Objective:To investigate the effect of microRNA (miR)-146b-5p on the proliferation and apoptosis of gallbladder cancer cells through Robo1.Methods:59 cases of gallbladder cancer and its adjacent tissues surgically removed from the First Affiliated Hospital of Zhengzhou University from January 2017 to December 2021were selected as the research subjects. The expression levels of miR-146b-5p in tumor and adjacent tissues was analyzed by fluorescence quantitative polymerase chain reaction (PCR). The expression levels of Robo1 protein in tumor and adjacent tissues was analyzed by Western blotting. Using transfection reagents to transfect miRNA control and miR-146b-5p inhibitors into the human gallbladder cancer cell line GBC-SD, they were named miRNA control group and miR-146b-5p KD group, respectively. The cell viability and proliferation ability of the two groups were analyzed using methyl thiazolyl tetrazolium (MTT) and clone formation experiments. The migration, invasion, and epithelial mesenchymal transformation abilities of the two groups of cells were analyzed by wounding heal, transwell and Western blotting. Analyze the target genes of miR-146b-5p using bioinformatics and dual luciferase reporter genes. The expression level of target genes was analyzed by Western blotting. The comparison of measurement data adopts t-test. Results:The expression level of miR-146b-5p in gallbladder cancer tissue(1.01±0.16) was significantly lower than that in adjacent tissues of gallbladder cancer (1.94±0.30, t=20.860, P<0.05). As compared with cells in the miR-146b-5p KD group, The light absorption value of cells in the miRNA control group was significantly higher (2.22±0.10 vs. 1.76±0.06, t=9.294, P<0.05), the clone formation rate was significantly higher [(72.85±5.91)% vs. (55.56±6.53)%, t=4.806, P<0.05], the scratch healing rate was significantly higher [(72.85±5.91)% vs. (55.56±6.53)%, t=4.806, P<0.05], the migration number of cells was significantly higher [(110.83±16.18) cells vs. (75.66±11.70) cells, t=4.313, P<0.05], the expression level of E-cadherin protein was significantly lower (0.94±0.06 vs. 1.30±0.17, t=5.081, P<0.05), the expression levels of N-Cadherin and SNAIL proteins were significantly higher (1.28±0.10, 0.91±0.09 vs. 0.84±0.08, 0.52±0.10, t=8.436、7.256, P<0.05), the difference were statistically significant. Robo1 is the target gene for miR-146b-5p. The expression level of Robo1 protein in miRNA control group cells(1.19±0.16) was significantly higher than that in miR-146b-5p KD group cells (0.64±0.11, t=6.881, P<0.05). The expression level of Robo1 protein in gallbladder cancer tissue (1.06±0.13) was significantly higher than that in adjacent tissue (0.60±0.11, t=21.000, P<0.05). Conclusion:MiR-146b-5p is involved in the proliferation and apoptosis of gallbladder cancer cells, mainly through targeting the Robo1 protein.

目的 研究香叶木素(diosmetin,Dio)在肝脏缺血/再灌注(ischemia/reperfusion,I/R)中的作用及其可能的机制.方法 32只小鼠随机分为假手术组、I/R组、低剂量Dio组和高剂量Dio组,每组8只.I/R组行肝I/R术,假手术组行假手术.低和高剂量Dio组行肝I/R术前30 min分别腹腔注射10 mg·kg-1和40 mg·kg-1 Dio.再灌注后24 h,采集血液和肝脏样本.ELISA检测血清中白介素(IL)-1β、IL-6、乳酸脱氢酶(LDH)和天冬氨酸氨基转移酶(AST)的水平.测定肝匀浆中丙二醛(MDA)、活性氧(ROS)、还原性谷胱甘肽(GSH)和总胆红素(TBIL)的水平.HE染色观察肝损伤情况.免疫组化染色观察肝组织中IL-1β的表达.Western blot检测肝组织中cleaved-caspase-3、p-NF-κB p65和p-p38蛋白表达.结果 与假手术组比较,I/R组小鼠肝损伤程度明显增加,血清中IL-1β、IL-6、LDH和AST水平、肝组织中MDA和ROS水平以及cleaved-caspase-3、p-NF-κB p65和p-p38蛋白水平均明显升高,而GSH和TBIL的水平明显下降,差异均具有统计学意义.与I/R组比较,低和高剂量Dio组中上述指标均得到明显改善(P<0.05),其中高剂量组的效果优于低剂量组.结论 Dio预处理可减轻肝I/R损伤,这一作用可能与其减弱肝I/R诱导的氧化应激以及炎症相关通路有关.

Objective:To investigate the effect of long non-coding RNA (lncRNA) NEAT1 targeting microRNA (miR)-149-5p on the proliferation, migration and invasion of glioma cells and its molecular mechanism.Methods:Lipofectamine 2000 was used for cell transfection to construct si-NEAT1 group, overexpression miR-149-5p group and miR-149-5p inhibition group. The inhibition rate of cell proliferation was detected by cell counting kit-8 (CCK-8) method, the targeted regulation of miR-149-5p by LNC NEAT1 was analyzed by double luciferase reporter gene analysis, the number of migrating and invasive cells was detected by Transwell chamber method, and the protein table was detected by Western blotting.Results:The level of NEAT1 in U251 cells (35.33±7.57) increased significantly ( t=13.613, P<0.01), while miR-149-5p (0.41±0.07) decreased significantly ( t=12.111, P<0.01). In si-NEAT1 group, the inhibition rate of cell proliferation increased significantly [(29.48±2.67)%, t=13.809, P<0.01], while the number of migrating cells (35.33±7.57) and invasive cells (27.33±6.81) decreased significantly ( t=4.395, 4.962, P<0.05), and the levels of phosphorylated protein kinase B (p-Akt) and phosphorylated mammalian target of rapamycin (p-mTOR) decreased significantly ( t=6.959, 8.661, P<0.05). Overexpression of miR-149-5p also significantly increased the inhibition rate of cell proliferation [(29.32±6.41)%, t=6.922, P<0.05], decreased the number of migrating cells (35.67±6.51) and invasive cells (28.00±8.89, t=4.392, 4.471, P<0.05), and decreased the levels of p-Akt and p-mTOR ( t=4.337, 9.981, P<0.05). NEAT1-WT activity was inhibited by miR-149-5p ( t=15.251, P<0.01). Transfection of pc-DNA-NEAT1 could significantly reduce miR-149-5p ( t=8.178, P<0.01), while transfection of si-NEAT1 significantly increased miR-149-5p ( t=5.988, P<0.05). Compared with si-NEAT1+ anti NC group, the expression of cell proliferation inhibition rate in si-NEAT1+ anti-miR-149-5p group decreased significantly ( t=7.955, 13.261, P<0.05), while the number of migrating cells and invasive cells, and p-Akt and p-mTOR levels increased significantly ( t=4.841, 3.784, 5.589, 4.572, P<0.05). Conclusion:Lnc NEAT1 targeting miR-149-5p and regulating Akt/mTOR signaling pathway can promote the proliferation and metastasis of glioma cells. Inhibiting the expression of miR-149-5p can reverse the effect of inhibiting the expression of NEAT1 on the proliferation and metastasis of glioma cells.

Objective:To explore the effects of microRNA-200c (miR-200c) on epithelial-mesenchymal transition (EMT) of human pancreatic cancer stem cells.Methods:CD24 + CD44 + ESA + cells were sorted from PANC-1 cell line by flow cytometry. The miR-200c precursor fragment and negative control fragment were transfected into pancreatic cancer stem cells. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of E-cadherin, vimentin, zinc finger E-box binding protein 1 (ZEB1) and miR-200c. Transwell invasion assay was used to detect the invasiveness. Results:CD24 + CD44 + ESA + cells (0.8%) were isolated in PANC-1 cells. The relative miR-200c expression level in pancreatic cancer stem cell line (0.15±0.01) was significantly lower than that in PANC-1 cell line (1.00±0.09, t=3.306, P<0.05). The transmembrane number of pancreatic cancer stem cells [(321.00±7.62) cells] was greater than that of PANC-1 cells [(70.00±16.47) cells, t=2.152, P<0.05]. The relative miR-200c expression level in pancreatic cancer stem cell line in miR-200c precursor transfected group (3.70±0.42) was significantly higher than that in negative control group (0.22±0.21, t=2.073, P<0.05). The mRNA expression of E-cadherin, vimentin, ZEB1 in miR-200c precursor transfected group was (3.12±0.02) ×10 -4, 0.36±0.15, (0.38±0.07) ×10 -4, and that in the negative control group was (1.31±0.05) ×10 -4, 1.16±0.08, (1.13±0.03) ×10 -4. There was significant difference between the two group ( t=1.941, 2.165, 2.308, P<0.05). The transmembrane number in miR-200c precursor transfected group [(80.00±5.35) cells] was less than that in negative control group [(310.00±19.41) cells, t=2.613, P<0.05]. Conclusion:Overexpression of miR-200c in the PCSCs could inhibite EMT and reverse invasiveness.

目的 探讨肢体缺血预处理对大鼠肝脏缺血再灌注损伤的保护作用.方法 将32只Wistar大鼠随机分为假手术组(SO)、肝脏缺血再灌注组(IR)、肝脏缺血预处理组(IPC+IR)和肢体缺血预处理组(LIPC+IR).观察术后各组大鼠血液中炎症因子(IL-6,TNF-α)及氧化应激水平的差异;同时观察各组大鼠术后生存率及肝脏酶学水平的差异.结果 LIPC组及IPC组大鼠术后血清AST、ALT均较IR组明显降低,术后第7天存活率较IR组明显提高,术后血清TNF-α、IL-6均较IR组明显降低,差异均有统计学意义(P<0.05).LIPC组与IPC组比较无统计学意义(P>0.05).LICP及ICP组大鼠术后体内MDA水平均较IR组降低,SOD水平均较IR组显著升高,差异均有统计学意义(P<0.05).结论 肢体缺血预处理能减轻大鼠肝脏缺血再灌注损伤,可能与减轻肝脏氧化应激水平有关.

目的:探讨miR-1228-5p在原发性肝细胞癌组织和血清中的表达及意义.方法:采用miRNA快速提取试剂盒分别抽取患者和健康体检者血清中的总miRNA及患者肝癌组织和癌旁正常肝组织中的总miRNA,采用实时荧光定量PCR检测总miRNA中miR-1228-5p的相对表达量;分析miR-1228-5p的相对表达量与患者年龄、性别、家族史、肿瘤最大径和临床分期的相关性;此外,用ROC曲线下面积分析miR-1228-5p在原发性肝细胞癌和健康人群中的诊断价值.结果:原发性肝细胞癌患者血清中miR-1228-5P的表达量明显高于健康体检者(P＜0.05),并且miR-1228-5P在原发性肝细胞癌组织中的表达量明显高于其癌旁正常肝组织(P＜0.05);miR-1228-5p表达水平与患者的年龄、性别及家族史无关(P＞0.05),而与患者的肿瘤最大径和临床分期有关(P＜0.05);miR-1228-5p鉴别原发性肝细胞癌与健康人群中的敏感度和特异度分别为71％和93％.结论:miR-1228-5p在肝癌患者中的表达水平高于健康人群,且其在肝癌组织中的表达水平高于癌旁正常肝组织.miR-1228-5p的表达水平与患者的肿瘤最大径和临床分期相关.此外,miR-1228-5p具有诊断原发性肝细胞癌的潜在价值.

Objective To explore the expression of microRNA (miRNA,miR)-449a in liver cancer,the effect on migration and invasion,and the possible regulatory mechanism.Methods Eighteen cases of liver cancer tissues and adjacent tissues were collected.The expression of miR-449a in liver cancer tissues and adjacent tissues,4 kinds of human liver cancer cell lines,and human normal hepatocytes was detected by reverse transcriptase-polymerase chain reaction (RT-PCR).The human liver cancer cell lines were transfected with miR-449a mimic and the negative control (NC) plasmid.The expression of miR-449a after transfection was detected by RT-PCR.The cell number of invasion and migration after transfection in the two groups was measured by Transwell assays.The expression of CXC chemokine ligand 5 (CXCL5) protein in the cells was detected by Western blotting.Results As compared with the adjacent tissues,the expression of miR-449a in liver cancer tissues was significantly reduced (0.957 ±0.165 vs.0.462 ±0.221,t =4.137,P =0.021).As compared with the human normal hepatocytes,the expression of miR-449a in four kinds of human liver cancer cell lines was significantly reduced too (P =0.009,0.008,0.005,0.020),the difference being statistically significant (BEL-7402:6.873 ± 1.293,HepG2:7.246 ± 1.320,NC:1.000,P =0.012,0.017).Two kinds of human liver cancer cell lines with the middle expression of miR-449a were chosen,and the expression of miR-449a after transfection was statistically increased,and the cell number of invasion (BEL-7402 vs.NC:21.57±13.32 vs.79.84± 11.68;HepG2 vs.NC:45.32 ±12.65 vs.92.21 ±15.43,t=24.331,21.109,P=O.011,0.019) and migration (BEL-7402 vs.NC:10.25±3.21 vs.26.63±6.48;HepG2 vs.NC:23.44 ± 10.08 vs.65.65 ± 12.26,t =11.039,12.224,P =0.010,0.008) after transfection was significantly reduced.The expression of CXCL5 proteinafter transfection was statistically significantly reduced (BEL-7402 vs.NC:0.367 ±0.083 vs.0.965 ±0.110;HepG2 vs.NC:0.163 ±0.043 vs.0.949±0.141,t =20.176,25.158) (P=0.021,0.016).Conclusion The miR-449a in liver cancer is expressed weakly,and can serve as a tumor suppressor to influence the migration and invasion of liver cancer cells through targeting CXCL5.

Objective To investigate the protective effect of paeoniflorin on acute pancreatitis with lung injury and its effect on nuclear factor-κB (NF-κB) signaling pathway.Methods Rats were randomly divided into 3 groups (n=26 each): the sham operation group, severe acute pancreatitis (SAP) group, and the paeoniflorin group.After the model of ASP was established, the serum amylase (AMY), interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) expression levels were detected by enzyme linked immunosorbent assay (ELISA).The NF-κB mRNA expression level was detected by real-time fluorescent quantitative polymerase chain reaction(FQ-PCR), and the wet/dry ratio and pathological changes of lung tissue were measured and compared.Results The expression level of AMY [(4 267±1 459), (2 681±822) vs.(1 482±5 240) U/L] in peripheral blood, MPO [(5.47±1.28), (4.39±0.57) vs.(2.17±0.37) U/g] in the lung, and the wet/dry ratio (5.13±0.62, 4.54±0.34 vs.4.41±0.21) in the lung tissue in SAP group and the paeoniflorin group were significantly higher than in the sham operation group at 6 h after the operation, and the changes were even more significant in 12 h sub-groups.Pathological score of pancrea tissue (7.44±1.57 vs.11.30±2.27, P=0.021) and lung tissue (0.83±0.82 vs.1.47±1.31, P=0.029), aortic blood TNF-α [(83.44±15.28) vs.(120.05±25.88) ng/L, P=0.042], IL-1β [(46.33±10.03) vs.(84.75±17.83) ng/L, P=0.036], IL-6 [(72.77±18.51) vs.(92.81±19.92) ng/L, P=0.046] protein levels and NF-κB mRNA in paeoniflorin group were significantly lower than in SAP group 12 h after the operation (P=0.002).Conclusion Peoniflorin may have the positive protection on lung injury in SAP through inhibition of the NF-κB signaling pathway and thus reducing the release of inflammatory factors such as IL-1β, IL-6 and TNF-α.

Objective To improve the anti-tumor effect in vitro and in vivo,a bispecific antibody (CT-BiAb)was constructed.Methods CT-BiAb was obtained by overlapping polymerase chain reaction (PCR) between variable region of Trastuzumab,G4S linker and variable region of Cetuximab.Pichia pastoris was used to express.The binding activity of CT-BiAb to PANC-1 was detected by flow cytometry.Methyl thiazol tetrazolium (MTT) assay was used to detect the inhibition of cell growth.And flow cytometry was used to detect the apoptosis of PANC-1 after treatment with CT-BiAb.Finally,mouse tumor model was generated by endermic injecting PANC-1 cells,and we used this model to detect the antitumor activity of CT-BiAb.Results CT-BiAb was purified and the Western blot result showed that CT-BiAb has a correct molecular weight.CT-BiAb maintained binding activity to PANC-1 and the binding ratio was 50.9%.From the MTT assay,CT-BiAb restrained cell growth.From the apoptosis analysis,we found the cell apoptosis ratio of CT-BiAb (33.50±7.14)%was better than patented Trastuzumab (15.86±4.32)% (P=0.022) and Cetuximab (18.64±5.71)% (P=0.048).In vivo assay of the PANC-1 cell xenografts in nude mice showed that the tumor inhibitory rate of high dose group (73.76±10.21)% was better than patented Trastuzumab (32.55±14.42)% (P=0.001) and Cetuximab (52.63±8.47)% (P=0.006).Conclusion We established and express CT-BiAb,which maintained the binding activity of parent antibody.CT-BiAb provide rationale and drug development strategy for dual inhibition of human epidermal growth factor receptor-2 (Her-2) and epidermal growth factor receptor (EGFR) signaling in pancreatic carcinoma.

Objective To observe the effect of immunotherapy on hepatocellular carcinoma (HCC) by melanoma-associated antigen-A3 (MAGE-A3) activated dendritic cells (DC)-induced antigen-specific CD8+ cytotoxic T lymphocytes (CTL) and offer help to HCC treatment.Methods After MAGE-A3 activated DC biomarkers, interleukin (IL)-12, and IL-10 in DC were tested.MAGE-A3-DC was used to induce antigen-specific CD8+ CTL.The apoptosis of L02 cells and HepG2 cells was detected after treatment with CD8+ CTL.Interferon (IFN)-γ of CD8+ CTL was detected by enzyme-linked immunospot assay (ELISPOT).Subcutaneous tumor model of mouse HCC was established.The tumor volume of different groups was measured.Tumor tissue was further observed by pathological sections.Results MAGE-A3 protein stimulation significantly increased the levels of Human leukocyte antigen-DR (HLA-DR), DC83, DC86 and DC80 markers on the surface of DC (97.45±2.58 vs.48.33±1.68, 92.81±2.36 vs.52.92±1.90, 94.00±3.01 vs.53.97±2.05, 92.16±1.87 vs.34.13±1.32;t=35.680,P=0.013;t=29.440,P=0.021;t=24.580,P=0.012;t=56.690,P=0.009).The IL-12 level of MAGE-A3-DC was significantly higher than that of DC [(338.44±18.15) pg/ml vs.(243.23±16.56) pg/ml;t=8.670,P=0.005], but IL-10 level of MAGE-A3-DC was significantly lower than that of DC [(207.21±10.89) pg/ml vs.(327.58±14.36) pg/ml;t=14.930, P=0.009].The apoptosis of L02 cells treated with MAGE-A3-DC-CD8+ CTL was similar to that after treatment with DC-CD8+ CTL [(9.10±1.40)% vs.(9.71±1.58)%;t=0.650, P=0.120].The apoptostic rate of HepG2 cells treated with MAGE-A3-DC-CD8+ CTL was significantly higher than that after treatment with DC-CD8+ CTL [(58.84±5.27)% vs.(9.63±1.61)%;t=19.970, P=0.008].After therapy, tumor sizes V/V0 in MAGE-A3-DC-CD8+CTL group decreased significantly as compared with those of phosphate buffer (PBS) group and DC-CD8+ CTL group (5.43±1.22 vs.21.81±2.01 vs.22.85±2.40;t=22.030, P=0.010;t=20.460, P=0.012).Hematoxylin and eosin (HE) staining showed that nuclear condensation, increased cell gap, vacuoles, and edema were observed in tumor tissues after treatment with MAGE-A3-DC-CD8+ CTL, but pathological changes of tumor tissues were not observed in PBS group and DC-CD8+ CTL group.Conclusion MAGE-A3 protein can stimulate the maturation of DC that induce the production of MAGE-A3 specific CD8+CTL.MAGE-A3-DC-CD8+CTL have specific tumor killing ability.

Objective To investigate the clinical significance and interrelation of long-chain non-coding RNA (lcnRNA) HOXA transcript at the distal tip (HOTTIP) and metabotropic glutamate receptor 1 (mGluR1) in pancreatic cancer patients.Methods Pancreatic cancer samples,which were treated in our hospital were collected.The expression levels of HOTTIP and mGlUR1 mRNA were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in fresh pancreatic cancer tissue,and those of HOTTIP and mGluR1 protein by Western blotting and immunohistochemistry.Moreover,the relationship between the expression of HOTTIP,mGlUR1 and clinicopathological features was analyzed.Results The levels of HOTHP and mGlUR1 mRNA and protein were significantly up-regulated in pancreatic cancer tissues as compared with normal pancreatic tissue (P =0.000).The expression of HOTTIP protein was positive in 32 samples (68.09％) and mGlUR1 protein was positive in 30 samples (63.83％).The positive rate of HOTTIP and mGlUR1 in adjacent tissues was 31.91％ and 36.17％,respectively,and the difference was also statistically significant (P =0.000).The expression of HOTTIP and mGluR1 was correlated with lymph node metastasis,differentiation degree and TNM stage (P =0.000,0.004,0.000).The survival time of HOTTIP and mGlUR1 positive patients was significantly shorter than that of negative patients (P =0.029,0.047).Cox regression multivariate analysis showed that lymph node metastasis,differentiation degree,TNM stage,HOTTIP and mGlUR1 expression were independent prognostic factors for pancreatic cancer (P =0.022,0.048,0.026,0.000,0.002).In addition,HOTTIP and mGlUR1 expression showed a significant positive correlation in cervical cancer patients (r =0.689,P =0.002).Conclusion The positive expression of HOTTIP and mGlUR1 is associated with the occurrence,progression and prognosis of pancreatic cancer.

Objective:To investigate the improvement of the perioperational insulin resistance of people underwent hepatectomy combined with anti - viral treatment. Methods:110 patients were randomly divided into treatment group (60 cases)and control group(50 cases)according to the anti - viral treatment(Entecavir,0. 5mg/ d). The data of FPG,FINS,IL - 6 and TNF - α were measured perioperationally,and HOMA - IR was calculated to eveluate the insu-lin resistance changes. Results:The postoperational serum level of IL - 6,TNF - α were significantly elevated in both groups,and the treatment group had a significant smaller evluated range compared with the control group(P < 0. 05). Patients in both groups presence insulin resistance before operation,the postoperational data of FPG,FINS and HOMA- IR were increased in both groups,but the insulin resistance level and the complication rates of treatmen group were significantly improved than the control group(P < 0. 05). Conclusion:Anti - viral therapy may improve perioperative insulin resistance in patients with hepatectomy.

Objective To explore the therapeutic effect of wortmannin on acute pancreatitis (AP) with renal dysfunction and on phosphatidylinositol 3 kinase (PI3K)/protein kinase B (PKB) signaling pathway.Methods Eighty healthy male SD rats were randomly divided into normal control group,sham operation group (SO group),AP group and wortmannin group.SO group,AP group and wortmannin group were further divided into 3,6 and 12 h sub-groups (n =8 each).The AP model was established by bile duct retrograde injection.Wortmannin was intraperitoneally injected 4 h before operation.Serum urea nitrogen,creatinine,amylase,pathological findings of the kidney and pancreas,and PKB,p-PKB and tumor necrosis factor-α (TNF-α) proteins in the kidney and pancreas were observed.Results Histopathological damage of the kidney and pancreas was severer,blood urea nitrogen [(9.81 ± 1.28) and (77.49±1.17) vs.(5.33 ±0.32) mmol/L],creatinine [(62.19 ±5.84) and (55.12 ±5.27) vs.(45.13 ±3.01) μmol/L] and amylase levels [(2 931 ±619) and (2 061 ±897) vs.(677 ± 120) U/L] were significantly higher in AP group and wortmannin group than in SO group 3 h after operation (P ＜ 0.05),more significantly in 6 h and 12 h sub-groups.Histopathological damage of the kidney and pancreas was severed,blood urea nitrogen [(16.51 ± 2.00) vs.(21.16 ± 2.57) mmol/L],creatinine [(94.63±11.30) vs.(116.24± 14.82) μmol/L],amylase levels [(3264±932) vs.(7725± 1 517) U/L],p-PKB (1.01 ± 0.24 vs.1.23±0.30) andTNF-αproteins (1.11±0.29vs.1.33± 0.37) levels were significantly lower in wortmannin group than in AP group at 12 h after operation (P ＜ 0.05).Conclusion Activation of PI3K/PKB signaling pathway plays a very important role in the occurrence and development of AP with renal function dysfunction.PI3K inhibitors (wortmannin) can significantly alleviate the renal function damage induced by AP.

Objective To explore the effect of microRNA (miRNA,miR)-200c in reversing chemo-resistance of human pancreatic cancer stem cells.Methods CD24 + CD44 + ESA + cells were sorted from PANC-1 cell line by FACS.miR-200c precursor fragment and negative control fragment were transfected into pancreatic cancer stem cells.The real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect the miR-200c expression.MTT assay was used to measure drug sensitivity to gemcitabine.Results CD24+ CD44 + ESA + cells (0.8％) were isolated in PANC-1 cells.The relative miR-200c expression level in pancreatic cancer stem cell line (0.15 ± 0.01) was significantly lower than that in PANC-1 cell line (1.00 ± 0.09,P ＜ 0.05).The half inhibition concentration (IC50) of gemcitabine was significantly higher in pancreatic cancer stem cells group [(19.15 ± 1.53) μmol/L] than that in PANC-1 cells group [(0.86 ±0.18) μmol/L] (P＜0.05).The relative miR-200c expression level in pancreatic cancer stem cell line in miR-200c precursor transfected group [(3.70 ± 0.42)] was significantly higher than that in negative control group (0.22 ±0.21,P ＜0.05).The IC50 of gemcitabine was significantly lower in miR-200c precursor transfected group [(0.92 ± 0.13) μ mnol/L] than that in negative control group [(17.94 ± 1.36) μmol/L,P ＜ 0.05].Conclusion miR-200c could reverse chemo-resistance of human pancreatic cancer stem cells to gemcitabine.

Objective To investigate the sensitivity of human pancreatic cancer stem cells to chemotherapy and the expression of microRNA (miRNA,miR)-200c in cancer stem cells.Methods CD24 + CD44 + ESA + cells were sorted from PANC-1 cell line by fluorescence-activated cell sorter (FACS).The stem like properties of this subpopulation were assessed by non-obese diabetic (NOD)/ severe combined immunodeficiency (SCID) xenograft transplantation experiment.Sensitivity to gemcitabine and apoptosis ratio of pancreatic cancer stem cells and PANC-1 cells were detected by methyl thiazol tetrazolium (MTT) assay and FACS respectively.The real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to detect miR-200c expression in pancreatic cancer stem cells and PANC-1 cells.Results CD24 + CD44 + ESA + cells (0.8％) were isolated in PANC-1 cells.NOD/SCID xenografl transplantation experiment confirmed that the sub-group had the characteristics of cancer stem cells.The half inhibition concentration (IC50) of gemcitabine was significantly higher in pancreatic cancer stem cells group [(19.15 ± 1.53) μmol/L] than in PANC-1 cells group [(0.86 ± 0.18) μmol/L] (P ＜ 0.05).Mter the interference of gemcitabine (1,10 μmol/L),the apoptosis ratio was significantly lower in pancreatic cancer stem cells group [(0.69 ±0.09)％ and (0.90 ±0.13)％] than that in PANC-1 cells group [(60.54 ± 3.73) ％ and (91.76 ± 5.07) ％] (P ＜ 0.05).The relative miR-200c expression level in cancer stem cell line [(0.15 ±0.01)] was significantly lower than that in PANC-1 cell line [(1.00 ± 0.09)] (P ＜ 0.05).Conclusion The expression of miR-200c was significantly reduced in pancreatic cancer stem cells which were significantly chemo-resistant,and miR-200c may play an important role in the chemo-resistance of pancreatic cancer stem cells.

Objective:To investigate the expression and action of microRNA-200c (miRNA-200c) in pancreatic cancer stem cells. Methods:Pancreatic cancer stem cells were sorted from human pancreatic cancer PANC-1 cells by FACS using CD24+CD44+ESA+as marker, and their stem cell properties were assessed by xenogratf tumor assay in NOD/SCID mice. hTe miRNA-200c expression and invasion ability in PANC-1 cells, pancreatic cancer stem cells and pancreatic cancer stem cells transfected with miRNA-200c precursor sequence or negative control sequence were determined by RFQ-PCR method and Transwell invasion assay, respectively. Results:The CD24+CD44+ESA+cells (accounting for 0.8%) sorted from PANC-1 cells presented tumor stem cell properties, and the volume of the xenograft tumor after their subcutaneous transplantation in mice was significantly larger than that after PANC-1 cells transplanted at the same time [(1 725.14±261.29) mm3 vs. (479.65±99.67) mm3, P<0.05]. In pancreatic cancer stem cells compared with PANC-1 cells, the miRNA-200c expression level was significantly decreased (0.15±0.01 vs. 1.00±0.09, P<0.05), and transmembrane cell number was signiifcantly increased (321±7.62 vs. 70±16.47, P<0.05), but the miRNA-200c expression level was signiifcantly increased and transmembrane cell number was signiifcantly decreased in pancreatic cancer stem cells atfer transfection with miRNA-200c precursor sequence (both P<0.05). Conclusion:MiRNA-200c expression is reduced in pancreatic cancer stem cells, and miRNA-200c has inhibitory effect on growth and invasiveness of pancreatic cancer stem cells.