沉默STAT3基因对人胰腺癌细胞体内生长能力的影响

Objective To investigate the effect and mechanism of RNAi-mediated STAT3 gene silence on human pancreatic cancer cells growth in vivo. Methods STAT3 shRNA expression vector was stably transfected to SW1990 cells. STAT3 and p-STAT3 protein was examined using Western blot. The growth ability of SW1990 cells in vivo was determined in a subcutaneous tumor model of nude mice. Western blot was performed to detect the protein expression of Bcl-xL and cyclin D1. Results The protein expression of STAT3 and p-STAT3 decreased by 90% and 92% by stable transfection of STAT3 shRNA expressing vectors(P ＜0. 05). Inhibition of STAT3 with RNAi significantly inhibited the growth ability of SW1990 cells in vivo( P ＜ 0. 05 ). The tumor weight significantly decreased( P ＜ 0. 05 ). Moreover, the relative Bcl-xL and cyclinD1 protein expression in SW1990-RNAi cells reduced by 56% and 50% compared with that of the parental SW1990 cells, respectively (P ＜ 0. 05). Conclusions Inhibition of STAT3 with RNAi significantly inhibits the growth ability of pancreatic cancer cells through down-regulating Bcl-xL and cyclin D1.     

沉默STAT3基因对人胰腺癌细胞体内生长能力的影响

Objective To investigate the effect and mechanism of RNAi-mediated STAT3 gene silence on human pancreatic cancer cells growth in vivo. Methods STAT3 shRNA expression vector was stably transfected to SW1990 cells. STAT3 and p-STAT3 protein was examined using Western blot. The growth ability of SW1990 cells in vivo was determined in a subcutaneous tumor model of nude mice. Western blot was performed to detect the protein expression of Bcl-xL and cyclin D1. Results The protein expression of STAT3 and p-STAT3 decreased by 90% and 92% by stable transfection of STAT3 shRNA expressing vectors(P ＜0. 05). Inhibition of STAT3 with RNAi significantly inhibited the growth ability of SW1990 cells in vivo( P ＜ 0. 05 ). The tumor weight significantly decreased( P ＜ 0. 05 ). Moreover, the relative Bcl-xL and cyclinD1 protein expression in SW1990-RNAi cells reduced by 56% and 50% compared with that of the parental SW1990 cells, respectively (P ＜ 0. 05). Conclusions Inhibition of STAT3 with RNAi significantly inhibits the growth ability of pancreatic cancer cells through down-regulating Bcl-xL and cyclin D1.     

白细胞介素6促进人胰腺癌细胞的侵袭及机制探讨

目的 应用白细胞介素6(IL-6)作用于人胰腺癌细胞株SW1990和Capan-2,观察侵袭能力的变化并探讨其机制.方法 使用IL-6处理人胰腺癌细胞株SW1990和Capan-2,MTT法检测细胞增殖,免疫细胞化学和Western印迹检测P-STAT3的表达,荧光定量PCR、Western印迹检测VEGF、MMP-2 mRNA和蛋白表达,体外侵袭检测细胞的侵袭能力.结果 IL-6 100 μg/L作用人胰腺癌细胞株后,细胞增殖能力增强(P＜0.05);Western印迹和免疫细胞化学显示P-STAT3的表达增加;荧光定量PCR、Western印迹显示VEGF、MMP-2的mRNA和蛋白表达明显升高(P＜0.05);细胞侵袭能力增强.结论 IL-6通过激活STAT3信号转导通路,上调MMP-2和VEGF表达,增强胰腺癌细胞侵袭能力.

Abstract：

Objective To investigate the effects and mechanism of IL-6 on invasion and metastasis of human pancreatic cancer cells. Methods IL-6 was added into the culture media of human pancreatic cancer cells Capan-2 and SW1990. Cell growth was measured by MTT assay. Western blot and immunocytochemistry were performed to detect Phosphorylated STAT3 (P-STAT3) protein. VEGF and MMP-2 mRNA and protein expression were examined using fluorescence quantitative polymerase chain reaction (RT-PCR) and Western blot, respectively. The invasion ability of SW1990 and Capan2 cells was determined by cell invasion assay in vitro. Results 100 ng/mL IL-6 significantly promoted growth and invasion ability of Capan-2 and SW1990 cells (P＜0.05). The use of IL-6 not only markedly increased the protein expression of P-STAT3, VEGF and MMP-2, but also greatly increased the mRNA expression of MMP-2 and VEGF. Conclusions STAT3 signal transducer pathway activation with IL-6 can promote the invasion ability of pancreatic cancer cells in vitro through up-regulation of MMP-2 and VEGF expression. STAT3 signal transducer may provide a novel therapeutic target for the treatment of pancreatic cancer.     

二氢青蒿素对胰腺癌生长的抑制作用

Objective To investigate the anti-tumor activity of dihydroartemisinin in pancreatic cancer in vitro and in vivo. Methods For cultured cells,cell growth was determined by the MTT assay and apoptosis was evaluated by flow cytometry analysis stained with Annexin V-FITG/PI. The protein expression in BxPC-3 cells was analyzed by Western blot assay. BxPC-3 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors and the tumor volume was monitored after exposure to dihydroartemisinin. Ki-67 staining and TUNEL assay were used to assess tumor cell proliferation and apoptosis in tumor tissue. Results After treatment by dihydroartemisinin in vitro, the proliferative inhibition rates of pancreatic cancer cells BxPC-3 and AsPC-1 reached up to (76.2 ± 3.5) % and (79.5 ± 2.9) %, and the apoptosis rates were up to (55.5 ± 3.2)% and (40.0 ± 3.5)%, the differences were significantly (P ＜ 0.01) compared with control [(2.0 ± 1.3) % and (0.9 ± 0.4) %]. Dihydroartemisinin inhibited the growth of pancreatic xenograft tumors in nude mice. The proliferation index and apoptusis index were (49.1 ± 3.9)% and (50.2 ± 4.4)% respectively in dihydroarternisinin 50 mg/kg treatment group, compared to those of (72.1 ± 3.3) % and (9.4 ± 2.9) % in control, the differences were significantly (P ＜0.01). Western blot assay indicated that dihydroartemisinin up-regulates expression of proliferation-associated protein p21WAF1 and down-regulates expression of PCNA, increases expression of apoptosis-associated protein Bax and decreases expression of Bcl-2 and activates caspase-9 in BxPC-3 cells. Conclusions Dihydroartemisinin exerts anti-tumor activity in pancreatic cancer both in vitro and in vivo by proliferation inhibition and apoptusis induction. Dihydroartemisinin can be used as a potential anti-tumor drug in pancreatic cancer.     

二氢青蒿素对胰腺癌生长的抑制作用

Objective To investigate the anti-tumor activity of dihydroartemisinin in pancreatic cancer in vitro and in vivo. Methods For cultured cells,cell growth was determined by the MTT assay and apoptosis was evaluated by flow cytometry analysis stained with Annexin V-FITG/PI. The protein expression in BxPC-3 cells was analyzed by Western blot assay. BxPC-3 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors and the tumor volume was monitored after exposure to dihydroartemisinin. Ki-67 staining and TUNEL assay were used to assess tumor cell proliferation and apoptosis in tumor tissue. Results After treatment by dihydroartemisinin in vitro, the proliferative inhibition rates of pancreatic cancer cells BxPC-3 and AsPC-1 reached up to (76.2 ± 3.5) % and (79.5 ± 2.9) %, and the apoptosis rates were up to (55.5 ± 3.2)% and (40.0 ± 3.5)%, the differences were significantly (P ＜ 0.01) compared with control [(2.0 ± 1.3) % and (0.9 ± 0.4) %]. Dihydroartemisinin inhibited the growth of pancreatic xenograft tumors in nude mice. The proliferation index and apoptusis index were (49.1 ± 3.9)% and (50.2 ± 4.4)% respectively in dihydroarternisinin 50 mg/kg treatment group, compared to those of (72.1 ± 3.3) % and (9.4 ± 2.9) % in control, the differences were significantly (P ＜0.01). Western blot assay indicated that dihydroartemisinin up-regulates expression of proliferation-associated protein p21WAF1 and down-regulates expression of PCNA, increases expression of apoptosis-associated protein Bax and decreases expression of Bcl-2 and activates caspase-9 in BxPC-3 cells. Conclusions Dihydroartemisinin exerts anti-tumor activity in pancreatic cancer both in vitro and in vivo by proliferation inhibition and apoptusis induction. Dihydroartemisinin can be used as a potential anti-tumor drug in pancreatic cancer.     

二氢青蒿素对胰腺癌生长的抑制作用

Objective To investigate the anti-tumor activity of dihydroartemisinin in pancreatic cancer in vitro and in vivo. Methods For cultured cells,cell growth was determined by the MTT assay and apoptosis was evaluated by flow cytometry analysis stained with Annexin V-FITG/PI. The protein expression in BxPC-3 cells was analyzed by Western blot assay. BxPC-3 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors and the tumor volume was monitored after exposure to dihydroartemisinin. Ki-67 staining and TUNEL assay were used to assess tumor cell proliferation and apoptosis in tumor tissue. Results After treatment by dihydroartemisinin in vitro, the proliferative inhibition rates of pancreatic cancer cells BxPC-3 and AsPC-1 reached up to (76.2 ± 3.5) % and (79.5 ± 2.9) %, and the apoptosis rates were up to (55.5 ± 3.2)% and (40.0 ± 3.5)%, the differences were significantly (P ＜ 0.01) compared with control [(2.0 ± 1.3) % and (0.9 ± 0.4) %]. Dihydroartemisinin inhibited the growth of pancreatic xenograft tumors in nude mice. The proliferation index and apoptusis index were (49.1 ± 3.9)% and (50.2 ± 4.4)% respectively in dihydroarternisinin 50 mg/kg treatment group, compared to those of (72.1 ± 3.3) % and (9.4 ± 2.9) % in control, the differences were significantly (P ＜0.01). Western blot assay indicated that dihydroartemisinin up-regulates expression of proliferation-associated protein p21WAF1 and down-regulates expression of PCNA, increases expression of apoptosis-associated protein Bax and decreases expression of Bcl-2 and activates caspase-9 in BxPC-3 cells. Conclusions Dihydroartemisinin exerts anti-tumor activity in pancreatic cancer both in vitro and in vivo by proliferation inhibition and apoptusis induction. Dihydroartemisinin can be used as a potential anti-tumor drug in pancreatic cancer.     

抑制信号转导与转录激活因子-3基因表达对人胰腺癌细胞转移能力的影响

目的 探讨RNA干扰(RNAi)技术抑制信号转导与转录激活因子-3(STAT3)表达对人胰腺癌细胞株SW1990体内转移能力的影响及其机制.方法 构建STAT3短发卡RNA(shRNA)表达载体,稳定转染SW1990细胞.应用RT-PCR方法 观察STAT3 mRNA表达的改变,EMSA方法 检测STAT3-DNA结合活性的改变.应用裸鼠急性血路转移实验检测细胞体内转移能力的变化,并通过RT-PCR方法 检测基质金属蛋白酶-2(MMP-2)和血管内皮生长因子(VEGF)mRNA表达的改变.结果 STAT3 shRNA表达载体稳定转染SW1990细胞可显著抑制STAT3 mRNA表达和STAT3-DNA结合活性(P＜0.05);裸鼠急性血路转移实验显示,RNAi技术抑制STAT3后,SW1990细胞体内转移能力明显下降(P＜0.05);RT-PCR结果 显示,RNAi技术抑制STAT3后,SW1990细胞中MMP-2和VEGF的mRNA表达明显减低(P＜0.05).结论 RNAi技术能有效抑制STAT3基因表达,并可通过下调MMP-2和VEGF表达抑制胰腺癌细胞体内转移能力.     

抑制信号传导与转录激活因子3对人胰腺癌体外血管生成的影响

目的　探讨RNA干扰(RNAi)抑制信号传导与转录激活因子3(STAT3)对人胰腺癌体外血管生成的影响及其机制.方法 RNAi抑制胰腺癌细胞株SW1990细胞中STAT3、噻唑蓝(MTT)和流式细胞技术分别检测胰腺癌细胞上清液对人脐静脉内皮细胞(HUVEC)细胞增殖和细胞周期的影响.体外迁移实验检测胰腺癌细胞上清液诱导的HUVEC迁移能力;酶联免疫吸附试验(ELISA)检测胰腺癌细胞上清液中血管内皮生长因子(VEGF)蛋白表达.结果　MTT和流式细胞仪结果显示RNAi抑制STAT3后,HUVEC增殖能力下降,24、48、72 h的细胞增殖率分别为(1.19±0.11)％、(1.62±0.15)％、(1.95±0.18)％;细胞周期阻滞于G0/G1期,为(80.95±7.49)％.体外迁移实验显示RNAi抑制STAT3后,HUVEC迁移能力明显减弱.ELISA显示RNAi抑制STAT3后,VEGF蛋白表达下降60％.结论　RNAi抑制STAT3可以通过下调VEGF,抑制胰腺癌细胞体外血管生成能力.     

靶向STAT3的慢病毒表达载体的构建及其对血管平滑肌细胞增殖凋亡的影响

目的 构建大鼠STAT3基因的shRNA慢病毒表达载体,并观察其对大鼠血管平滑肌细胞增殖和凋亡的影响.方法 针对STAT3基因的不同部位设计4对shRNA的寡核苷酸片段,克隆到慢病毒载体PLKO.1中,构建靶向STAT3基因的慢病毒载体PLK0.1-STAT3-shRNA,检测并筛选最佳抑制效率的shRNA干扰载体.并将其转染大鼠血管平滑肌细胞,用噻唑蓝法和流式细胞仪检测沉默STAT3基因后对血管平滑肌细胞增殖和凋亡能力的影响.结果　靶向STAT3慢病毒表达载体构建成功.转染PLKO.1-STAT3-shRNA后,STAT3蛋白表达明显下降,其中以PLKO.1-STAT3-S1最为明显,达到90%以上;转染PLKO.1-STAT3-S1的细胞增殖能力(A值=0.25±0.05)明显低于未转染组(A值=0.62±0.12)和阴性对照组细胞(A值=0.59±0.11)(P＜0.05);而早期细胞凋亡率(26.9±2.8)%和晚期细胞凋亡率(9.5±1.6)%均明显高于未转染组和阴性对照组(P＜0.01).结论　成功构建并筛选最佳抑制效率的靶向STAT3慢病毒表达载体PLKO.1-STAT3-S1,该载体能有效抑制大鼠血管平滑肌细胞增殖,并促进细胞凋亡.

Abstract：

Objective To construct a recombinant short hairpin RNA (shRNA) lentiviral vector carrying STAT3 gene in rats, and to investigate its effects on proliferation and apoptosis of vascular smooth muscle cells by silencing STAT3. Methods Four oligonucleotides targeting STAT3 gene were synthesized and cloned into lentivirus vector PLKO. 1. The shRNA lentiviral vector with best transfection efficiency was detected and identified, which was transfected into vascular smooth muscle cells in rats, and its effects on proliferation and apoptosis of vascular smooth muscle cells were measured by MTT and flow cytometry after silencing STAT3. Results The recombinant lentivirus vector PLKO. 1-STAT3-shRNA was constructed successfully. PLKO. 1-STAT3-shRNA knocked down the expression of STAT3 protein dramatically, especially PLKO. 1-STAT3-S1, whose transfection efficiency was more than 90%. The proliferation capacity of vascular smooth muscle cells transfected with PLKO. 1-STAT3-S1 (A value =0. 25 ±0. 05 ) was significantly lower than no-transfected group (A value =0. 62 ±0. 12) and negative control group (A value =0. 59 ±0. 11 )(P ＜ 0. 05). Meantime the early apoptosis rate (26. 9 ± 2. 8 ) % and late apoptosis rate (9. 5 ± 1.6 ) % in PLKO. 1-STAT3-shRNA-transfected group were significantly higher than in no-transfected group and negative control group (P ＜ 0. 01 ). Conclusion The recombinant lentivirus shRNA vector targeting STAT3,PLKO. 1-STAT3-S1, with best transfection efficiency, is constructed successfully. PLKO. 1-STAT3-S1 can inhibit the proliferation of vascular smooth muscle cells, and promote the cell apoptosis. This study lays the foundation for further studying on targeting treatment of vascular restenosis.     

Impact of STAT1-siRNA transfection on the expressions of STAT3 and transforming growth factor β1 in human glomerulur mesangial cells induced by high glucose

Objective To observe the cell proliferation and the protein expression of STAT1,phosphorylation of STAT1 (p-STAT1), STAT3, p-STAT3 and transforming growth factor β1 (TGF-β1) in human glomerulur mesangial cells (HMCs) induced by high glucose after STAT1-siRNA transfection. Methods Three STAT1-siRNA sequences were designed and synthetized. HMCs in 6-well plate were transiently transfected with STAT1-siRNA using Lipofectamine 2000. After transfection for 48 h or 72 h, STAT1 mRNA and protein expression were detected by real-time PCR and Western blotting, respectively, to choose the effective sequence in later experiments. After transfection for 24 h and stimulated with 25 mmol/L glucose for 24 h, 48 h, 72 h, cell proliferation was measured by MTT assays, the protein expressions of STAT1, p-STAT1, STAT3 and p-STAT3 were detected by Western blotting, the expression of TGF-β1 was detected by ELISA in each group. Results High glucose could stimulate HMCs proliferation. The protein expressions of p-STAT1, p-STAT3 and TGF-β1 were increased in the group stimulated by high glucose (P＜0.05). The protein expressions of p-STAT3 and TGF-β1 were further increased in HMCs induced by high glucose after STAT1-siRNA transfection (P＜0.05). Conclusions Under high glucose conditions, JAK-STAT signal transduction pathway of HMCs can be activated, then it is far greater when HMCs are induced by high glucose after STAT1-siRNA transfection. The secretion of TGF-β1 is increased in HMCs under the state of high glucose, and it is further increased after STAT1-siRNA transfection, which is related to the kidney fibrosis.     

JAK2-STAT3 pathway regulates the expression of complement factor B in autosomal dominant polycystic kidney disease

Objective To investigate the role of JAK2-STAT3 pathway in the expression of complement factor B (CFB) in autosomal dominant polycystic kidney disease (ADPKD). Methods Renal tissue samples of patients with ADPKD after nephrectomy were collected. Normal renal tissue samples as control were taken from patients after radical nephrectomy. Renal tissue samples of Han: SPRD Cy/+ rats (ADPKD model) and wild-type Han: SPRD +/+ rats were also collected at 4, 8, 16 week. Han:SPRD Cy/+ rat renal tubular epithelial cells (16 w) were primarily cultured in vitro, then stimulated with the JAK2 inhibitor (WP1066) and STAT3 inhibitor (pyrimethamine) for 24 h respectively. Western blotting was used to detect the expression of p-JAK2, JAK2, p-STAT3, STAT3, CFB protein. Results Compared with control group, the protein expressions of p-JAK2, p-STAT3, STAT3, CFB significantly increased in the renal tissue of ADPKD patients (all P＜0.05). The protein expressions of p-JAK2, JAK2, p-STAT3, STAT3 and CFB also significantly increased in the renal tissue of Cy/+ rats compared with wild-type rats (all P＜0.01). When the Cy/+ renal tubular epithelial cells were treated with WP1066, the expressions of p-JAK2, p-STAT3, CFB were suppressed (P＜0.05) and the degree of inhibition was correlated with the WP1066 dose. Pyrimethamine inhibited the protein expressions of p-STAT3 and CFB in the tubular epithelial cells of Cy/+ rats (all P＜0.05) and the degree of inhibition was correlated with the pyrimethamine dose. Conclusions The JAK2-STAT3 pathway is abnormally activated in ADPKD and increases the protein expression of CFB. CFB protein level is correlated with the progress of ADPKD, suggesting that it may take part in the growth and development of ADPKD vesicles.     

胰岛素调节人胰腺癌细胞ASPC-1中HIF-1α的表达研究

目的 探讨胰岛素对胰腺癌细胞株ASPC-1中缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)表达的影响.方法 将ASPC-1细胞分为5组:常氧组,常氧加胰岛素组,缺氧组,同一缺氧时间不同胰岛素浓度组以及相同胰岛素浓度不同缺氧时间组,实时定量PCR检测HIF-1α基因表达,免疫细胞化学与Western blot检测HIF-1α蛋白表达水平的变化.Transwell实验检测加入胰岛素后肿瘤细胞侵袭能力的变化.结果 常氧下ASPC-1细胞即有一定水平HIF-1α表达,加入胰岛素刺激后HIF-1α蛋白表达随着胰岛素浓度的升高和作用时间的延长而逐渐升高,与对照组相比差异有统计学意义(P＜0.05);缺氧条件下HIF-1α表达较常氧时升高(P＜0.05);相同缺氧时间使用不同浓度胰岛素刺激时,高浓度胰岛素刺激下HIF-1α的表达较单纯缺氧更高(P＜0.05),低浓度胰岛素对HIF-1α无明显作用(P＞0.05);使用相同胰岛素处理后再进行缺氧处理,随着时间延长HIF-1α的表达先升高后下降,但各处理组较对照组差异均有统计学意义(P＜0.05).Transwell实验显示胰岛素可增强胰腺癌细胞的侵袭能力(P＜0.05).结论 胰岛素可上调胰腺癌细胞ASPC-1中HIF-1蛋白表达,并且这种作用具有一定的剂量依赖性和时间依赖性.胰岛素还可以增加ASPC-1的侵袭能力.

Abstract：

Objective To investigate the effect of insulin on the expression of hypoxia-inducible factor-1α in human pancreatic cancer cell line ASPC-1. Methods We divided ASPC-1 cells into five groups: normoxia; normoxia stimulated with insulin; hypoxia; hypoxia pretreated with different concentration of insulin; hypoxia of different time points pretreated with same concentration of insulin. Real-time PCR was used to test the expression of HIF-1α mRNA. Immunohistochemistry was used to examine the expression of HIF-1α in ASPC-1 of insulin treated cancer cells. Western blot was used to determine the expression of HIF-1 α protein in those cells. Transwell was used to test whether insulin could enhance the invasion ability of ASPC-1 pancreatic cancer cells. Results Insulin promotes HIF-1α protein expression. ASPC-1 cells expressed low levels of HIF-1α protein under normoxic condition. After stimulated with insulin, the expression of HIF-1 α protein significantly increased (P ＜ 0. 05 ). After treated with hypoxia, the expression of HIF-1α protein also increased(P ＜ 0. 05 ). Low concentrations of insulin didn't increase the expression of HIF-1α under hypoxic environment ( P ＞ 0. 05 ), while high concentration of insulin could increase its expression(P ＜ 0. 05). When ASPC-1 cells pretreated with insulin suffered from hypoxia, the expression of HIF-1α first increased then decreased moderately( P ＜0. 05). Insulin could enhance the invasion ability of pancreatic cancer cells( P ＜ 0. 05 ). Conclusions Insulin mediates the expression of HIF-1α protein in human pancreatic cancer ASPC-1 cells with the characteristics of dose and time dependency. Insulin could enhance the invasion ability of ASPC-1 cells.