。

[摘 要] 目的：检测lncRNA LOC440173在NSCLC组织和细胞中的表达及探讨其对癌细胞恶性生物学行为的影响。方法：选取河北医科大学第四医院生物标本库中2014至2017年手术切除的72例NSCLC患者的癌及癌旁组织标本，应用qPCR法检测NSCLC组织和癌旁组织中，以及6种NSCLC细胞株（H520、H358、A549、HCC827、H1703和H1299）中LOC440173的表达水平；构建LOC440173的敲低及过表达载体，分别转染H520和H1703细胞，应用MTS、克隆形成及Transwell小室迁移和侵袭实验分别检测敲低及过表达LOC440173对NSCLC细胞增殖、迁移及侵袭能力的影响，qPCR法检测LOC440173对于EMT过程相关标志物（E-cadherin、N-cadherin及vimentin）mRNA表达水平的影响，WB法检测其对E-cadherin、N-cadherin蛋白表达的影响。结果：LOC440173在NSCLC组织中的表达明显高于癌旁组织（P<0.01），并与淋巴结转移、组织学分化程度、TNM分期和肿瘤大小有关联（P<0.05或P<0.01）。敲低LOC440173可以抑制H520细胞的体外增殖、迁移和侵袭（P<0.05或P<0.01），过表达LOC440173可显著促进H1703细胞的增殖、迁移和侵袭（P<0.05或P<0.01）。在转录水平上，敲低LOC440173后，E-cadherin的表达水平升高，间充质相关标志物N-cadherin、vimentin的表达水平降低（P<0.05或P<0.01）；而过表达LOC440173后，E-cadherin的表达水平降低，间充质相关标志物N-cadherin、vimentin的表达水平升高（P<0.05或P<0.01）。在转录后水平上，LOC440173负向调节E-cadherin蛋白的表达、正向调节N-cadherin的蛋白表达（均P<0.05）。结论：LOC440173在NSCLC组织中的异常高表达可能与NSCLC的发生发展有关，LOC440173可显著提高NCSCL细胞的体外增殖、迁移、侵袭能力，且其作用机制可能与调控EMT相关基因表达有关。

[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。

[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.

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. ·

Objective: To investigate the expression of lncRNA LINC00886 in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines, and its effects on proliferation, migration and invasion of Eca109 cells. Methods: The cancer tissues and corresponding para-cancerous tissues of 69 ESCC patients were collected in the biological specimen bank of the Fourth Hospital of Hebei Medical University from June 2014 to December 2016; the ESCC cell lines Eca109, TE13, TE1, Kyse150, Yes-2 and Kyse170 were also collected. LINC00886 gene expression in ESCC tissues and cell lines was detected by qPCR. Eca109 cells were transfected with pIRES2-LINC00886 and pIRES2-NC, respectively, and the overexpression efficiency of LINC00886 gene in Eca109 cells was detected by qPCR; MTS, clone formation assay, wound-healing assay and Transwell invasion assay were respectively used to detect the effect of LINC00886 over-expression on proliferation, migration and invasion ability of Eca109 cells. Results: The expression of LINC00886 gene in ESCC tissues was significantly lower than that in para-cancerous tissues (P<0.01), and its expression level was associated with tumor TNM stage and lymph node metastasis (both P<0.05). The expression level of LINC00886 gene in ESCC cell lines was also lower than that of the control group (all P<0.01). Compared with control group, the expression level of LINC00886 gene was significantly higher in Eca109 cells transfected with pIRES2-LINC00886 (both P<0.05). Compared with the control group, LINC00886 overexpression significantly inhibited the proliferation, migration and invasion abilities of Eca109 cells (all P<0.01). Conclusion: The decreased expression of LINC00886 gene may be related to the occurrence and development of ESCC. Over-expression of LINC00886 gene inhibits the proliferation, migration and invasion abilities of ESCC cells.