miRNA-26b-3p regulates proliferation and migration of esophageal squamous cell carcinoma cells by targeting STAT3 / 中国肿瘤生物治疗杂志

@#[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。

Analysis of the population structure and genetic diversity of the red swamp crayfish (Procambarus clarkii) in China using SSR markers

BACKGROUND: Procambarus clarkii produces high-quality, delicious meat that is high in protein, low in fat, and rich in calcium and phosphorus. It has become an important aquatic resource in China. Our objectives are (i) to analyze the level of genetic diversity of P. clarkii populations; (ii) to explore the genetic differentiation (Gst); and (iii) to propose appropriate strategies for the conservation. RESULTS: In this study, Shannon's index (I) and Nei's gene diversity index (H) for P. clarkii were high (I = 0.3462 and H = 0.2325 on average and I = 0.6264, H = 0.4377 at the species level) based on the SSR markers. The expected heterozygosity value of 17 microsatellite loci in 25 crayfish populations was 0.9317, the observed heterozygosity value was 0.9121, and the observed number of alleles per locus was 2.000; and the effective number of alleles per locus was 1.8075. Among the P. clarkii populations, the inbreeding coefficient within populations (Fis) was 0.2315, overall inbreeding coefficient (Fit) was 0.4438, genetic differentiation coefficient among populations (Fst) was 0.3145 and gene differentiation (Gst) was 0.4785 based on SSR analyses. The cluster analysis results obtained by unweighted pair-group method with arithmetic mean (UPGMA) analysis, principal coordinate analysis (PCoA) and STRUCTURE analysis were similar. A mantel test showed that the isolation-by-distance pattern was not significant. CONCLUSIONS: The high Gst among P. clarkii populations is attributed to genetic drift and geographic isolation. The results indicated that more P. clarkii populations should be collected when formulating conservation and aquaculture strategies.

Endothelialization of acellular scaffold from sterilized bovine pericardium / 中国组织工程研究

BACKGROUND: In the research of tissue engineering, there are inevitably various kinds of microorganisms attached to animal tissues, and asepsis is a basic requirement of clinical application of tissue engineering materials. OBJECTIVE: To investigate the effect of 75% ethanol sterilization on the properties and biocompatibility of bovine pericardium. METHODS: Bovine pericardial tissue was sterilized with sterile PBS (control group), PBS containing 1% antibiotic (penicillin/streptomycin/ amphotericin B solution), chlorhexidine and 75% ethanol. LB solid medium was used to evaluate the bactericidal effect of four methods. VB staining was used to evaluate the effect of four sterilization treatments on the tissue structure of bovine pericardium. The cytotoxicity of four sterilized extracts was determined by the CCK-8 assay. The bovine pericardium was sterilized with 75% ethanol and then used to make acellular scaffold, which was co-cultured with human umbilical vein endothelial cells to observe the effect of cell adhesion and endothelialization. RESULTS AND CONCLUSION: (1) The bovine pericardium treated with 75% ethanol and chlorhexidine for 24 hours met the requirements of complete sterilization, and significant colony formation was observed in 1% antibiotic-treated and control groups. (2) VB staining revealed that the collagen fibers of bovine pericardium treated with 75% ethanol, chlorhexidine and 1% antibiotic were arranged in wavy pattern, with compact structure, less elastic fiber content but clear structure.(3) Bovine pericardium treated with 75% ethanol did not affect the proliferation activity of L929 cells, and the cell survival rate within 1-3 days was more than 100%. Chlorhexidine-sterilized bovine pericardium had strong cytotoxicity, leading to cell death. (4) Human umbilical vein endothelial cells grew and adhered normally on the surface of acellular scaffold. During the 20-day implantation period, the highest number of cells adhering to acellular scaffold appeared on days 8-12. These results suggest that 75% ethanol could effectively eliminate all microorganisms attached to the bovine pericardium without affecting the histological integrity and biocompatibility.

Minimal invasive microscopic tooth preparation based on endodontic, periodontal and functional health / 华西口腔医学杂志

Tooth preparation is the primary and core operation technique for dental esthetic restoration treatment, due to its effect of providing restoration space, bonding interfaces and marginal lines for dental rehabilitation after tooth tissue reduction. The concept of microscopic minimal invasive dentistry put forward the issue of conducting high-quality tooth preparation, conserve tooth-structure, protect vital pulp and periodontal tissue simultaneously. This study reviewed the concepts, physiology background, design and minimal invasive microscopic tooth preparation, and in the meantime, individualized strategies and the two core elements of tooth preparation (quantity and shape) are listed.

Screening and identification of MAGE-A11 related genes based on DNAmicroarray / 中国肿瘤生物治疗杂志

@# 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.

Expression and clinical significance of melanoma antigen A gene family in peripheral blood of esophageal carcinoma patients / 中国肿瘤生物治疗杂志

@# 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.

miR-124 regulates autophagy to inhibit invasion and migration of esophageal cancer KYSE170 cells by targeting BECN1 / 中国肿瘤生物治疗杂志

@# 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.

miR-92b suppresses growth and invasion of esophageal cancer Eca109 cells by regulating EZH2 gene expression / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the regulating effects of miR-92b on the expression of EZH2 (enhancer of zeste homolog 2) gene and the proliferation and invasion abilities of esophageal cancer (EC) cells. Methods: Fifteen cases of esophageal cancer tissues that preserved in the research center of the Fourth HospitalAffiliated to Heibei Medical University from January 2016 to January 2017 were selected for this study. The bioinformatics tool was used to predict the possible miRNAs that might target EZH2. The mimics of predicted miRNAs were transfected into human esophageal carcinoma cell lines Eca109, respectively. Then the regulation effect of miRNAs on EZH2 gene expression was validated by real-time PCR, Western blotting and dual luciferase reporter experiment. In the meanwhile, EZH2 over-expression plasmids were co-transfected into esophageal carcinoma Eca109 cells, and the effects of miRNAs and EZH2 expression changes on the proliferation, apoptosis , invasion and migration of esophageal carcinoma cells were detected by CCK-8 method, Flow Cytometry, Transwell Invasion and migration assay, respectively. Results: Bioinformatics analysis showed that miR-92b, let7a and miR-25 could combine with potential binding sites at 3’-terminal non-translation region of EZH2 gene. Real-time PCR results showed that only miR-92b was able to regulate the expression of EZH2, and miR-92b was negatively correlated to EZH2 in esophageal cancer (P<0.01). Compared with mimic-NC, the expression of EZH2 mRNA, protein and luciferase activity in Eca109 cells after miR-92b mimic transfection was significantly down-regulated (both P<0.01). However, miR-92b mimic transfection had no effect on the apoptosis of Eca109 cells. Moreover, the proliferation, invasion and migration of Eca109 cells were significantly inhibited after transfection with miR-92b-mimic (P<0.01). In addition, after co-transfection with EZH2 over-expression plasmids, the effects of miR-92b-mimic on the proliferation, invasion and migration of Eca109 cells were significantly weakened (P<0.01). Conclusion: miR-92b can inhibit the proliferation,invasionandmigrationofesophagealcarcinomacells,anditsmechanismmayberelatedtoitstargetregulationofEZH2. ··

Expression of circular RNA ciRS-7 in tissues of sophageal squamous cell carcinoma and its effect on cell biological characteristics of TE1 cells / 中国肿瘤生物治疗杂志

@#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.

Identification of reference genes for gene expression normalization in safflower (Carthamus tinctorius)

ABSTRACT Safflower (Carthamus tinctorius L., Asteraceae) is an important oil crop and medicinal plant. Gene expression analysis is gaining importance in the research of safflower. Quantitative PCR has become a powerful method for gene study. Reference genes are one of the major qualification requirements of qPCR because they can reduce the variability. To identify the reference genes in safflower, nine candidate genes of the housekeeping genes were selected from the EST library of safflower constructed by our lab: CtACT (actin), CtGAPDH (glyceraldehyde 3-phosphate dehydrogenase), CtE1F4A (elongation factor 1 alpha), CtTUA (alpha-tubulin), CtTUB (beta-tubulin), CtPP2A (serine/threonine-protein phosphatase), CtE1F4A (eukaryotic initiation factor 4A), CtUBI (Ubiquitin), and Ct60S (60S acidic ribosomal protein). Expression stability was examined by qPCR across 54 samples, representing tissues at different flowering stages and two chemotype of safflower lines. We assessed the expression stability of these candidate genes by employing four different algorithms (geNorm, NormFinder, ΔCt approach, and BestKeeper) and found that CtUBI and Ct60S were the highly ranked candidate genes. CtUBI and Ct60S were used as reference genes to evaluate the expression of CtFAD2-10 and CtKASII. Our data suggest CtUBI and Ct60S could be used as internal controls to normalize gene expression in safflower.

VEGF Pathway-targeted Therapy for Advanced Renal Cell Carcinoma: A Meta-analysis of Randomized Controlled Trials / 华中科技大学学报（医学）（英德文版）

Immunotherapy which has been in practice for more than 20 years proves effective for the treatment of metastatic renal cell carcinoma (mRCC).Anti-angiogenesis-targeted therapy has recently been identified as a promising therapeutic strategy for mRCC.This study was aimed to evaluate the effectiveness of vascular endothelial growth factor (VEGF) pathway-targeted therapy for mRCC by comparing its effectiveness with that of immunotherapy.The electronic databases were searched.Randomized controlled trials (RCTs) on comparison of VEGF inhibiting drugs (sorafenib,sunitinib and bevacizumab) with interferon (IFN) or placebo for mRCC treatment were included.Data were pooled to meta-analyze.A total of 7 RCTs with 3451 patients were involved.The results showed that anti-VEGF agents improved progression-free survival (PFS) and offered substantial clinical benefits to patients with mRCC.Among them,sunitinib had a higher overall response rate (ORR) than IFN (47％ versus 12％,P＜0.000001).Bevacizumab plus IFN produced a superior PFS [risk ratio (RR):0.86,95％ confidence interval (CI):0.76-0.97; P=0.01] and ORR (RR:2.19; 95％ CI:1.72-2.78; P＜0.00001) in patients with mRCC over IFN,but it yielded an increase by 31％ in the risk of serious toxic effects (RR:1.31; 95％ CI:1.20-1.43; P＜0.00001) as compared with IFN.The overall survival (OS) was extended by sorafenib (17.8 months) and sunitinib (26.4 months) as compared with IFN (13 months).It was concluded that compared with IFN therapy,VEGF pathway-targeted therapies improved PFS and achieved significant therapeutic benefits in mRCC.However,the risk to benefit ratio of these agents needs to be further evaluated.

Inhibiting PI3K/Akt Pathway Increases DNA Damage of Cervical Carcinoma HeLa Cells by Drug Radiosensitization / 华中科技大学学报（医学）（英德文版）

This study examined the role of PI3K/Akt pathway in radiosensitization of DNA damage of cervical carcinoma cells.The 50% inhibition concentration(IC50)of cisplatin and docetaxel in HeLa cells was detected by Mono-nuclear cell direct cytotoxicity assay(MTT)in vitro.HeLa cells were treated by cisplatin/docetaxel of 10 percent of IC20 alone or combined with LY294002 for 24 h,and then radiated by different doses of X-ray.The cell survival ratio was obtained by means of clone formation.One-hit multi-target model was fitted to the cell survival curve to calculate dose quasithreshold (Dq),mean lethal dose(D0),2Gy survival fraction(SF2)and sensitization enhancement ratio(SER).The pAkt and total Akt expression was detected by Western blotting and DNA damage by neutro-comet electrophoresis.The HeLa cells were randomly divided into 7 groups in terms of different treatments: Control; radiation treatment(RT)group; LY294002+RT group; cisplatin+RT group; docetaxel+RT group; LY294002+cisplatin+RT group; LY294002+docetaxel+RT group.The apoptosis ratio of each group was measured by flow cytometry.The results showed that docetaxel and cisplatin significantly enhanced the phosphorylation of Akt in radiation-treated HeLa cells.The Dq,Do and SF2 in LY294002-contained groups were lower than those in docetaxel or cisplatin+RT group.The SER in the LY294002+docetaxel+RT group was 1.35 times that of the docetaxel+RT group,and that in the LY294002+cisplatin+RT group 1.26 times that of the cisplatin+RT group.The Comet electrophoresis showed that tail distance in the LY294002+cisplatin+RT group or LY294002+do-cetaxel+RT group was longer than in the cisplatin+RT group or docetaxel+RT group.The apoptosis ratio in the LY294002+cisplatin+RT group or LY294002+docetaxel+RT group was higher than in the cisplatin+RT group or docetaxel+RT group.It was concluded that inhibiting PI3K/Akt pathway can increase the effect of docetaxel and cisplatin on the radiosensitivity of HeLa cells and DNA damage resulted from radiation.

Intracranial Hypertension Syndrome in Systemic Lupus Erythema-tosus:Clinical Analysis and Review of the Literature / 华中科技大学学报（医学）（英德文版）

In order to better understand the clinical manifestation of systemic lupus erythematosus (SLE) with intracranial hypertension syndrome (IHS),we analyzed the clinical features and treatment of a typical SLE patient with IHS.SLE is one of the most unpredictable autoimmune diseases in-volving multiple organ systems that is defined clinically and associated with antibodies directed against cell nuclei.IHS is an uncommon manifestation of neuropsychiatric SLE (NPSLE) and is characterized by an elevated intracranial pressure,papilledema,and headache with occasional ab-ducens nerve paresis,absence of a space-occupying lesion or ventricular enlargement,and normal cerebrospinal fluid chemical and hematological constituents.IHS has been reported in a few sporadic cases in patients with SLE worldwide,but rarely has been reported in China.In this study,a 34-year-old female SLE patient with IHS was.reported and pertinent literature reviewed.The clinical presentation,image logical features,and investigatory findings were discussed.