Lentivirual vector-mediated doxycycline-inducible iASPP gene targeted RNA interference in hepatocellular carcinoma

Background and Objective:iASPP, an inhibitory member of the apoptosis-stimulating proteins of p53 (ASPP) family, has been found to be up-regulated in various human tumor types.This study was to construct an efficient doxycycline-regulated, lentiviral vector-mediated knockdown system for iASPP that will allow for inducible down-regulation of iASPP gene expression and preliminary functional analysis.Methods:A pair of complementary oligos with hairpin structures targeting the iASPP gene and a negative control ...     

靶向糖基转移酶shRNA对前列腺癌细胞增殖能力的影响

目的 抑制前列腺癌细胞pc-3中Grit-V的表达,研究下调Grit-V后对前列腺癌细胞pc-3增殖能力的影响.方法 设计靶向针对Gnt-V基因的shRNA,将其转染进前列腺细胞pc-3,利用G418筛选出稳定转染的细胞.通过RT-PCR检测转染前后细胞中Grit-V mRNA含量变化,以及cck-8法检测shRNA表达质粒对前列腺癌癌细胞增殖能力的影响.结果 Gnt-V shRNA表达质粒成功地转染前列腺癌细胞,mRNA表达量下降了73%.Cck-8法证明和对照组相比,pc-3Cat-V/1079的增殖能力降低.结论shRNA表达质粒可下调前列腺癌pc-3中Cat-V mRNA的表达.增殖实验证明下调Cnt-V表达后,细胞增殖能力减弱.     

抑制整合素α9基因的表达对黑色素瘤细胞B16F1的生长和肺转移的影响

目的 观察shRNA干扰整合素α9（ITGA9）的表达对黑色素瘤细胞B16F1的生长和肺转移的影响。方法 用RNA干扰技术下调B16F1中ITGA9的表达,建立小鼠皮下成瘤和肺转移模型,观察肿瘤生长情况,计数肺转移灶数量。结果 ITGA9-shRNA转染组的肿瘤生长速度减慢（P＜0.05）,实验终点,该组肿瘤平均体积与scramble-shRNA组相比下降36%;肺转移灶数量显著减少（P＜0.05）。结论 下调ITGA9的表达可抑制黑色素瘤细胞B16F1在小鼠体内的生长和肺转移。ITGA9可能成为黑色素瘤的治疗靶点。     

Tumor-Derived Transforming Growth Factor-β is Critical for Tumor Progression and Evasion from Immune Surveillance

Tumors have evolved numerous mechanisms by which they can escape from immune surveillance. One of these is to produce immunosuppressive cytokines. Transforming growth factor-β(TGF-β) is a pleiotropic cytokine with a crucial function in mediating immune suppression, especially in the tumor  microenvironment. TGF-β produced by T cells has been demonstrated as an important factor for suppressing antitumor immune responses, but the role of tumor-derived TGF-β in this process is poorly understood. In this study, we demonstrated that knockdown of tumor-derived TGF-β using shRNA resulted in dramatically reduced tumor size, slowing tumor formation, prolonging survival rate of tumor-bearing mice and inhibiting metastasis. We revealed possible underlying mechanisms as reducing the number of myeloid-derived suppressor cells (MDSC) and CD4+Foxp3+ Treg cells, and consequently enhanced IFN-γ production by CTLs. Knockdown of tumor-derived TGF-β also significantly reduced the conversion of naïve CD4+ T cells into Treg cells in vitro. Finally, we found that knockdown of TGF-β suppressed cell migration, but did not change the proliferation and apoptosis of tumor cells in vitro. In summary, our study provided evidence that tumor-derived TGF-β is a critical factor for tumor progression and evasion of immune surveillance, and blocking tumor-derived TGF-β may serve as a potential therapeutic approach for cancer.     

Short-Hairpin RNA-Mediated MTA2 Silencing Inhibits Human Breast Cancer Cell Line MDA-MB231 Proliferation and Metastasis

Objective: To observe the effects of metastasis-associated tumor gene family 2 (MTA2) depletion on humanbreast cancer cell proliferation and metastasis. Methods: A short-hairpin RNA targeting MTA2 was chemicallysynthesized and transfected into a lentivirus to construct Lv-shMTA2 for infection into the MDA-MB231human breast cancer cell line. At 48 hours after infection cells were harvested and mRNA and protein levels ofMTA2 were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting,respectively. Cell viability and metastasis were assessed by CCK-8, wound-healing assay and Transwell assay,respectively. In addition, a xenograft model of human breast cancer was constructed to investigate cancerouscell growth and capacity for metastasis. Results: After infection with Lv-shMTA2, mRNA and protein levelsof MTA2 was significantly reduced (p<0.05) and MDA-MB231 cell proliferation and metastasis were inhibited(p<0.05). In addition, mean tumor size was smaller than that in control group nude mice (p<0.05) and numbersof metastatic deposits in lung were lower than in control group mice (p<0.05). Depletion of MTA2 affectedMMP-2 and apoptosis-related protein expression. Conclusions: For the first time to our knowledge we showedthat MTA2 depletion could significantly inhibit human breast cancer cell growth and metastasis, implying thatMTA2 might be involved in the progression of breast cancer. The role of MTA2 in breast cancer growth andmetastasis might be linked with regulation of matrix metalloproteinase and apoptosis.     

AEG-1表达下调对脑胶质瘤U373细胞的放射增敏效应

目的:探讨AEG-1基因表达下调对人脑胶质瘤细胞U373放射敏感性的影响。方法:以人MTDH/AEG-1（NM-178812）为靶标设计shRNA序列,慢病毒介导将AEG-1 shRNA转染至胶质瘤U373细胞中。荧光定量PCR及Western blot测定转染前后AEG-1 mRNA及蛋白的表达;克隆形成实验评估AEG-1基因下调后U373细胞放射敏感性;流式细胞术检测AEG-1下调后U373细胞凋亡及细胞周期分布。结果:通过慢病毒介导的shRNA转染,构建了AEG-1表达稳定下调的U373-shAEG-1细胞系,有效抑制了胶质瘤U373细胞中AEG-1的表达(抑制率84%,P<0.05),增加了凋亡细胞的比例（13.07%±0.28%,P<0.05）,并提高细胞周期中S期细胞比例（58.18％,P<0.01）,且AEG-1基因表达下调后U373细胞的D0值 （1.60Gy） 和Dq值 （1.06Gy）均明显低于空白对照组及阴性对照组细胞（P<0.05）。结论:下调AEG-1可以增强人脑胶质瘤U373细胞的放射敏感性,其机制与诱导细胞凋亡及干预细胞周期分布有关。     

Hoxa10真核表达载体增强K562细胞对柔红霉素敏感度的研究

目的构建高效干扰Hoxa10基因表达的shRNA真核载体,探讨其对人慢性髓系白血病细胞株K562对化疗药物柔红霉素（daunorubicin,DNR）敏感度的影响。方法设计合成针对Hoxa10的特异性shRNA寡核苷酸链,构建真核表达载体pGPHI/GFP/Neo-Hoxa10并测序,应用阳离子脂质体转染K562细胞。实验分三组：正常对照组、阴性对照组、实验组（分别为正常K562细胞、阴性对照质粒转染K562细胞、pGPHI/GFP/Neo-Hoxa10转染K562细胞）,三组细胞均加入不同浓度的DNR。RT-PCR检测Hoxa10 mRNA的表达,应用MTT检测各组细胞对DNR的敏感度,流式细胞术检测细胞凋亡率。结果成功构建pGPHI/GFP/Neo-Hoxa10载体并转染K562细胞,该载体能有效降低Hoxa10mRNA表达水平ODR=（38.864±4.488）%;MTT结果显示Hoxa10重组载体可显著降低K562细胞对DNR的IC50（P<0.05),对DNR的敏感度能提高约3.58倍。流式细胞术结果显示,该载体下调Hoxa10的表达后,细胞的凋亡率显著增加,可达（16.207±4.891）%（P<0.05),且联合DNR后细胞凋亡率明显提高,凋亡率达（76.887±0.967）%（P<0.05)。结论本实验构建的靶向Hoxa10的真核表达载体对K562细胞中Hoxa10的表达有明显抑制作用,并能明显增强其对DNR的化疗敏感度。     

Inhibiting calcium-activated chloride channel ANO1/TMEM16A suppresses migration of tumor epithelial cells

Uncontrolled cell migration is a common feature of tumor metastasis and formation. Understanding the molecular targetscritically involved in cell migration process can lead to the development of potentially novel therapeutic strategies for controlling invasion of tumor cells. In this study, we showed that calcium-activated chloride channel ANO1/TMEM16A played an important role in cell migration and inhibition of ANO1 channel function suppressed the migration of tumor epithelial cells. Silencing ANO1 by small hairpin RNA (shRNA) resulted in suppression of cell migration and invasiveness in cancer cell lines. In addition, pharmacological inhibition of ANO1 by the channel specific inhibitor T16Ain-A01 significantly slowed down the migration andinvasion of tumor epithelial cells in a dose-dependent manner. Taken together, our findings have demonstrated that calcium-activated chloride channel ANO1 contributes to cell migration, and specific ANO1/TMEM16A inhibitors can be the promising candidate to develop new therapies for cancer metastasis.     

GALNT12 对急性髓系白血病细胞生长影响的研究

目的 探索GALNT12的表达对急性髓系白血病细胞生长的影响。方法 通过对数据库中急性髓系白血病(AML)病人相关数据进行分析,明确GALNT12 mRNA的表达水平与AML病人的生存率之间的关系。构建GALNT12shRNA慢病毒载体并包装GALNT12shRNA慢病毒,以此为工具构建GALNT12沉默的白血病THP-1细胞系。采用杂乱序列(shScramble)的慢病毒感染的THP-1细胞系做对照,进而观察GALNT12沉默的白血病THP-1细胞和shScramble的慢病毒感染白血病THP-1细胞生长的差异。采用流式细胞分选术收集GFP阳性细胞并用于观察GALNT12对 AML细胞生长的影响。结果 对三个数据库中AML病人的数据进行统计分析结果显示,GALNT12的表达与AML患者的生存周期呈显著的负相关;且GALNT12在急性髓系白血病THP-1细胞系中高表达。本研究成功构建了GALNT12沉默的白血病THP-1细胞系。 研究发现,GALNT12 shRNA1干扰的THP-1细胞与shScramble相比,生长速度明显减慢。结论 本研究证实GALNT12的表达能够促进急性髓系白血病细胞的生长,有利于急性髓系白血病的发生发展,也为寻找治疗急性髓系白血病的有效分子靶点增添了新的内容。     

靶向survivin shRNA在肿瘤治疗中的研究进展

生存素在细胞凋亡抑制中起重要作用,越来越多的研究者将其作为肿瘤基因治疗的理想靶点。应用RNA干扰技术靶向抑制生存素的表达,从而抑制肿瘤细胞增殖、促进肿瘤细胞凋亡、抑制肿瘤血管生成、增强肿瘤细胞对放化疗的敏感性。