Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma

Double-stranded RNA (dsRNA) plays a major role in RNA interference (RNAi), a process in which segments of dsRNA are initially cleaved by the Dicer into shorter segments (21-23 nt) called small interfering RNA (siRNA). These siRNA then specifically target homologous mRNA molecules causing them to be degraded by cellular ribonucleases. RNAi down regulates endogenous gene expression in mammalian cells. Vascular endothelial growth factor (VEGF) is a key molecule in vasculogenesis as well as in angiogenesis. Tumor growth is an angiogenesis-dependent process, and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. To investigate the feasibility of using siRNA for VEGF in the specific knockdown of VEGF mRNA, thereby inhibiting angiogenesis, we have performed experiments with a DNA vector based on a siRNA system that targets VEGF (siVEGF). It almost completely inhibited the expression of three different isoforms (VEGF120, VEGF164 and VEGF188) of VEGF mRNA and the secretion of VEGF protein in mouse squamous cell carcinoma NRS-1 cells. The siVEGF released from cationized gelatin microspheres suppressed tumor growth in vivo. A marked reduction in vascularity accompanied the inhibition of a siVEGF-transfected tumor. Fluorescent microscopic study showed that the complex of siVEGF with cationized gelatin microspheres was still present around the tumor 10 days after injection, while free siVEGF had vanished by that time. siVEGF gene therapy increased the fraction of vessels covered by pericytes and induced expression of angiopoietin-1 by pericytes. These data suggest that cationized-gelatin microspheres containing siVEGF can be used to normalize tumor vasculature and inhibit tumor growth in a NRS-1 squamous cell carcinoma xenograft model.     

Notch3-siRNA增强结肠癌细胞对托泊替康的化疗敏感性

目的：探讨下调Notch3表达是否可增强结肠癌SW620细胞对化疗药托泊替康（topotecan）的敏感性。方法：将Notch3siRNA转染到SW620细胞中,Westernblotting检测转染后SW620细胞中Notch3的表达水平。转染后不同时间加入托泊替康,MTr法检测SW620细胞的增殖;Hoechst33342染色和流式细胞仪检测SW620细胞的凋亡;Caspase-3活化试剂盒检测SW620细胞中caspase-3的活化。结果：Notch3siRNA转染可明显抑制SW620细胞中Notch3蛋白的表达;托泊替康作用于Notch3siRNA转染组细胞的IC50较对照CtrlsiRNA转染组显著降低（P〈0．05）;流式细胞仪检测显示沉默Notch3的表达可显著增强托泊替康诱导的结肠癌细胞凋亡（P〈0．05）和caspase-3活化（P〈0．05）。结论：siRNA沉默Notch3的表达可增强SW620细胞对托泊替康的化疗敏感性。     

miR-92a 在结直肠癌中的表达及其对肿瘤血管生成的作用*

目的:探讨miR-92a 在结直肠癌中的表达及其对肿瘤血管新生功能的影响和作用机制。方法:采用qRT-PCR方法检测广东医学院附属深圳南山医院2014年6 月至2015年12月经手术切除的25例结直肠癌组织和对应癌旁组织及4 种结直肠癌细胞（HCT 116、SW620、SW480、HT29）中miR-92a 的表达;免疫组织化学法检测结直肠癌和癌旁组织中CD31阳性表达的微血管密度（microvesseldensity,MVD）,Pearson相关性分析探讨miR-92a 表达与肿瘤血管新生MVD的相关性。通过转染miR-92a-mimic、inhibitor 上调或抑制结直肠癌细胞HCT 116、SW620 中miR-92a 的表达水平,采用小管形成实验检测miR-92a 不同表达对HUVEC小管形成的影响,免疫印迹法检测对其下游潜在靶点PTEN的蛋白表达的影响。结果:结直肠癌组织miR-92a 的表达水平显著高于对应癌旁组织（P < 0.01）;4 种人结肠癌细胞系miR-92a 的表达水平均显著高于正常肠上皮组织（P < 0.05）;结直肠癌组织CD31阳性微血管密度显著高于癌旁组织（P < 0.01）,miR-92a 表达水平与结直肠癌血管新生MVD呈显著正相关（r = 0.580,P = 0.01）;上调miR-92a 表达的HCT 116 细胞培养上清液可以显著促进HUVEC小管形成（P < 0.05）;上调miR-92a 表达可以显著抑制HCT116 细胞中PTEN蛋白表达水平（P < 0.01）。 结论:miR-92a 在结直肠癌细胞和组织中高表达,与肿瘤血管新生增加密切相关;miR-92a 可能通过抑制PTEN的表达发挥促进结直肠癌血管新生的生物学功能。      

siRNA 沉默HIF-1α在缺氧状态下对食管鳞癌细胞VEGF表达的影响

 目的 观察体外乏氧培养条件下食管鳞癌细胞系EC9706中HIF-1α和VEGF的表达，探讨HIF-1α在低氧条件下对食管鳞癌血管生成的调控作用。方法 CoCl2化学缺氧法模拟肿瘤缺氧环境，RT-PCR、免疫组化法和免疫印迹法分别检测缺氧状态下HIF-1α和VEGF在mRNA和蛋白水平的表达。采用化学合成小干扰RNA（siRNA）介导的RNA干扰技术（RNAi）用siRNA转染EC9706细胞。观察转染后HIF-1α沉默效果。结果 低氧条件下，EC9706细胞HIF-1amRNA水平稳定，蛋白表达显著升高，而VEGFmRNA和蛋白的表达均显著升高。SiRNA转染EC9706后能够显著下调HIF-1α的基因表达，同时VEGF基因的表达也受到明显抑制。结论 缺氧促使EC9706细胞HIF-1α在蛋白水平表达升高，并通过转录激活VEGF的机制调控食管鳞癌血管生成。     

Tanshinone II-A Inhibits Angiogenesis through Down Regulation of COX-2 in Human Colorectal Cancer

Angiogenesis plays a significant role in colorectal cancer (CRC) and cyclooxygenase-2 (COX-2) appears tobe involved with multiple aspects of CRC angiogenesis. Our aim was to investigate the inhibitory effects of TanII-A (Tanshinone II-A, Tan II-A) on tumor growth in mice, as well as alteration of expression of COX-2 andVEGF in CRC. We established the mice xenograft model of C26 CRC cell line, and injected 0.5, 1, 2mg/kg ofTan II-A and 1mg/kg of 5-FU in respectively in vivo. Then, we assayed tumor weight and volume, and evaluatedmicrovascular density and expression of VEGF. COX-2 promoter and COX-2 plasmids were transfected intoHCT-116 cells, followed by detection of COX-2 promoter activity by chemiluminescence, and detection of COX-2mRNA expression by fluorescence quantitative PCR. Taken together, the results showed Tan II-A could inhibittumor growth and suppress the VEGF level in vivo. HCT-116 cell experiments showed marked inhibitory effectsof Tan II-A on COX-2 and VEGF in a dose-dependent manner. The results indicate that Tan II-A can effectivelyinhibit tumor growth and angiogenesis of human colorectal cancer via inhibiting the expression level of COX-2and VEGF.     

RNA干扰MDM2下调人乳腺癌细胞中VEGF的合成并抑制肿瘤血管的生成

目的 探讨小干扰RNA（small interfering RNA, siRNA）抑制人乳腺癌细胞中鼠双微粒体2 （murine double minute 2, MDM2）的表达对癌细胞中血管内皮生长因子（vascular endothelial growth factor, VEGF）合成以及裸鼠移植瘤组织内血管生成的影响。方法 根据MDM2已知的cDNA序列设计并转录合成特异性siRNA,转染高表达MDM2的人乳腺癌MDA-MB-468细胞,低氧培养后应用蛋白质印迹法和ELISA法检测肿瘤细胞及其上清液中VEGF的水平。构建裸鼠乳腺癌移植瘤模型,观察MDM2沉默后肿瘤生长情况,蛋白质印迹法、免疫组织化学及ELISA方法检测荷瘤组织和裸鼠血清标本中的VEGF含量,并以CD34标记血管内皮细胞计数微血管密度（microvessel density, MVD）。结果 siRNA抑制MDM2表达后,MDA-MB-468细胞分泌的VEGF蛋白显著减少（P=0.006）。裸鼠移植瘤模型显示,封闭MDM2表达使荷瘤组织生长变慢（P=0.008）,且荷瘤小鼠血清VEGF水平明显减低（P=0.008）,荷瘤组织内MVD也明显降低（P=0.003）。结论 MDM2 siRNA能有效减低乳腺癌细胞中VEGF的合成,并抑制裸鼠移植瘤组织内新生血管的生成,为MDM2/VEGF途径抗肿瘤血管生成作用的研究及靶向药物开发提供了新的思路。     

Colon cancer cell-derived tumor necrosis factor-alpha mediates the tumor growth-promoting response in macrophages by up-regulating the colony-stimulating factor-1 pathway

The interplay between malignant and stromal cells is essential in tumorigenesis. We have previously shown that colony-stimulating factor (CSF)-1, matrix metalloprotease (MMP)-2, and vascular endothelial growth factor (VEGF)-A production by stromal cells is enhanced by CSF-1-negative SW620 colon cancer cells. In the present study, the mechanisms by which colon cancer cells up-regulate host factors to promote tumorigenesis were investigated. Profiling of tumor cell cytokine expression in SW620 tumor xenografts in nude mice showed increased human tumor necrosis factor (TNF)-alpha mRNA expression with tumor growth. Incubation of macrophages with small interfering (si) RNAs directed against TNF-alpha or TNF-alpha-depleted SW620 cell conditioned medium versus SW620 cell conditioned medium failed to support mouse macrophage proliferation, migration, and expression of CSF-1, VEGF-A, and MMP-2 mRNAs. Consistent with these results, human TNF-alpha gene silencing decreased mouse macrophage TNF-alpha, CSF-1, MMP-2, and VEGF-A mRNA expression in macrophages cocultured with human cancer cells. In addition, inhibition of human TNF-alpha or mouse CSF-1 expression by siRNA reduced tumor growth in SW620 tumor xenografts in mice. These results suggest that colon cancer cell-derived TNF-alpha stimulates TNF-alpha and CSF-1 production by macrophages, and that CSF-1, in turn, induces macrophage VEGF-A and MMP-2 in an autocrine manner. Thus, interrupting tumor cell-macrophage communication by targeting TNF-alpha may provide an alternative therapeutic approach for the treatment of colon cancer.     

长链非编码RNA AFAP1-AS1通过PTEN/p-AKT信号通路调控结直肠癌细胞增殖的分子机制研究

目的 探讨长链非编码RNA(lncRNA)肌动蛋白纤维相关蛋白1-反义RNA1(Actin filament-associatedprotein1-antisenseRNA1,AFAP1-AS1)调控结直肠癌(Colorectal cancer,CRC)细胞增殖的分子机制。方法 收集2014年7月—2018年6月深圳市第二人民医院消化内科和胃肠外科诊治的38例正常人及38例CRC患者的粪便标本,用Real time-PCR检测lncRNA AFAP1-AS1表达,同时检测人正常结肠上皮细胞株NCM460、人结直肠癌细胞株SW620和HCT116中lncRNA AFAP1-AS1的表达;将靶向siRNA转染到CRC细胞株SW620和HCT116中抑制AFAP1-AS1的表达;通过MTT测定si-AFAP1-AS1转染对CRC细胞增殖的影响;应用免疫印迹检测Cleaved caspase 3、Bcl-2、Bax、p-AKT、total-AKT和PTEN的水平。结果 与正常人相比,CRC患者粪便中AFAP1-AS1的表达显著上升(P＜0.01);与NCM460细胞相比,SW620和HCT116细胞中AFAP1-AS1的表达也显著升高(P＜0.01);si-AFAP1-AS1转染抑制SW620和HCT116的细胞生长(P＜0.01)。与NCM460细胞相比,si-AFAP1-AS1干扰上调了SW620和HCT116细胞中Cleaved caspase 3和促凋亡蛋白Bax的表达水平(P＜0.05),下调了抗凋亡蛋白Bcl-2的表达水平(P＜0.001);此外,si-AFAP1-AS1干扰降低了CRC细胞中p-AKT的蛋白水平,并增加了PTEN的表达(P＜0.01)。结论 正常人和CRC患者粪便中lncRNA AFAP1-AS1表达的差异有助于CRC的早期诊断,且lncRNA AFAP1-AS1在CRC细胞中表达上调,并通过PTEN/p-AKT信号通路调节CRC细胞的增殖和凋亡。lncRNA AFAP1-AS1有望成为CRC早期诊断的分子靶标。     

miR-23b-3p在结直肠癌中的表达及其靶向KLF3抑制结直肠癌细胞侵袭和迁移能力的机制研究

目的:研究miR-23b-3p在结直肠癌中的表达情况及对结直肠癌SW620细胞侵袭和迁移的影响机制。方法:收集2018年01月至2020年12月我院胃肠外科手术切除的58例结直肠癌组织及癌旁组织标本,以及结直肠癌细胞株SW480、SW620、HCT116、HT-29、Lovo和人正常结直肠黏膜细胞FHC,采用qPCR法检测miR-23b-3p和KLF3相对表达水平。采用脂质体转染技术将miR-23b-3p mimics、mimics-NC转染至SW620细胞,采用Transwell实验和划痕实验检测其侵袭和迁移能力的改变。利用生物信息学软件预测并通过双荧光素酶报告基因实验验证miR-23b-3p和KLF3的结合位点。将KLF3过表达质粒（pcDNA3.1-KLF3）或空载质粒（Vector）单独或联合miR-23b-3p mimics转染至SW620细胞,采用Transwell实验和划痕实验检测其侵袭和迁移能力的改变,采用qPCR和Western Blot实验检测SW620细胞中KLF3 mRNA及蛋白的表达。结果:miR-23b-3p在结直肠癌组织中的表达水平显著低于癌旁组织（P＜0.05）,并与患者TNM分期和远处转移有关（均P＜0.05）;miR-23b-3p在结肠癌细胞系中的表达水平均显著低于FHC细胞,上调miR-23b-3p表达能显著抑制SW620细胞的侵袭和迁移能力。KLF3在结直肠癌组织和细胞中高表达,与miR-23b-3p在结直肠癌组织中的表达呈负相关（r=-0.326,P=0.013）,双荧光素酶报告基因实验证实miR-23b-3p直接靶向调节KLF3的表达, KLF3过表达能促进SW620细胞的侵袭和迁移,同时转染miR-23b-3p mimics可下调KLF3蛋白和mRNA表达,逆转KLF3过表达对SW620细胞侵袭和迁移能力的促进作用。结论:miR-23b-3p在结直肠癌中低表达并与肿瘤患者TNM分期及远处转移相关,miR-23b-3p靶向调控KLF3表达抑制结直肠癌SW620细胞的侵袭和迁移能力。     

腺病毒介导VEGF-siRNA对荷人骨肉瘤裸鼠移植瘤生长的影响

目的:观察腺病毒介导的VEGF-siRNA对荷人骨肉瘤细胞株MG63裸鼠移植瘤生长的影响。方法:将Ad-VEGF-siRNA感染MG63,用噻唑蓝(thiazolyl blue,MTT)法检测细胞体外增殖活力,反转录-聚合酶链反应(reverse transeription-poly-merase chain reaction,RT-PCR)法检测VEGF抑制效果。建立裸鼠移植瘤模型,定期瘤内注射Ad-VEGF-siRNA,观察裸鼠致瘤率、肿瘤体积、抑瘤率及免疫组化法检测肿瘤组织中bcl-2的表达。结果:感染Ad-VEGF-siRNA能明显抑制MG63细胞的生长,瘤灶内注射Ad-VEGF-siRNA后裸鼠移植瘤的质量和体积均显著低于对照组(P<0.01)。TUNEL染色显示肿瘤细胞凋亡增加,免疫组化结果提示肿瘤组织中bcl-2表达明显减少(P<0.01)。结论:Ad-VEGF-siRNA可有效而特异地阻断VEGF基因表达,抑制裸鼠移植瘤生长,促进肿瘤细胞的凋亡。     

转凝蛋白在结直肠癌组织中的表达及其对SW480细胞恶性生物学行为的影响

目的:探讨转凝蛋白(transgelin,TAGLN)在结直肠癌(colorectal cancer,CRC)组织中的表达及其对SW480细胞增殖、迁移及侵袭的影响.方法:选取郑州大学附属肿瘤医院2015年5月至2016年8月收治的97例CRC患者的癌及配对的癌旁组织标本,以及人CRC细胞系SW620、SW480、HC...     

Secretogranin II impairs tumor growth and angiogenesis by promoting degradation of hypoxia‐inducible factor‐1α in colorectal cancer

Distant metastasis is a major cause of death in patients with colorectal cancer (CRC) but the management of advanced and metastatic CRC still remains problematic due to the distinct molecular alterations during tumor progression. Tumor angiogenesis is a key step in tumor growth, invasion and metastasis. However, the signaling pathways involved in angiogenesis are poorly understood. The results of the present study showed that secretogranin II (SCG2) was significantly downregulated in malignant CRC tissues, and higher expression of SCG2 was correlated with longer disease‐free survival and overall survival of CRC patients. The results of an animal study showed that ectopic expression of SCG2 significantly inhibited CRC tumor growth by disrupting angiogenesis. Furthermore, the inhibition of expression of vascular endothelial growth factor (VEGF) by SCG2 and rescue of VEGF effectively blocked SCG2‐induced inhibition of angiogenesis. Investigations into the underlying mechanism suggested that SCG2 promoted degradation of hypoxia‐inducible factor (HIF)‐1α by interacting with the von Hippel–Lindau tumor suppressor in CRC cells. Blocking of degradation of HIF‐1α effectively attenuated the SCG2‐mediated decrease in expression of VEGF in CRC cells. Collectively, these results demonstrated that treatment with SCG2 effectively inhibited CRC tumor growth by disrupting the activities of HIF‐1α/VEGF, thereby clarifying the anti‐tumor and anti‐angiogenesis roles of SCG2 in CRC, while providing a novel therapeutic target and a potential prognostic marker of disease progression.     

Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells

Vascular endothelial growth factor (VEGF) is associated with tumor angiogenesis and poor prognosis in human colorectal cancer (CRC). VEGF receptor-1 (VEGFR-1 or Flt-1) is a high-affinity receptor for VEGF and is typically considered specific to endothelial cells. Here we report the expression and function of VEGFR-1 in CRC cell lines. VEGFR-1 was expressed in all CRC cell lines studied as determined by RT-PCR, Western blot analysis, FACS, and ELISA. Treatment of the human CRC cell lines HT-29 and SW480 with VEGF-A (a ligand for both VEGFR-1 and -2) or VEGF-B (a ligand specific for VEGFR-1) led to activation of Erk-1/2, SAPK/JNK, and translocation of the p65 subunit of nuclear factor-kappaB into the nucleus. Both VEGF-A and -B led to significant induction of cell motility and invasiveness of CRC cells. Stimulation of cells with VEGF-A or -B also led to larger and more numerous colonies in soft agar. However, activation of VEGFR-1 did not increase CRC cell proliferation. In contrast to the previous paradigm that VEGFRs are not present on tumor cells of epithelial origin, we found that VEGFR-1 is present and functional on CRC cells, and activation by VEGF family ligands can activate processes involved in tumor progression and metastasis.