[摘 要] 目的：观察miR-26b-3p在食管鳞状细胞癌（ESCC）组织中的表达水平及其对ESCC细胞增殖、侵袭和迁移能力的影响，并探讨其分子调控机制。方法: 选取河北医科大学第四医院2018年4月1日至2018年12月25日手术切除的ESCC组织及相应癌旁组织各60例，利用qPCR法检测ESCC组织、癌旁组织和ESCC细胞中miR-26b-3p的表达。选取miR-26b-3p表达水平较低的ESCC细胞TE1和KYSE150，转染miR-26b-3p mimic，并设立阴性对照。利用细胞增殖实验、划痕愈合实验和Transwell实验检测过表达miR-26b-3p对TE1和KYSE150细胞增殖、迁移和侵袭能力的影响。荧光素酶报告基因实验检测miR-26b-3p与STAT3的3'UTR靶点部位的结合情况。随后同时转染miR-26b-3p mimic和pcDNA3.0-STAT3，利用细胞增殖实验、划痕愈合实验和Transwell实验检测STAT3是否可逆转过表达miR-26b-3p对细胞增殖、迁移和侵袭能力的影响。qPCR和WB法检测甲基化酶抑制剂5-Aza-DC对ESCC细胞甲基化的影响和miR-26b-3p与STAT3表达的影响。结果: miR-26b-3p在ESCC组织中的表达低于癌旁组织（P<0.01），其在ESCC细胞TE1、KYSE150和LYSE170中表达水平显著低于人正常食管上皮细胞HEEC（均P<0.01）。与miR-26b-3p NC组相比，miR-26b-3p mimic转染可明显上调TE1和KYSE150细胞中miR-26b-3p的表达（均P<0.01），但可明显抑制两种细胞的增殖、迁移和侵袭能力（P<0.05或P<0.01）。荧光素酶报告基因实验结果表明，在TE1和KYSE150细胞中，miR-26b-3p明显抑制了野生型STAT3载体的荧光素酶活性（P<0.05或P<0.01），而突变型的荧光素酶活性不受影响。同时转染miR-26b-3p mimic和pcDNA3.0-STAT3可部分逆转miR-26b-3p mimic对TE1和KYSE150细胞增殖、迁移和侵袭能力的抑制作用（P<0.05或P<0.01）。5-Aza-DC处理后，TE1和KYSE150细胞中miR-26b-3p表达上调（均P<0.01）、STAT3 mRNA和蛋白水平降低（P<0.05），miR-26b-3p呈现去甲基化状态。结论: miR-26b-3p的启动子高甲基化导致其在ESCC组织和细胞中的表达下调，其作为抑癌因子可通过靶向STAT3而抑制ESCC细胞的增殖、侵袭和迁移能力。

[Abstract] Objective: To investigate the expression of long non-coding RNA (lncRNA) DiGeorge syndrome critical region gene 5 (DGCR5) in esophageal squamous cell carcinoma (ESCC) tissues, and to analyze its relationship with clinicopathological features and prognosis of ESCC patients. Methods: The expression of DGCR5 in ESCC data set from TCGA database was analyzed by bioinformatics method. Sixty pairs of ESCC tissues and para-cancerous tissues resected at the Fourth Hospital of Hebei Medical University from August 2016 to March 2017 were collected for this study. The expression of DGCR5 in ESCC tissues was detected by qPCR. The correlation between the expression of DGCR5 and the clinicopathological features and prognosis of ESCC patients was analyzed. Results: TCGAdatabase analysis showed that the expression of DGCR5 in ESCC tissues was significantly higher than that in normal esophageal tissues (P<0.01). The expression of DGCR5 in ESCC tissues was significantly higher than that in para-cancerous tissues (P<0.01). The expression level of DGCR5 was significantly correlated with TNM staging and lymph node metastasis in ESCC patients (all P<0.05). Kaplan-Meier univariate analysis showed that the 2-year survival rate of ESCC patients with high DGCR5 expression was significantly lower than that of patients with low expression (P<0.05). Conclusion: DGCR5 is highly expressed in ESCC tissues and is closely related to TNM staging, lymph node metastasis and poor prognosis, which may serve as a molecular marker for early diagnosis and prognosis prediction of ESCC.

glioma; U87 cell; U251 cell; MAGE-A1; MAGE-A3; DNA methylation; histone acetylation To detect the expressions of melanoma antigen genes MAGE-A1 and MAGE-A3 in glioma tissues and to explore their clinical significance. Methods: Seventy-eight surgically resected glioma specimens and 15 normal brain tissue samples from donors suffered traffic accidence were collected at the Department of Neurosurgery, the Fourth Hospital of Hebei Medical University between January 2006 and January 2010, and the mRNAexpressions of MAGE-A1 and MAGE-A3 in collected tissues were detected with RT-PCR; their associations with the overall survival of patients were also analyzed.The promoter methylation status of the two genes was observed with methylation specific PCR, and the relationship between the gene expressions and promoter methylation status was analyzed. The expressions of MAGE-A1 and MAGE-A3 genes in U251 and U87 glioma cell lines were detected by RT-PCR before and after the treatment with DNA methyltransferase inhibitor 5-aza-CdR and/or histone deacetylase inhibitor trichostatin A (TSA). Results:The positive expression rates of MAGE-A1 and MAGE-A3 genes in glioma tissues were 65.34% and 38.46%, respectively; however, the two genes were not detected in 15 cases of normal brain tissues.The 5-year overall survival of patients in MAGEA1 positive expression group was shorter than that of negative expression group (P<0.05). There was significant correlation between the mRNA expressions of two genes and their promoter methylation status (all P<0.01). There was no mRNA expressions of MAGEA1 and MAGE-A3 in U87 cells untreated with 5-Aza-CdR and TSA, but a small amount of MAGE-A1 mRNA and MAGE-A3 mRNA were detected in U251 cells. TSA alone could not activate the expression of MAGE-A1 and MAGE-A3 genes. 5-Aza-CdR alone or in combination with TSA could activate the expression of both genes, and the combined effect was better than that of single administration. Conclusion: There are different degrees of MAGE-A1 and-A3 expression in glioma tissues, and the expression of MAGE-A1 is a negative prognostic factor for glioma patients. DNApromoter methylation and histone acetylation are important mechanisms of the activation of MAGE-A1 and MAGE-A3 expression.

Objective: To explore the expressions of melanoma antigen (MAGE) -A9, -A11 and Ki67 in laryngeal squamous cell carcinoma (LSCC) tissues, and to analyze their correlation with clinicopathological features and the prognosisof LSCC patients. Methods: A total of 73 pairs of LSCC tissuesand corresponding para-cancerous tissues resected from LSCC patients, who were treated at the Fourth Hospital of Hebei Medical University from 2012 to 2014,were collected for this study. At the same time, testicular tissues from 3 patients with prostate cancer after castration were selected as positive control. The protein expressions of MAGE-A9, MAGE-A11 and Ki67 in LSCC tissues and its para-cancerous tissues were detected by immunohistochemistry. Results: The expression rates of MAGEA9, MAGE-A11 protein and Ki67 in LSCC tissues were 47.94% (35/73), 49.32% (36/73) and 46.58% (34/73) respectively, which were significantly higher than those in para-cancerous tissues. The protein expressions of MAGE-A9 and MAGE-A11 were correlated with clinical stage and lymphatic metastasis of LSCC (P<0.05). The expression of Ki67LI was correlated with tumor size, clinical stage and lymphatic metastasis of LSCC (P<0.05). The correlation analysis showed that the expressions of MAGE-A9 and MAGE-A11 were positively correlated with Ki67 (r=0.258, P＝0.027; r=0.672, P=0.001). Kaplan-Meier survival curve analysis showed that the survival rates of patients with high expression of MAGE-A9 protein (P=0.009), MAGE-A11 protein (P=0.031) and Ki67LI (P=0.040) were significantly lower than those with low expressions. And the survival time of patients with both high expressions of MAGE-A9 and Ki67LI (P=0.001) or both high expressions of MAGE-A11 and Ki67 (P=0.001) was significantly shorter than that of patients with low expression (both or single). Univariate and multivariate Cox regression analysis further indicated that MAGE-A9 protein (P=0.028) and MAGE-A11 protein (P=0.042) were independent prognostic factors for overall survival of LSCC patients. Conclusion: MAGE-A9, MAGE-A11 and Ki67 are tumor-associated antigens of LSCC, which can be used as prognostic indicators for LSCC.

Objective: To investigate the expressions of programmed death ligand 1（PD-L1）in triple-negative breast cancer (TNBC) and its correlation with angiogenesis. Methods: 120 cases of TNBC patients who underwent surgery in the Fourth Hospital of Hebei Medical University from March 1, 2011 to June 1, 2012 were collected. The tumor tissues of patients were surgically resected and confirmed by pathology. PD-L1 protein expression in TNBC tissues of 120 patients was detected by tissue microarray combined with immunohistochemistry, and its relationship with various clinical indicators was analyzed. Blood vessels and lymphatic vessels were labeled withCD34andD2-40todetectmicrovesseldensity(MVD)andlymphaticvesseldensity(LVD)inTNBC.Results:Thepositiveexpression rate of PD-L1 in the tumor cells and interstitial infiltrating lymphocytes fromTNBC was 56.7% (68/120); No correlation was found between PD-L1 protein expression and the gender, age, histological grade, clinical stage, or tumor size of patients with TNBC (P>0.05), but related to the lymph node metastasis (P<0.05) and vascular thrombus (P<0.05). TNBC with high PD-L1 expression exhibited high incidence of lymph node metastasis and formation of vascular thrombus, and the expression of PD-L1 was positively correlated with MVD (r=0.500, P=0.02) as well as LVD (r=0.662, P=0.01). Log-Rank test showed that the survival time of TNBC patients with positive PD-L1 protein expression was significantly shorter than that of patients with negative expression (P<0.05). Cox multivariate analysis suggested that PD-L1 protein expression could be an independent prognostic factor for TNBC overall survival. Conclusion: PD-L1 plays an important role in TNBC angiogenesis and lymphangiogenesis, and is closely related to TNBC invasion and metastasis; blocking PD1/PD-L1 signal pathway is expected to be an effective new strategy for TNBC treatment.

Objective: To screen related genes of melanoma-associated antigen-A11 (MAGE-A11) in breast cancer cells based on highthroughput DNAmicroarray technology, and to validate from the aspects of quantity and function. Methods: DNAmicroarray was used to screen the differently-expresseddown-stream mRNAs of MAGE-A11 in breast cancercelllines (MCF-7, MDA-MB-231 and BT-549). Cluster analysis was applied on representative genes and quantitative RT-PCR was used to validate. CCK-8, scratch wound healing assay and Transwell assaywere used to detect the effect of MAGE-A11 on the proliferation,migration and invasion of breast cancer cells. Results: Over-expression of MAGE-A11 caused the differential expression of 1608 down-stream genes in 3 breast cancer cell lines, which was associated with various cell functions such as protein ubiquitination,cell proliferation and apoptosis, tumor invasion and metastasis.qRT-PCR validated that the expression of ZNF-451, CENPTJ, CDK13, API5 and LMO7, which were highly expressed in microarray, were also significantly higher than those in control group (P<0.01);in addition, SHPRH, PML, MARK2, LIMA1 and ANGPTL4, which were low-expressed in microarray, were also significantly lower than those in control group (P<0.01). MAGE-A11transfection directly increased the proliferation of breast cancer MCF-7, MDA-MB-231 and BT-549 cells at 72 h (all P<0.01); compared with control group after transfectionexhibited obvious wound healing at 48 h (P<0.05 or P<0.01) and significantly increased trans-membrane cell numbers (all P<0.01). Conclusion: Many differentially expressed genes related to ubiquitination, cell proliferation and apoptosis, tumor invasion and migration were screened in MCF-7, MDA-MB-231 and BT-549 breast cancer cells. Among them, 10 typical differentially expressed genes were identified in terms of quantity and function.

Objective: To investigate the effect of miR-124 on the invasion and migration of esophageal cancer KYSE170 cells by regulating autophagy. Methods: miR-124 mimic was transfected into esophageal cancer KYSE170 cells. Transwell assay was used to detect the change of invasion and migration ability of cells. Dual luciferase reporter gene assay was used to verify the targeted regulation of BECN1 (Beclin1) by miR-124, and Western blotting was used to analyze the expressions of BECN1, P62 and LC3 protein. siRNA targeting BECN1 was transfeted into KYSE170 cells, and then the cell invasion and migration ability was calculated by Transwell assay. The expressions of BECN1, P62 and LC3 protein were detected by Western blotting. miR-124 mimic and BECN1 over-expression plasmid were co-transfected into KYSE170 cells, and then Transwell assay was used to detect the changes of cell invasion and migration ability, and Western blotting to examine the expression levels of autophagy-related gene. Results: The invasion and migration ability of KYSE170 cells were significantly inhibited after transfection with miR-124 mimic (All P<0.05). The expression of autophagyrelated protein P62 was increased, and the expression of BECN1 and LC3 was significantly decreased (All P<0.01); in addition, the activity of luciferase reporter gene was also significantly reduced (P<0.01). Silencing BECN1 expression inhibited the invasion and migration of esophageal cancer KYSE170 cells (P<0.01). However, after co-transfection with BECN1 over-expression plasmids, the effects of miR-124 mimic on the autophagy, invasion and migration of esophageal carcinoma KYSE170 cells were significantly weakened (P<0.01), it was also accompanied with lower P62 expression, and higher LC3 expression (P<0.01). Conclusion: miR-124 mimic can inhibit the invasion and migration of esophageal carcinoma cells. The mechanism may be related to the autophagy-related gene BECN1 expression.

Objective: To investigate the effect of interleukin-8 (IL-8) on esophageal cancer cell line KYSE170, and to preliminarily explore its mechanism. Methods: siRNA targeting IL-8 was in vitro synthesized and transfected into KYSE170 cells by lipofectamine 2000. The efficiency of silencing was determined by Real-time PCR, Western blotting and ELISA. Morphological changes of KYSE170 cells were observed microscopically. Scratch assay was performed to observe the cell migration ability. CCK-8 assay was used to detect the cell proliferation ability. Western blotting was used to detect the expressions of IL-8 receptor and JAK2-STAT3 signaling pathway related proteins. Results: Compared with the negative control group, the mRNA and protein expressions of IL-8 in KYSE170 cells were all significantly decreased after IL-8 silencing (P<0.01), and IL-8 secretion was significantly reduced (P<0.01).After IL-8 gene silencing, the migration capacity of KYSE170 cells was significantly weakened (P<0.01), while no significant changes in cell proliferation was detected. The expression of IL-8 receptor 2 (CXCR2) and transfer-related protein WASF3 were significantly decreased (P< 0.05), while the expression of IL-8 receptor 1 (CXCR1) was not significantly changed; the expressions of p-JAK2 and p-STAT3 protein in JAK2-STAT3 signaling pathway were significantly decreased (all P<0.01). Conclusion: Knock-down of IL-8 inhibits the migration of esophageal cancer KYSE170 cells, and the mechanism may be related with the alteration of CXCR2 and its downstream JAK2STAT3 signaling pathway.

Objective: To evaluate the expression of melanoma antigen A family（MAGE-As）in the peripheral blood of patients with esophageal carcinoma (EC), and to analyze its correlations to the clinicopathological features and the prognosis of EC patients. Methods: mRNA expression of MAGE-As in peripheral blood from 153 EC patients and 30 healthy donors was detected using multiplex semi-nested PCR. In addition, restriction endonuclease treatment was used to determine the expression of MAGE-As family members, including MAGE-A1, A2, A3, A4 and A6. Results: The positive expression of MAGE-As was observed in 30 of 153 EC patients (19.61%) in peripheral blood. The positive expression rate of MAGE-A1, A2, A3, A4, A6 was 10.46% (16/153), 16.34%(25/153), 9.8% (15/153), 11.11% (17/153) and 18.30%(28/153), respectively. Additionally, the expression of MAGE-As was positively associated with clinical stage, lymphatic metastasis and distant metastasis (all P<0.05). The positive expressions of MAGE-As and its sub-type genes were all associated with low 5-year overall survival of ES patients (all P<0.05). Expression of MAGE-As, tumor volume, lymphatic metastasis and distant metastasis can be used as independent prognostic factors for the survival of EC patients (all P<0.01). Conclusion: The expression of MAGE-As in peripheral blood of EC patients was associated with the prognosis of EC, and may be used as an important indicator for the prognosis of esophageal carcinoma.

Objective: To investigate the expression of ciRS-7 in esophageal squamous cell carcinoma (ESCC) and its effect on the cellular proliferation, migration and invasion. Methods: The cancer tissues and paired adjacent normal tissues from 60 ESCC patients treated in the Fourth Hospital of Hebei Medical University between May, 2016 andApril, 2017 were selected for this study. The expressions of ciRS-7 were detected by qRT-PCR. After over-expressing or silencing of ciRS-7, the proliferation of ESCC cell line TE1 was measured by CCK-8 assay; and the migration and invasion were tested by wound healing assay and Transwell invasion assay，respectively. Finally, the effect was validated via animal experiment. Results: CiRS-7 was highly expressed in ESCC tissues (P<0.05), and its expression level was closely related to pathological grade and lymph node metastasis (P<0.05). Over-expression of ciRS-7 significantly increased the proliferation, migration and invasion (all P<0.05) of TE1 cells; while silencing of ciRS-7 remarkably suppressed the proliferation, migration and invasion (all P<0.05). Conclusion: CiRS-7 was up-regulated in ESCC and could enhance ESCC cell proliferation, migration and invasion, suggesting that ciRS-7 could be used as a potential target for the diagnosis and treatment of ESCC.