目的 探讨钾通道四聚化结构域5(KCTD5)对胰腺癌细胞的生物学行为的影响.方法 生物信息学检测KCTD5在胰腺癌中184例mRNA及211例蛋白质中的表达水平,观察KCTD5的表达对胰腺癌患者预后的影响,进一步对数据库中所得数据进行单因素Cox回归分析.定量聚合酶链反应(qPCR)、蛋白免疫印迹法检测KCTD5在胰腺癌细胞株中的表达水平.转染KCTD5小干扰RNA至SW1990胰腺癌细胞中,分为si-NC、si-KCTD5-1、si-KCTD5-2组.细胞计数实验(CCK-8)实验检测细胞增殖能力;Transwell实验检测转染后胰腺癌细胞迁移及侵袭能力.结果 生物信息学结果表明KCTD5在胰腺癌中的mRNA及蛋白表达水平显著高于正常组织;单因素分析及生存分析结果表明KCTD5可作为胰腺癌患者一种独立危险因素;qPCR结果表明KCTD5在正常人胰腺导管上皮细胞HPDE中的表达水平为1.10±0.18,在胰腺癌细胞株中的表达水平分别为SW1990(2.44±0.19)、PANC-1(1.65±0.15)、ASPC-1(2.15±0.12),KCTD5 在胰腺癌细胞株中的表达水平明显升高(t=12.84,5.76,12.16,P 均＜0.05);转染后,CCK-8 实验表明 72 h 后 si-KCTD5-1、si-KCTD5-2、si-KCTD5-3 组吸光度值分别为 2.93±0.88、1.76±0.97、3.03±0.99,明显低于对照组(4.20±1.02,t=2.31、4.25、2.02,P 均＜0.05);降低 KCTD5 表达后 Transwell 实验中胰腺癌细胞的迁移能力和侵袭能力明显降低.结论 胰腺癌中表达升高的KCTD5可促进胰腺癌细胞的增殖、迁移和侵袭能力,降低其表达后胰腺癌细胞上述能力降低.

目的 探讨长链非编码RNA HOTAIR在肝细胞癌(HCC)组织中的表达及对HCC细胞奥沙利铂耐药的影响.方法 实时荧光定量反转录聚合酶链反应(RT-qPCR)检测HOTAIR在47例HCC肿瘤组织和2株细胞系的表达水平,分析HOTAIR对HCC患者的总生存期(OS)的影响.应用小干扰RNA(si-HOTAIR)对HOTAIR进行敲低,对照组转染空白质粒(si-NC),流式细胞技术、细胞活力检测试剂盒检测细胞的凋亡比例和奥沙利铂(OxP)的半数抑制浓度(1Cs0)值.结果 HOTAIR在HCC肿瘤组织的表达水平高于癌旁组织,差异有统计学意义(t=5.976,P＜0.001),HOTAIR高表达的患者OS较低表达的患者缩短(P＜0.01).OxP干预HepG2和Huh7细胞,si-HOTAIR 组凋亡率分别为(40.83±0.89)％和(41.30±1.50)％,高于 si-NC 组[(13.40±0.36)％、(15.50±0.80)％],差异有统计学意义(t=28.64,P＜0.001;t=15.46,P＜0.001).HOTAIR 敲低后,OxP 对 HepG2 和 Huh7 细胞的 IC50 分别从(31.64±1.07)μmol/L 和(28.36±0.45)μmol/L下降至(15.09±0.83)μmol/L 和(14.13±1.36)μmol/L,差异有统计学意义(t=12.25,P＜0.01;t=9.97,P＜0.05).结论 HOTAIR在HCC组织中高表达,HOTAIR表达高的患者预后较差,敲低HOTAIR可以提高HCC细胞对OxP的敏感性.

Objective:To investigate the effects of long non-coding RNA Hox transcript antisense RNA (lncRNA HOTAIR) on proliferation and migration ability of pancreatic cancer cells by targeting microRNA-520g-3p (microRNA-520g, miR-520g-3p).Methods:Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of miR-520g-3p and HOTAIR in pancreatic cancer cells. HOTAIR small interfering RNA was transfected into SW1990 pancreatic cancer cells. The normal control (NC) group, siHOTAIR-1 group and siHOTAIR-2 group were set up. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation ability. The scratch healing experiment was used to detect cell migration ability. The dual luciferase reporter gene experiment was used to verify the relationship between miR-520g-3p and HOTAIR targeting regulation. The quantitative data between the two groups were compared by t test. Results:qRT-PCR showed that the level of HOTAIR mRNA in SW1990 was significantly higher than that in human normal pancreatic ductal epithelial cells human pancreatic duct epithelial (HPDE) (3.08±0.33 vs. 1.57±0.17, t=29.830, P<0.01). The expression of miR-520g-3p in SW1990 was significantly lower than that in HPDE (0.93±0.11 vs. 2.63±0.24, t=22.410, P<0.01). HOTAIR small interfering RNA was transfected into SW1990, and at72 h, A value in the NC group was significantly higher than that in siHOTAIR-1 group and siHOTAIR-2 group (3.52±0.99 vs. 1.92±0.23, 2.36±0.58, t=6.340, 5.150, P<0.01). At 48 h after down-regulating the HOTAIR expression, the percentage of wound healing area in the NC group was significantly higher than that in the siHOTAIR-1 group and the siHOTAIR-2 group [(85.37±9.82)% vs. (50.56±5.82)%, (35.28±6.86)%, t=7.760, 14.750, P<0.01]. The dual luciferase reporter gene indicated that miR-520g-3p could significantly reduce the luciferase activity of wild-type HOTAIR vector. After down-regulating the expression level of miR-520g-3p, the proliferation and migration ability of SW1990 cells was significantly increased as compared with that in the control group ( t=13.190, 12.480, P<0.01). Conclusion:HOTAIR can influence the proliferation and migration ability of pancreatic cancer cells by targeting and negatively regulating miR-520g-3p.

Intrahepatic cholangiocarcinoma (ICC) is a highly lethal hepatobiliary neoplasm, which originates from the bile ducts proximal to the second-order division. ICC can be anatomically divided into two subtypes: the large duct type (mucin-production ICC, muc-ICC) and the small duct type (mixed-ICC) origins from hepatic progenitor cells (HPCs). The immunoreactivity of S100P and neural cell adhesion molecule (NCAM) are useful biomarkers to distinguish the two subtypes. In this study, we report a difficult-to-diagnose case of metastatic retroperitoneal tumor of occult hepatolithiasis-associated ICC. Besides, this case was both positive for S100P and NCAM, considered as a rare muc-ICC with the HPCs features. Tumor whole exome sequencing detection results by Genetron (China) revealed that there were 41 gene mutations in this patient. The SMAD4-p.His530ThrfsTer47 and KRAS-p.Gly12Val mutation might promote the occurrence and distant metastasis of the tumor.

目的 探究长链非编码RNA(LncRNA)锌指结构反义转录本1(ZFAS1)靶向微小RNA(miR)-373导致肝癌细胞顺铂(DDP)耐药的机制.方法 HepG2细胞设正常培养组、si-NC组、si-ZFAS1组,实时荧光定量PCR(qPCR)检测细胞中ZFAS1、miR-373表达水平.在si-NC组、si-ZFAS1组基础上分别添加0、50、100、200、300μmol/L DDP处理细胞12 h,CCK-8检测细胞增殖情况,Transwell检测细胞侵袭情况,蛋白免疫印迹检测细胞中基质金属蛋白酶(MMP)2、MMP9蛋白表达水平.双荧光素酶验证ZFAS1与miR-373的靶向关系.在si-ZFAS1组基础上添加inhibitor miR-373,检测细胞中MMP2、MMP9蛋白表达水平.结果 si-ZFAS1组细胞中ZFAS1表达水平均低于正常培养组和si-NC组,miR-373表达水平均高于正常培养组和si-NC组(P<0.05).si-NC组和si-ZFAS1组经不同浓度顺铂处理后细胞增殖率、侵袭数量、侵袭蛋白MMP2、MMP9表达水平基本呈降低趋势;si-ZFAS1组上述指标分别低于其对应浓度的si-NC组(P<0.05).Starbase分析发现,miR-373与ZFAS1存在互补的结合位点并经双荧光素酶验证.si-ZFAS1+inhibitor miR-373组细胞MMP2、MMP9蛋白表达水平高于si-ZFAS1组和si-ZFAS1+inhibitor NC组(P<0.05).结论 ZFAS1可能降低肝癌细胞DDP耐药,其机制可能与调控miR-373有关.

Objective:To detect the expression of receptor for activated C kinase 1 (RACK1) in cholangiocarcinoma cells, and to explore its effect on the biological phenotype of cholangiocarcinoma cells.Methods:Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the expression level of RACK1 in human normal bile duct epithelial cells HIBEC, 2 cholangiocarcinoma cell lines RBE and HUH28. The t test was used to analyze the difference in the RACK1 expression. Lentivirus was used to construct a stable low-expression strain of RACK1, which was divided into sh-RACK1-1, sh-RACK1-2 and negative control (NC-RACK1) group. The transfection efficiency was tested by qPCR. The invasion and migration abilities of cholangiocarcinoma cells were tested by Transwell experiment. T test was used to analyze the migration and invasion ability of cholangiocarcinoma cells after knocking down RACK1. Results:The qPCR results showed that the expression level of RACK1 in RBE and HUH28 cholangiocarcinoma cell lines (3.45±0.92, 2.96±0.33) was significantly higher than that in HIBEC cells (1.73±0.58, t=21.965, 24.153, P<0.01). Western blotting showed that the expression level of RACK1 in RBE and HUH28 cells was significantly higher than that in HIBEC cells. The results of Transwell migration experiment suggested that the number of cells penetrating the membrane in the sh-RACK1-1 and RBE RACK1-2 groups in RBE cells was [(261.89±8.65), (231.07±9.73)/field of view], significantly less than that in the NC-RACK1 group [(430.19±10.15)/field of view, t=30.684, 24.380, P<0.01]. The number of cells penetrating the membrane in the sh-RACK1-1 and sh-RACK1-2 groups in HUH28 cells [(253.57±7.28)/field of view, (210.11±6.09)/field of view] was significantly less than in the NC-RACK1 group [(424.78±8.03)/field of view] ( t=35.442, 36.781, P<0.01). The results of the invasion experiment showed that the number of cells in the sh-RACK1-1 and sh-RACK1-2 groups in RBE cells was [(52.15±1.43) and (60.58±2.01)/field of view] was significantly lower than that in NC-RACK1 group [(125.39±4.03)/field of view, t=9.935, 12.566, P<0.05]. The number of cells passing through the membrane in the sh-RACK1-1 and sh-RACK1-2 groups [(60.91±1.30) and (51.19±0.96)/field of view] in HUH28 cells was less than in the NC-RACK1 group [(150.88±7.28)/field of view] ( t=13.554, 15.328, P<0.01). Conclusion:RACK1 is up-regulated in cholangiocarcinoma cells. The down-regulation of the RACK1 expression can inhibit the migration and invasion of cholangiocarcinoma cells. RACK1 can promote the occurrence and development of cholangiocarcinoma.

Objective:To explore the expression and clinicopathological significance of vacuolar protein sorting 72 (VPS72) in liver cancer tissues and corresponding adjacent tissues, and observe its influence on cell proliferation and migration.Methods:From September 2018 to September 2020, 60 cases of liver cancer tissues and corresponding adjacent tissues from Henan University Henan Provincial People′s Hospital were selected. The liver cancer tissues were divided into center group, and the specimens 2 cm away from the edge of the cancer tissue served as the paracancerous group. Western blotting and immunofluorescence methods were used to detect the expression level and distribution of VPS72 protein in center group and paracancerous group, and its relationship with age, gender, tumor size, number, stage, and degree of differentiation, the presence and absence of vascular tumor thrombus, capsule invasion and other clinicopathological parameters was analyzed. human hepatoma cells-7 (Huh-7) cells with VPS72 silencing by small interfering RNA were divided into experimental group and control group. The scratch assay and Transwell assay were used to verify the effect of VPS72 on the ability of migration. Cell counting kit-8 (CCK-8) experiment and plate cloning experiment were done to detect the ability of proliferation in Huh-7 cells.comparisons between groups were performed by paired-sample t and χ2 test. Results:The relative expression of VPS72 in cancer tissues was (0.900±0.511), significantly higher than that in adjacent tissues (0.729±0.643) ( t=2.027, P<0.05). The expression of VPS72 was significantly correlated with tumor differentiation, size, vascular tumor thrombus, cell proliferation antigen-67 (Ki-67) and staging ( χ2=5.621, 4.855, 4.275, 8.418, 7.202, P<0.05). The scratch assay showed that the number of migrating cells at24 and 48 hin the experimental group [(38.330±3.512), (66.670±5.508)] was significantly less than that in the control group [(78.670±2.517), (165.670±5.033)] ( t=14.162, 16.275, P<0.05). Transwell experiment showed that the number of invasive cells in the experimental group (32.333±3.512) was significantly les than that in the control group (52.667±2.082) ( t=7.625, P<0.05). The CCK-8 experiment showed that the absorbance (A) value (0.235±0.047) at 0 h in the experimental group had no significant difference from the control group (0.237±0.068) ( t=0.020, P>0.05). The A values at24 and 48 h in the experimental group [(0.539±0.300), (0.815±0.038)] were significantly lower than those in the control group [(0.683±0.100), (1.415±0.131)] ( t=4.654, 7.373, P<0.05). The plate cloning experiment showed that the number of cell clusters in the experimental group (50.222±6.222) was significantly reduced as compared with the control group (71.560±7.038) ( t=7.875, P<0.01). Conclusion:The expression of VPS72 protein in liver cancer tissue is obviously high, and it is obviously related to clinicopathological parameters. Silencing the expression of VPS72 can significantly inhibit the ability of proliferation and migration of Huh-7 cell lines.

Objective:To observe the expression of B-cell lymphoma 6 (BCL6) corepressor (BCOR) in hepatocellular carcinoma (HCC) tissues and its influence on the proliferation and migration of HCC cell.Methods:Western blotting was used to detect the expression of BCOR in 60 cases of HCC tissues and their adjacent normal tissues. The BCOR overexpression vector and empty vector were constructed and stably transfected into HCC cells by lentivirus packaging to form an experimental group and a control group respectively. The expression of BCOR was detected by immunoblotting assay. The proliferation of experimental group and control group after transfection was detected by cell counting kit-8 (CCK-8) assay. The migration ability was detected by scratch test. The ability of invasion was detected by Transwell assay. The t test was used for comparison between groups. Results:The expression level of BCOR in paracancer tissue was (1.316±0.642) times higher than that in HCC tissues and the difference was statistically significant ( t=2.689, P<0.05). Western blotting results showed that BCOR expression in HCC cells was (1.867±0.661) times lower than that in normal human liver cells, and the difference was statistically significant ( t=2.941, P<0.01). The expression level of BCOR in the experimental group was (3.038±1.007) times higher than that in the control group, and the difference was statistically significant ( t=7.246, P<0.01). The CCK-8 assay showed that the absorbance value in the experimental group (0.604±0.043) had no significant change at 0 h compared with that in the control group (0.619±0.014), and the difference was not statistically significant ( t=0.934, P>0.05). The absorbance values in the experimental group at 24, 48 and 72 h were (0.644±0.028), (0.788±0.097) and (1.139±0.253) respectively, significantly lower than those in the control group [(0.860±0.057), (1.250±0.282) and (1.934±0.326), t= 9.545, 4.385 and 5.444, P<0.01]. The scratch test showed that the number of migrating cells in the experimental group at 24 48 and 72 h [(128.333±2.082), (230.667±2.517) and (324.667±4.509)] was significantly less than that in the control group [(161.333±4.933), (415.667±1.528) and (686.667±8.505)], and the difference was statistically significant ( t = 10.675, 108.844 and 65.134, P<0.05). The Transwell experiment showed that the number of perforating cells in the experimental group was (56.833±9.368), significantly less than that in the control group (207.333±27.267), and the difference was statistically significant ( t=12.786, P<0.05). Conclusion:The expression of BCOR in HCC tissues is lower than that in normal tissues. Overexpression of BCOR can inhibit the proliferation and migration of HCC.

Various surgical methods impact the prognosis of patients with hepatocellular carcinoma (HCC) differently. However, clinical guidelines remain inconsistent and the relative importance of predictors of survival outcomes requires further evaluation. The present study aimed to rank the importance of predictive factors that impact the survival outcomes of patients with HCC and to compare the prognosis associated with different surgical methods based on data obtained from the Surveillance, Epidemiology and End Results database. To achieve these aims, the present study used a random forest (RF) model to detect important predictive factors associated with survival outcomes in patients with HCC. Cox regression analysis was used to compare different surgery methods. The variables included in the Cox regression model were selected based on the Gini index calculated by the RF model. Using the RF model, the present study demonstrated that surgery method, tumor size and age were the first, second and third most important factors associated with HCC prognosis, respectively. Overall, patients who underwent local tumor destruction [(hazard ratio (HR)=0.48; 95% confidence interval (CI), 0.45-0.51; P<0.001)], wedge or segmental resection (HR, 0.31; 95% CI, 0.29-0.33; P<0.001), lobectomy (HR, 0.29, 95% CI, 0.27-0.31; P<0.001) or liver transplantation (HR, 0.16; 95% CI, 0.14-0.17; P<0.001) demonstrated improved overall survival time compared with those treated with surgery, with a gradual decreasing trend observed in HRs. The present study demonstrated that the surgical method used is the most important predictor of the survival outcomes of patients with HCC. Liver transplantation resulted in the best prognosis for patients with HCC, except for those with undifferentiated tumors or distant metastasis.

High rate of glycolysis supports hepatocellular carcinoma (HCC) cell growth even in a hypoxic environment. However, the mechanism underlying glycolysis under hypoxia remains largely unknown. Long noncoding RNAs (lncRNAs) play essential roles in regulating glucose metabolism in cancers. This study aimed to explore the role of lncRNA homeobox transcript antisense RNA (HOTAIR) in HCC glycolysis under hypoxia. Thirty-eight HCC patients were recruited. HepG2 and Huh7 cells were used for study in vitro. The expression levels of HOTAIR, microRNA-130a-3p (miR-130a-3p) and hypoxia inducible factor 1 alpha (HIF1A) were measured by quantitative real-time polymerase chain reaction and western blot, respectively. The glycolysis under hypoxia (1 % O2) condition was investigated by glucose consumption, lactate production and hexokinase 2 (HK2) level. The target interaction between miR-130a-3p and HOTIR or HIF1A was analyzed by bioinformatics analysis, luciferase assay, RNA pull-down and RNA immunoprecipitation. We found that HOTAIR expression was enhanced in HCC tissues and cells. Under hypoxia condition, HOTAIR expression was increased and its knockdown inhibited glycolysis in HCC cells. HOTAIR was validated as a decoy of miR-130a-3p and miR-130a-3p deficiency reversed the suppressive effect of HOTAIR silence on glycolysis under hypoxia. HIF1A was indicated as a target of miR-130a-3p and miR-130a-3p overexpression repressed glycolysis under hypoxia by targeting HIF1A. Moreover, HIF1A expression was regulated by HOTAIR and miR-130a-3p. In conclusion, knockdown of HOTAIR suppressed glycolysis by regulating miR-130a-3p and HIF1A in HCC cells treated by hypoxia, elucidating a novel mechanism in HCC glycolysis.

Objective To examine the expression of microRNA (miRNA,miR)-19b in pancreatic ductal adenocarcinoma (PDAC) cell lines,and the functional role of miR-19b in proliferation,migration and invasion of PDAC cells.Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression level of miR-19b in human pancreatic ductal epithelial cell line (HPDE),human PDAC cell lines (PANC1,MiaPaCa2 and BxPC3) and huamn PDAC metastasis cell line (COLO357) and its fast-growing variant FG.The expression of miR-19b was inhibited or overexpressed in PDAC cell lines.Transwell assay and Matrigel assay were carried out to examine the migration and invasion capabities of PDAC cells with miR-19b silencing or overexpressing.Methyl thiazol tetrazolium (MTT) and colony formation assays were performed on PDAC cell lines with miR-19b silencing or overexpression.Western blotting was used to detect the expression of proliferation markers (p21 and p27) in PDAC cell lines after miR-19b was silenced or overexpressed.Results The results of RT-qPCR showed the expression level of miR-19b in human PDAC cell lines PANC1,MiaPaCa2,BxPC3 and metastatic cell line COLO357 and FG (198.45 ± 21.22,221.37 ± 18.59,182.51 ± 13.48,317.64 ± 15.20,331.34± 17.18) was significantly higher than that in normal human pancreatic ductal HPDE (98.21 ± 14.54,P ＜ 0.05).Transwell cell migration assay and Matrigel cell invasion assay showed that the migration and invasion ability of FG cells after miR-19b silencing [(22.54 ± 2.87) ％,(28.87 ± 5.62) ％] was significantly decreased as compared with the control group [(99.65 ± 3.07) ％,(98.73 ±4.38)％,P ＜0.01].In contrast,the migration and invasion ability of BxPC3 cells overexpressing miR-19b [(317.53 ± 10.21) ％,(286.77 ± 12.83) ％] was significantly higher than that of the control group [(97.89 ±5.31)％,(98.25 ±7.57)％,P ＜0.01].The colony formation and MTT assays showed that the proliferation ability of PDAC cells after miR-19b was silenced was significantly decreased as compared with the control group (P ＜ 0.01).The result of Western blotting showed that the expression of proliferation markers (p21 and p27) in PDAC was increased by (3.25 ± 0.27) and (5.38 ±0.33) times respectively as compared with the control group after miR-19b silencing (P ＜0.01).When miR-19b was overexpressed,the expression of p21 and p27 decreased by (2.68 ±0.15) and (3.27 ±0.27) times respectively as compared with the control group (P ＜ 0.01).Conclusion MiR-19b could promote the migration and invasion of PDAC cells and increase the proliferation capability.

Objective To investigate the expression of S-phase kinase-associated protein 1 (SKP1) in intrahepatic cholangiocarcinoma and to investigate its effects on proliferation,migration and invasion of intrahepatic cholangiocarcinoma.Methods Western blotting assay was used to determine the expression difference of SKP1 in intrahepatic cholangiocarcinoma and normal tissues.The SKP1 interference vector and empty vector were constructed and stably transfected into intrahepatic cholangiocarcinoma cells by lentivirus packaging to form an experimental group and a control group.The expression of SKP1 was detected by immunoblotting assay.The proliferation of experimental group and control group after transfection was detected by cell counting kit-8 (CCK-8) assay.The migration ability was detected by scratch test.The invasion ability of Transwell was detected by Transwellassay.Results The expression of SKP1 in intrahepatic cholangiocarcinoma was (1.701 ± 0.215) times,and the difference was statistically significant (t =8.512,P ＜ 0.05).Lentiviral transfection of RBE cells decreased the expression of SKP1,and the Western blotting assay decreased the experimental group by (3.632 ± 0.335) times,and the difference was statistically significant (t =11.376,P ＜ 0.01).The CCK-8 experiment showed that the relative survival rates of the experimental group at 24,48,and 72 hours were (53.136 ± 0.314)％,(42.661 ± 0.248) ％ and (35.037 ± 0.203) ％,respectively,and the differences were statistically significant (t =8.594,8.899,9.210,P ＜ 0.01).The scratch test showed that the number of migrated cells in the experimental group at 24 and 48 hours (41.198 ±3.092) and (64.773 ±6.437) were significantly lower than those in the control group (113.487 ± 3.514) and (168.767 ± 8.151),and the difference was statistically significant (t =5.430,6.224,P ＜ 0.05).The Transwell experiment showed that the number of perforated cells in the experimental group was (34.185 ± 3.124),which was significantly lower than that in the control group (121.234 ± 6.814),and the difference was statistically significant (t =9.918,P ＜ 0.05).Conclusion The expression of SKP1 in intrahepatic cholangiocarcinoma is higher than that in normal tissues.Decreasing SKP1 expression can inhibit the proliferation,migration and invasion of intrahepatic cholangiocarcinoma.

Objective To determine whether G protein-coupled receptor 30 (GPR30) agonist G-1 can inhibit the activation of nucleotide oligomerization domain-like receptor family,pyrin domain containing 3 (NLRP3) inflammasomes and the maturation of interleukin-1β (IL-1β).Methods The peritoneal macrophages were extracted from C57BL/6,and then pretreated with 10,30,50 μmol/L G-1.After induction with 50 ng/ml lipopolysaccharide (LPS),macrophages were collected.Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression of NLRP3,apoptosis-related microparticle protein (ASC),and IL-1β,and the expression levels of NLRP3,ASC and pro-cysteinyl aspartate-specific protease (Caspase)-1 proteins were detected by Western blotting.The macrophages were pretreated with 1,5,15,30,50 μmol/L G-1,and cells were induced with 50 ng/ml LPS and 5 mmol/L adenosine triphosphate (ATP).The levels of IL-1β and TNF-α in supernatants were determined by enzyme linked immunosorbent assay (ELISA).Results There was no significant difference in the expression of NLRP3,ASC and IL-1β mRNA in macrophages between LPS group and G-1 plus LPS group.There was no significant difference in the expression of NLRP3,ASC and pro-Caspase-1 proteins between LPS group and G-1 plus LPS group.The ELISA results showed that with the increase of G-1 concentration,the content of IL-1β (3 062.42 ± 12.10,1 709.29 ± 25.31,653.27 ± 35.66,144.23 ± 4.18,98.40 ± 2.02) decreased gradually in G-1 plus LPS group as compared with the LPS + ATP group (2 714.73 ± 107.05),and the difference was statisti cally significant when G-1 concentration ≥5 μmol/L (P ＜0.05),while the level of TNF-α in G-1 plus LPS group [(361.93 ± 10.66),(394.62 ± 20.46),(444.17 ± 14.80),(444.73 ± 19.21) and (366.41 ±27.04)] showed no significant difference from the LPS + ATP group (412.38 ± 1.59) (P ＞ 0.05).Conclusion GPR30 agonist G-1 can specifically inhibit the activation of NLRP3 inflammasome and the maturation of IL-1β.

Objective To study the effects of puerarin on the proliferation and apoptosis of human cholangiocarcinoma (RBE) cells and the expression of related genes.Methods RBE cells cultured were treated with puerarin at different concentrations (0,100,250,500,1 000,1 500 μg/ml).And then methyl thiazol tetrazolium (MTr) assay was used to measure cell viability.Flow cytometry (FCM) was used to detect the apoptosis of RBE cells treated with puerarin at (500,1 000 μg/ml) concentrations.The expression levels of B cell lymphoma/leukemia-2 (bcl-2) and bcl-2 associated X protein (bax) genes were detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR).Western blotting was used to detect the expression of apoptosis-related proteins.Results MTT assay revealed that puerarin (500,1 000,1 500 pg/ml) inhibited the proliferation of RBE cells in a time-and dose-dependent manner,with inhibition rate at 12,24 and 48 h being (8.14 ± 1.85) ％,(12.57 ± 3.10)％ and (18.26 ±2.74)％ (P=0.000,0.005,0.003),(22.38±2.77)％,(29.14 ±3.85)％and (37.43±2.11)％ (P=0.005,0.007,0.002),and (35.47 ±3.75)％,(40.81 ±2.52)％ and (52.29 ± 3.98) ％ (P =0.007,0.000,0.002) respectively.After RBE cells were treated with puerarin at (500,1 000 μg/ml) for 24 h,the apoptosis rate [(14.21 ± 2.53)％ and (22.87 ± 2.74)％] was increased greatly as compared with the control group [(3.246 ± 2.25) ％] (P =0.013,0.008).The expression of bax was up-regulated (2.37 ± 0.17 and 3.78 ± 0.41) times as compared with the control group (0.98 ± 0.04) (P =0.005,0.003),and the expression of bcl-2 was down-regulated (1.57 ± 0.14 and 2.86 ±0.55) times as compared with the control group (0.99 ±0.03) (P =0.002 and 0.008).The expression levels of Cleaved-Caspase-8,Cleaved-Caspase-9 and bax protein were increased (P =0.015,0.003 and 0.008) and those of bcl-2 protein were decreased (P =0.021).Conclusion Puerarin can inhibit the proliferation of RBE cells and promote the apoptosis.

Objective To analyze the expression of RNA-RGD1566401 (long non-coding RNA-RGD1566401,lncR-RGD1566401) in rat pancreatic acinar cells (AR42J) and the effect of lncR-RGD1566401 on apoptosis of AR42J cells.Methods The GFP LncR-RGD1566401 overexpressed lentivirus and special long non-coding RNA (lncRNA) Smart Silencer were designed and transfected into AR42J cells repectively for 24 h.The apoptosis of AR42J cells in different groups was established by inducing cells with 100 nmol/L Caerulin for 24 h.The flow cytometry was used to detect the apoptosis of AR42J cells in different groups.Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression level of lncR-RGD1566401 in AR42J cells of different groups after transfection.The expression of cysteinyl aspartate-specific protease (Caspase)-3 and p53 was detected by Western blotting.Results The apoptosis rate [(19.34 ± 0.54) ％] of Caerulin-induced AR42J cells was elevated as compared with the control group [(7.23 ± 0.33)％].The expression of lncR-RGD1566401 in the AR42J cells was increased by (5.43 ±0.31) times in the experimental group after treatment with Caerulin and the levels of Caspase-3 and p53 were increased obviously.LncR-RGD1566401 level was elevated by (8.24 ± 0.22) times after AR42J cells were transfected with lenti-lncRNA as compared with the vehicle groups (P =0.006).The AR42J cells showed more tendency to apoptosis [(38.20 ±0.37)％] than the vehicle groups [(22.60 ±0.96)％,P =0.016] and the levels of Caspase-3 and p53 were increased obviously.The AR42J cells that were transfected with Silencer-lncRNA expressed the lower level of IncR-RGD1566401 about [(0.54 ± 0.14),P =0.003] times and less apoptosis [(13.60 ±0.33)％] than the Silencer-NC groups [(19.60 ±0.46)％,P =0.012].Conclusion LncR-RGD1566401 is associated with the apoptosis of AR42J cells,and up-reg ulation of LncR-RGD1566401 can promote the apoptosis of AR42J cells.

AIM To elucidate the underlying mechanism that microRNA-22 (miR-22) promotes the apoptosis of rat pancreatic acinar cells (AR42J) and the elements that regulate the expression of miR-22. METHODS One hundred nanomoles per liter of caerulein (Cae) was administrated to induce the apoptosis of AR42J cells and the apoptosis rate was detected by flow cytometry analysis. An amylase assay kit was used to measure the amylase expression level in the supernatant. Quantitative real-time PCR (qRT-PCR) was adopted to measure miR-22 expression. We used online tools to predict the potential transcription promoter of miR-22 and the binding sites, which was further identified by using luciferase reporter analysis, chromatin immunoprecipitation (ChIP) and ChIP-qPCR assays. Then, a mimic of miR-22, Nr3c1 plasmid encoding the glucocorticoid receptor (GR), and si-Nr3c1 were used to transfect AR42J cells, respectively. The mRNA expression of miR-22, Nr3c1, and Erb-b2 receptor tyrosine kinase 3 (ErbB3) was confirmed by qRT-PCR and the apoptosis rate of AR42J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of ErbB3, GR, PI3k, PI3k-p85α, Akt, p-Akt, Bad, Bax, Bcl-xl, Bcl-2, and cleaved caspase3. RESULTS After inducing apoptosis of AR42J cells in vitro, the expression of miR-22 was significantly increased by 2.20 ± 0.26 and 4.19 ± 0.54 times, respectively, at 3 h and 6 h in comparison with the control group. As revealed by qRT-PCR assay, the expression of miR-22 was 78.25 ± 6.61 times higher in the miR-22 mimic group relative to the miRNA control group, accompanied with an obviously increased acinar cell apoptosis rate (32.53 ± 1.15 vs 18.07 ± 0.89, P = 0.0006). The upregulation of miR-22 could suppress its target gene, ErbB3, and the phosphorylation of PI3k and Akt. Furthermore, we predicted the potential transcription promoter of miR-22 and the binding sites using online tools. Luciferase reporter analysis and site-directed mutagenesis indicated that the binding site (GACAGCCATGTACA) of the GR, which is encoded by the Nr3c1 gene. Downregulation of the expression of GR could upregulate the expression of miR-22, which further promoted the apoptosis of AR42J cells. CONCLUSION GR transcriptionally represses the expression of miR-22, which further promotes the apoptosis of pancreatic acinar cells by downregulating the downstream signaling pathway.

针对格上基于多身份的全同态加密方案(mIBFHE)中陷门函数低效的问题,提出一种改进的格上mIBFHE方案.首先利用MP12陷门函数结合对偶RegeV算法构造出一种可转化的基于身份的加密(IBE)方案,并构造出一种支持标准模型下IBE方案转化的Mask系统;然后基于该系统利用特征向量思想将构造出的IBE方案转化为mIBFHE方案.对比分析结果表明,新方案较同类方案在陷门生成和原像采样阶段均有效率提升,且格的维数、密文和运算密文尺寸等明显缩短.在标准模型下,方案的安全性归约至格上容错学习问题的难解性,并包含严格的安全性证明.

Objective To examine the expression of miR-19b in pancreatic ductal adenocarcinoma (PDAC) cell lines, and invetigate the functional role of miR-19b in proliferation, migration and invasion of PDAC cells. Methods Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression level of miR-19b in human pancreatic ductal epithelial cell line (HPDE), human PDAC cell lines (PANC1, MiaPaCa2 and BxPC3) and huamn PDAC metastasis cell line COLO357 and its fast-growing variant FG. PDAC cell lines stably over-expressing of low expressing miR-19b were constructed. Transwell assay and Matrigel assay were carried out to examine the migration and invasion capabities of PDAC cells with miR-19b down-regulated or up-regulated. Methyl thiazol tetrazolium (MTT) and colony formation assay were performed on PDAC cell lines with miR-19b silenced or overexpressed. Results The results of RT-qPCR showed the expression level of miR-19b in human PDAC cell lines PANC1, MiaPaCa2, BxPC3 and metastatic cell line COLO357 and FG (198.45±21.22, 221.37±18.59, 182.51±13.48, 317.64±15.20, 331.34±17.18) was significantly higher than that in HPDE (98.21±14.54), (P=0.003, 0.005, 0.021, 0.001, 0.001) and the difference was statistically significant. Transwell cell migration assay and Matrigel cell invasion assay revealed that the migration and invasion ability of FG cells after miR-19b silencing [(22.54±2.87)%, (28.87±5.62)%] was significantly decreased as compared with the control group [(99.65±3.07)%, (98.73±4.38)%], (P=0.005, 0.002) and the difference was statistically significant. In contrast, the migration and invasion ability of BxPC3 cells overexpressing miR-19b [(317.53±10.21)%, (286.77±12.83)%] was significantly higher than that in the control group [(97.89±5.31)%, (98.25±7.57)%] (P=0.003, 0.007) and the difference was statistically significant. The colony formation and MTT assay showed that after miR-19b silencing, the expression of p21 and p27 in PDAC cells was significantly reduced as compared with the control group (P=0.003, 0.005) and the difference was statistically significant. The results of Western blotting indicated that the silencing of miR-19b resulted in the up-regulation of p21 and p27 in PDAC cells by (3.25±0.27, 5.38±0.33) as compared with the control group (P=0.003, 0.001) and the difference was statistically significant. The overexpression of miR-19b led to the down-regulation of p21 and p27 by (2.68±0.15, 3.27±0.27) as compared with the control group (P=0.006, 0.005) and the difference was statistically significant. Conclusion MiR-19b could promote the migration and invasion of PDAC cells and increase the proliferation capability. Key words: MicroRNA-19b; Pancreatic ductal adenocarcinoma; Metastsis; Proliferation

Objective To observe the effect of liver natural killer (NK) cells on a liver fibrosis in mice with obstructive jaundice.Methods Mice were randomly divided into 4 groups with 20 mice in each group: group A, operation + Poly I∶C group;group B, operation + phosphate buffered solution (PBS) group;group C, sham-operation + Poly I∶C group;group D, sham-operation + PBS group.Operation groups underwent common bile duct ligation (BDL) to establish obstructive jaundice models and sham-operation groups only underwent dissociation of common bile duct.Poly I∶C was injected to activate and induce the accumulation of NK cells in mouse liver.All the mice were sacrificed at day 14 after operation.The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), and indirect bilirubin (IBIL) were determined, and the liver tissue received hematoxylin and eosin (HE) stain and Masson stain.The levels of alpha-smooth muscle actin (α-SMA) was detected by histopathologic method.The liver NK 1.1, α-SMA, and tumor necrosis factor-α (TNF-α) mRNA expression was analyzed by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR).Results As compared with group B [(508.69±102.46), and (486.20±126.80) U/L] at day 14 after BDL, the serum ALT and AST levels were significantly increased in group A [(829.78±79.33), and (752.00±67.90) U/L].The results of HE stain and Masson stain showed that the degree of liver fibrosis in the group A was more severe than that in the group B.The liver histopathologic scores of α-SMA in each group were as follows: group A (+++), group B (++), group C (+), group D(+).As compared with group C (91.86±5.95, 1.21±0.31, and 1.51±0.03) and group D (1.01±0.03, 1.00±0.00, and 0.99±0.02), the expression levels of NK1.1, α-SMA, and TNF-α mRNA in group A (181.79±13.68, 239.67±12.09, and 12.39±0.87) and group B (168.69±12.90, 179.96±20.95, and 7.44±0.66) were significantly increased (P=0.032, P=0.030).As compared with group D, the expression of NK1.1 mRNA in group C was increased (P=0.029, P=0.031, P=0.027).Conclusion Poly I∶C activated and induced accumulation of NK cells in mouse livers, which aggravated liver fibrosis in mice with obstructive jaundice.

AIM:To investigate the expression of miR-29a in rat acute pancreatitis and its functional role in AR42J cell apoptosis.METHODS:Twelve SD rats were divided into a control group and an acute edematous pancreatitis (AEP) group randomly. AEP was induced by intraperitoneal injection of L-arginine (150 mg/kg) in the AEP group and equal volume of 0.9% NaCl was injected in the control group. The apoptosis of acinar cells in pancreatic tissue was determined by TUNEL assay. miRNA chip assay was performed to examine the expression of miRNAs in two groups. Besides, to further explore the role of miR-29a in apoptosis in vitro, recombinant rat TNF-α (50 ng/mL) was administered to treat the rat pancreatic acinar cell line AR42J for inducing AR42J cell apoptosis. Quantitative real-time PCR (qRT-PCR) was adopted to measure miR-29a expression. Then, miRNA mimic, miRNA antisense oligonucleotide (AMO) and control vector were used to transfect AR42J cells. The expression of miR-29a was confirmed by qRT-PCR and the apoptosis rate of AR42J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of activated caspase3. Moreover, we used bioinformatics software and luciferase assay to test whether TNFRSF1A was the target gene of miR-29a. After transfection, qRT-PCR and Western blot was used to detect the expression of TNFRSF1A in AR42J cells after transfection.RESULTS:The expression of miR-29a was much higher in the AEP group compared with the control group as displayed by the miRNA chip assay. After inducing apoptosis of AR42J cells in vitro, the expression of miR-29a was significantly increased by 1.49 ± 0.04 times in comparison with the control group. As revealed by qRT-PCR assay, the expression of miR-29a was 2.68 ± 0.56 times higher in the miR-29a mimic group relative to the control vector group, accompanied with an obviously increased acinar cell apoptosis rate (42.83 ± 1.25 vs 24.97 ± 0.15, P < 0.05). Moreover, the expression of miR-29a in the miRNA AMO group was 0.46 ± 0.05 times lower than the control vector group, and the cell apoptosis rate was much lower accordingly (17.27 ± 1.36 vs 24.97 ± 0.15, P < 0.05). The results of bioinformatics software and luciferase assay showed that TNFRSF1A might be a target gene of miR-29a. TNFRSF1A expression was up-regulated in the miR-29a mimic group, while the miR-29a AMO group showed the reverse trend.CONCLUSION:miR-29a might promote the apoptosis of AR42J cells via up-regulating the expression of its target gene TNFRSF1A.