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Design of functional small interfering rnas targeting amyotrophic lateral sclerosis-associated mutant alleles

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Chinese Medical Journal 2011年第1期124卷 106-110页

作者： GENG Chang-ming DING Hong-liu Department of Neurology Shanghai People's Liberation Army 85th Hospital Shanghai 200052 China Brigham and Women's Hospital Harvard Medical School BostonMA 02115 USA

Background rna interference （rnai） is a potential cure for amyotrophic lateral sclerosis （ALS） caused by dominant, gain-of-function superoxide dismutase 1 （SOD1） mutations. The success of such therapy relies on the functional small interfering rnas （sirnas） that can effectively deliver rnai. This study aimed to design the functional sirnas targeting ALS-associated mutant alleles. Methods A modified dual luciferase system containing human SOD1 mrna target was established to quantify sirna efficacy. Coupled with validated sirnas identified in the literature, we analyzed the rationale of sirna design and subsequently developed an asymmetry rule-based strategy for designing sirna. We then further tested the effectiveness of this design strategy in converting a naturally symmetric sirna into functional sirnas with favorable asymmetry for gene silencing of SOD1 alleles. Results The efficacies of sirnas could vary tremendously by one base-pair position change. Functional sirnas could target the whole span of SOD1 mrna coding sequence as well as non-coding region. While there is no distinguishable pattern of the distribution of nucleobases in these validated sirnas, the high percent of GC count at the last two positions of sirnas （P18 and P19） indicated a strong effect of asymmetry rule. Introducing a mismatch at position 1 of the 5＇ of antisense strand of sirna successfully converted the inactive sirna into functional sirnas that silence SOD1 with desired efficacy. Conclusions Asymmetry rule-based strategy that incorporates a mismatch into sirna most consistently enhances rnai efficacy and guarantees producing functional sirnas that successfully silence ALS-associated SOD1 mutant alleles regardless target positions. This strategy could also be useful to design sirnas for silencing other disease-associated dominant, gain-of-function mutant genes.

关键词： amyotrophic lateral sclerosis neurodegenerative disease," superoxide dismutase 1," rna interference rna, small interfering

Silencing effect of recombinant plasmids PPARγ-pSUPER-EGFP on the expression of peroxisome proliferator-activated receptor γ

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Chinese Journal of Traumatology 2005年第6期8卷 352-357页

作者：杨策王正国陈力勇周健朱佩芳蒋建新 State Key Laboratory of Trauma Burns and Combined Injury Department 4Institute of Surgery Research Daping HospitalThird Military Medical UniversityChongqing 400042China

To investigate the silencing effect of gene encoding peroxisome proliferator-activated receptor γ （PPAR-γ） on the expression of tumor necrosis factor alpha （TNFα） by constructing vectors for rna interference in RAW264.7 cells. Methods： The pSUPER-EGFP vectors were used to transcribe functional small interfering rna （ sirna ）. Four pairs of oligonucleotides （ 64 nt ） targeting PPAR-γ gene were inserted into the downstream of the HI promotor, with their veracity confirmed by double digestion and sequencing. Western blotting and immunofluorescence assay were used to examine the silencing effect of PPAR-γ gene in RAW264.7 cells. Meanwhile, the TNFα level was determined by Sandwich ELISA. Results ： Compared with other recombinant pSUPER- EGFP vectors （R-pSUPER. EGFP）, R-pSUPER. EGFP2 induced the best silencing effect on the expression of PPART in RAW264.7 cells, which played an obvious inhibitory role in down-regulating the TNFα expression after the curcumin and lipopolysaccharide （LPS） stimulation. Conclusions： PPARγ-pSUPER-EGFP inducing a silencing effect on the expression of PPART can efficiently play a negative role in controlling the inflammatory responses of RAW264.7 cells.

关键词： PPAR alpha rna, small interfering Tumor necrosis factor- alpha

Silencing of high mobility group A1 enhances gemcitabine chemosensitivity of lung adenocarcinoma cells

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Chinese Medical Journal 2011年第7期124卷 1061-1068页

作者： CAO Yuan-dong HUANG Pei-lin SUN Xin-chen MA Jun JIN Zhi-liang CHENG Hong-yan XU Rui-zhi LI Fan QIN Shu-kui DENG Yu-xia GE Xiao-lin Department of Oncology Zhong Da Hospital Affiliated to Southeast University Nanjing Jiangsu 210009 China Department of Pathology and Pathophysiology School of Biological Science and Medical Engineering Southeast University Nanjing Jiangsu 210009 China School of Medicine School of Biological Science and Medical Engineering Southeast University Nanjing Jiangsu 210009 China Department of Oncology the 8 lth Hospital of People's Liberation Army Nanjing Jiangsu 210034 China

Background The high mobility group A1 （HMGA1） proteins are architectural transcription factors found to be overexpressed in iung adenocarcinoma. Lentivirus-mediated rna interference （rnai） technology is a powerful tool for silencing endogenous or exogenous genes in human cancer cells. Our preliminary study shows that gemcitabine inhibits growth of the human lung cancer cell line SPCA-1 and induces apoptosis, and this effect might link with down-regulation of HMGA1 expression. This study aimed to investigate the chemosensitivity change of the lung adenocarcinoma cells SPCA-1 after HMGA1 inhibition by Jentivirus-mediated rnai.

关键词： rna, small interfering high mobility group AI proteins apoptosis chemosensitivity

Effect of blocking Ras signaling pathway with K-Ras sirna on apoptosis in esophageal squamous carcinoma cells

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Journal of Traditional Chinese Medicine 2013年第3期33卷 361-366页

作者： Xinjie Wang Yuling Zheng Qingxia Fan Xudong Zhang Department of Integrated Chinese and Western Medicine the First Affiliated Hospital Zhengzhou University Department of Tumor the First Affiliated Hospital Zhengzhou University

OBJECTIVE： To study the effect of rnai silencing of the K-Ras gene on Ras signal pathway activity in EC9706 esophageal cancer cells. METHODS： EC9706 cells were treated in the follow- ing six groups： blank group （no transfection）, nega- tive control group （transfection no-carrier）, trans- fection group （transfected with pSilencer-siK-ras）, taxol chemotherapy group, taxol chemotherapy plus no-carrier group, taxol chemotherapy plus transfection group. Immunocytochemistry, Reverse transcription-polymerase chain reaction and west- ern blotting were used to analyze the expression of MAPK1 （mitogen-activated protein kinases 1） and cyclin D1 in response to sirna （small interfering rna） transfection and taxol treatment. RESULTS： K-Ras （K-Ras gene） sirna transfection of EC9706 esophageal squamous carcinoma cells de- creased the expression of K-Ras, MAPK1 and cyclinD1 at the mrna and protein level. Reverse tran- scription-polymerase chain reaction indicated that the expression levels of MAPK1 and cyclin D1 mrnas were significantly lower in the transfection group than in the blank group （P〈0.05）. Western blotting showed that 72 h after EC9706 cell trans- fection, the expression levels of MAPK1 and cyclin D1 proteins had decreased in all groups, and the ex- pression levels in the transfection group were sig- nificantly inhibited as compared with the blank group. Apoptosis increased significantly in the transfection group or after addition of taxol as com- pared with the blank group and the no-carrier group. The degree of apoptosis in the taxol plus transfection group was more severe. CONCLUSION： Apoptosis increased significantly in EC9706 esophageal carcinoma cells after sirna-me- diated inhibition of Ras signaling, with the most ob- vious increase observed in the transfection plus tax- ol chemotherapy group. Ras knockdown therefore increased cellular sensitivity to the chemotherapeu- tic agent, taxol. Ras knockdown also down-regulat- ed the expression of the downstream genes, M

关键词： rna, small interfering Genes, ras Rassignal pathway Esophageal neoplasms Taxol Apoptosis Chemotherapy sensitivity