STAR Protocols
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2021
·
IF 0
Lidia Wrobel,Farah H. Siddiqi,David C. Rubinsztein
Summary We present a protocol for in vivo siRNA-mediated knockdown of a gene of interest in mouse liver using systemic delivery via intravenous injection. We describe a step-by-step protocol for delivery of siRNA particles, with tips on how to optimize dosage. We detail steps for feeding/starving cycles as well as for liver tissue isolation, followed by gene expression analysis, measured at the mRNA and protein levels. For complete information on the generation and use of this protocol, please refer to Wrobel et al. (2020) .
研究领域:
分子生物学
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遗传学
✓ 已验证
DOI
PubMed
JOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH
·
2021
·Vol. 47
·期 6
·2196-2203
·
IF 1.73
Lianhua Wang,Qiming Huang,Qingqing Lin,Lvxuan Chen,Qiyang Shi
Aim Endometrial cancer (EC) is a common type of malignant gynecological cancer. Small nucleolar RNA host gene 9 (SNHG9) has been discovered to serve a role in several types of cancer; however, the role of SNHG9 in EC remains unclear. The present study aimed to investigate the effects of lncRNA SNHG9 on cell proliferation and glycolysis in EC cells. Methods SNHG9 and hexokinase 2 (HK2) mRNA expression levels were measured by reverse transcription-quantitative PCR. Glucose consumption and lactate production were detected by the glycolysis cell-based assay kit. Cell Counting Kit-8 and colony formation assays were conducted to detect cell proliferation. The knockdown experiments of SNHG9 and HK2 were carried out by transfection of corresponding small interference RNAs (siRNA). The SNHG9-overexpressed plasmid was transfected into the cells to upregulate SNHG9. HK2 protein levels were analyzed by western blotting assay. Results SNHG9 expression levels were significantly upregulated in EC tissues and cells. The knockdown of SNHG9 subsequently effectively attenuated cell proliferation and glycolysis in vitro, while SNHG9 overexpression reported the opposite effects. Notably, the transfection of 2-DG partially reversed the promoting effect of SNHG9 on glycolysis. Downregulation of HK2 markedly decreased cell proliferation and glycolysis in EC cells antagonized SNHG9. Conclusion Either downregulation of SNHG9 or HK2 inhibits EC cell proliferation and glycolysis via repressing EC cell proliferation and glycolysis.
研究领域:
肿瘤学
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分子生物学
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癌症遗传学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Heliyon
·
2025
·
IF 3.6
Kan Liu,Jinlong Zhu,Yangyang Bao,Jin Fang,Shuihong Zhou,Jun Fan
Multiple studies have demonstrated a significant association between glucose transporter-1 (GLUT1) and the development and recurrence of adenoid cystic carcinoma (ACC). In this study, we investigated the impact of GLUT1 knockdown on adenoid cystic carcinoma. Our findings revealed that hypoxic conditions promoted the progression and autophagy of SACC83 and SACC-LM cell lines, an effect that was mitigated by GLUT1 knockdown. In vivo experiments showed that the combination of lentivirus-delivered GLUT1 shRNA and autophagy inhibitor chloroquine (CQ) produced the most substantial reduction in tumor volume, weight, Ki67 expression, and autophagy in tumor tissues. In conclusion, hypoxia facilitates ACC progression by upregulating GLUT1 expression. The suppression of GLUT1 expression and autophagy effectively inhibited ACC cell proliferation both in vitro and in vivo .
研究领域:
肿瘤学
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分子生物学
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细胞生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Indian Journal of Hematology and Blood Transfusion
·
2024
·
IF 0.7
Liu Xiaoyu,Yang Lin,Guan Kunping,Chen Zhen,Yang Hua
Purpose This study aims to investigate the increased toxicity of Cytarabine (Ara-c) by knockdown of chromosome segregation 1-like (CSE1L) in acute myeloid leukemia(AML) cells(Kasumi-1, U937, and THP-1 cells) and to explore its possible mechanisms. Methods Target gene silencing was achieved using the shRNA-mediated lentivirus method. Apoptosis was identified using the Annexin V PE/7-AAD double-staining assay. Cell viability was assessed with the Cell Counting Kit-8 (CCK-8) assay. Protein expression was detected by Western blotting. Results In vitro, knocking down CSE1L promoted caspase-3 and caspase-9 proteins expression and induced apoptosis in AML cells. Knockdown of CSE1L enhanced AML cells' sensitivity to Ara-c. knockdown of CSE1L reduced the expression levels of p-JKA2 and p-STAT3 proteins, while no significant difference was observed in the expression levels of total JAK2 and STAT3 proteins. Furthermore, JAK2 overexpression reversed the increase in Ara-c toxicity to AML cells caused by CSE1L knockdown. Conclusion In conclusion, our study reveals that CSE1L is a potential therapeutic target for overcoming Ara-c resistance in AML cells. Thus, we have gained new insights into the oncogenic process of CSE1L in AML cells and raised the prospect of knockdown of CSE1L in AML in combination with cytarabine-targeted therapy.
研究领域:
肿瘤学
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血液学
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分子生物学
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细胞生物学
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药理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
CARDIOVASCULAR PATHOLOGY
·
2017
·
IF 2.359
Lei Zhang,Hailing Cheng,Yuxia Yue,Shuangzhan Li,Daping Zhang,Ruili He
Background Long non-coding RNAs (lncRNAs) have been revealed to participate in the pathological events associated with atherosclerosis . However, the exact role of lncRNA taurine-up-regulated gene 1 (TUG1) and its possible molecular mechanism in atherosclerosis remain unidentified. Methods High-fat diet (HFD)-treated ApoE −/− mice were used as an in vivo model of atherosclerosis. Ox-LDL-induced macrophages and vascular smooth muscle cells (VSMCs) were employed as cell models of atherosclerosis. qRT-PCR was performed to detect the expression of TUG1 and miR-133a. Serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were analyzed by commercially available enzyme kits. Oil red O and hematoxylin and eosin (H&E) staining were conducted to examine atherosclerotic lesion. Luciferase reporter assay combined with RNA immunoprecipitation (RIP) was applied to confirm the interaction between TUG1, miR-133a and FGF1 . Cell proliferation ability was determined by Cell Counting Kit-8 (CCK-8) assay and trypan blue dye exclusion test. Cell apoptosis was evaluated with TUNEL assay. Expression and production of inflammatory cytokines was measured with western blot and ELISA analysis. Results TUG1 expression was up-regulated in HFD-treated ApoE −/− mice, as well as in ox-LDL-induced RAW264.7 and MOVAS cells. TUG1 knockdown inhibited hyperlipidemia , decreased inflammatory response, and attenuated atherosclerotic lesion in HFD-treated ApoE −/− mice. TUG1 could function as a molecular sponge of miR-133a to suppress its expression. TUG1 overexpression accelerated cell growth, improved inflammatory factor expression, and inhibited apoptosis in ox-LDL-stimulated RAW264.7 and MOVAS cells, while this effect was abated after transfection with miR-133 mimic. Moreover, fibroblast growth factor 1 (FGF1) was identified as a direct target of miR-133a. Restored expression of FGF1 overturned the effect of miR-133a on cell proliferation, inflammatory factor secretion and apoptosis in ox-LDL-treated RAW264.7 and MOVAS cells. Finally, TUG1 was revealed to up-regulate FGF1 expression by sponging miR-133a. Conclusion TUG1 knockdown ameliorates atherosclerosis by modulating FGF1 via miR-133a, raising the possibility of targeting TUG1 as an atheroprotective therapeutic strategy.
研究领域:
分子生物学
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心血管疾病
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遗传学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
MACROMOLECULAR BIOSCIENCE
·
2021
·Vol. 21
·期 5
·2100025
·
IF 4.979
Qinping Yang,Yanliang Dong,Xuanyu Wang,Zhihao Lin,Mingyu Yan,Weiwei Wang,Anjie Dong,Jianhua Zhang,Pingsheng Huang,Changrong Wang
pH-sensitive polyelectrolytes provide enormous opportunity for siRNA delivery. Especially, their tertiary amine structures can not only bind genes but also act as pH-sensitive hydrophobic structure to control genes release. However, the influence of molecular structures on siRNA delivery still remains elusive, especially for the asymmetric alkyl substituents of the tertiary amine groups. Herein, a library of N -methyl- N -alkyl aminoethyl methacrylate monomers (MsAM) with asymmetric alkyl substituents on the tertiary amine group is synthesized and used to prepare a series of tri-block polycationic copolymers poly(aminoethyl methacrylate)-block-poly ( N -methyl- N -alkyl aminoethyl methacrylate)-block-poly(ethylene glycol methacrylate) (PAMA-PMsMA-PEG). And the properties of these polycations and their self-assembled micelles are characterized, including molecular structure, proton buffering capacity, pH-sensitivity, size, and zeta potential. With the length increase of one alkyl substituent, the proton buffering capacity of both monomers and polycations is demonstrated to be narrowed down. The siRNA delivery efficiency and cytotoxicity of these micelles are also evaluated on HepG2 cells. In particular, poly(aminoethyl methacrylate)-block-poly( N -methyl- N -ethyl aminoethyl methacrylate)-block-poly(ethylene glycol methacrylate) (PAMA-PMEMA-PEG) elicited the best luciferase knockdown efficiency and low cytotoxicity. Besides, PAMA-PMEMA-PEG/siRRM2 also induced significant anti-tumor activity in vitro. These results indicated PAMA-PMEMA-PEG has potential for further use in the design of gene vehicles with the improved efficiency of siRNA delivery.
研究领域:
聚合物化学
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分子生物学
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基因治疗
✓ 已验证
DOI
PubMed
高影响因子
PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY
·
2023
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IF 4.7
Zhenyu Li,Mengling Chen,Wenjie Bai,Shuxia Zhang,Liwei Meng,Wei Dou,Jinjun Wang,Guorui Yuan
Carboxylesterases (CarEs) are a multifunctional superfamily of enzymes and play an important role in detoxification of various insecticides in insects. The oriental fruit fly, Bactrocera dorsalis , is one of the most destructive agricultural pests and has developed different degrees of resistance to organophosphates in field. However, the involvement of BdCarEs in tolerance or resistance to other alternative insecticides are still unclear. In the present study, 33 BdCarEs genes were identified based on the genome database of B. dorsalis . Phylogenetic analysis demonstrated that they were classified into nine clades, with abundance of α -esterases. Meanwhile, the sequence characterization and the chromosome distribution were also analyzed. The spatiotemporal expression analysis of BdCarEs genes suggested that the diversity of potential function in different physiological processes . With the exception of BdCarE21 , all BdCarEs genes responded to at least one insecticide exposure, and BdCarE20 was found to be up-regulated after exposure to all five tested insecticides individually. Eight BdCarEs genes were overexpressed in MR strain when compared to that in SS strain. Subsequently, knockdown the expression of representative BdCarEs genes significantly increased the susceptibility of the oriental fruit fly to corresponding insecticides, which indicated that the tested BdCarEs genes contributed to one or multiple insecticide detoxification. These findings provide valuable insights into the potential role in respond to tolerance or resistance to insecticides with different mode of action, and will facilitate development of efficiency management strategy for B. dorsalis .
研究领域:
昆虫学
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分子生物学
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基因组学
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农药科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2023
·
IF 6.1
Yun Huang,Junyue Zheng,Peizhuo Wu,Yu Zhang,Lihong Qiu
Cytochrome P450 CYP6B7 has previously been proved to be associated with fenvalerate-resistance in Helicoverpa armigera. Here, how CYP6B7 is regulated and involved in the resistance of H. armigera is studied. Seven base differences (M1–M7) were found in CYP6B7 promoter between a fenvalerate-resistant (HDTJFR) and a susceptible (HDTJ) strain of H. armigera. M1–M7 sites in HDTJFR were mutated into the corresponding base in HDTJ, and pGL3-CYP6B7 reporter genes with different mutation sites were constructed. Fenvalerate-induced activities of reporter genes mutated at M3, M4, and M7 sites were significantly reduced. Transcription factors Ubx and Br, whose binding sites contain M3 and M7, respectively, were overexpressed in HDTJFR. Knockdown of Ubx and Br results in significant expression inhibition of CYP6B7 and other resistance-related P450 genes, and increase of sensitivity of H. armigera to fenvalerate. These results indicate that Ubx and Br regulate the expression of CYP6B7 to mediate the fenvalerate-resistance in H. armigera.
研究领域:
昆虫学
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分子生物学
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遗传学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Synthetic Biology
·
2025
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IF 3.9
Hanqing Zhang,Luai R. Khoury,Peng Xu
Yarrowia lipolytica, a nonconventional yeast, has become an industrial workhorse to synthesize valuable compounds, including lipids, oleochemicals, and nutraceuticals. While the synthetic biology toolkits to engineer the genome and endogenous metabolic pathways are not as developed as Baker’s yeast, it has emerged as a promising microbial host for industrial applications. In this study, we examined the multiplexed editing capability of the CRISPR-AsCpf1 coupled with gRNAs generated from either a yeast native promoter or an orthogonal T7 promoter, which yielded 73.3% editing efficiency for up to four target genes and 100% editing efficiency for two genes. We also attempted two strategies to enhance homology-directed recombination (HDR) efficiency; only minor improvements were observed. We further demonstrated that CRISPR-dAsCpf1 with T7-driven gRNA achieved significant gene knockdown compared to the CRISPR-RfxCas13d system. Its knockdown efficiency was comparable to that of an antisense T7 promoter system. Taken together, this work provides a facile toolkit that enables efficient and multiplexed genome editing and transcriptional knockdown of critical genes by combining CRISPR-Cpf1 with an orthogonal T7 transcription system in Y. lipolytica.
研究领域:
合成生物学
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基因工程
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微生物生物技术
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL IMMUNOPHARMACOLOGY
·
2025
·
IF 4.7
Zhongcheng Zhu,Xiaolin Dou,Qizhen Chen,Yebin Lu
Acute pancreatitis (AP) has a high mortality, involving oxidative stress and mitochondrial damage. Nuclear receptor subfamily 4 group A member 3 (Nr4a3) and BTG anti-proliferation factor 2 (Btg2) have a close connection with mitochondrial damage. Here, we found that Nr4a3 expression was upregulated in pancreatic tissues of AP mice induced by cerulein. To elucidate Nr4a3's role, we injected adeno-associated virus 9 (AAV9) carrying Nr4a3-targeting short hairpin RNA into the pancreatic duct. This intervention reduced pancreatic edema, inflammatory infiltration, and acinar cell necrosis by suppressing pancreatic enzyme activation. Moreover, Nr4a3 knockdown improved mitochondrial function in pancreatic acinar cells, evidenced by increased mitochondrial membrane potential, enhanced ATP production, and decreased superoxide levels. It also inhibited cell apoptosis by suppressing the activation of the caspase pathway. In vitro, Nr4a3 knockdown in CCK-treated pancreatic acinar cells decreased oxidative stress, mitigated mitochondrial damage, and suppressed apoptosis, while Nr4a3 overexpression exacerbated cell injury. Further investigation identified Btg2 as a downstream target of Nr4a3. Btg2 expression was elevated in pancreatic acinar cells of AP mice, and siRNA-mediated Btg2 knockdown alleviated CCK-induced cell injury. Mechanistically, Nr4a3 promoted Btg2 transcription, thereby promoting mitochondrial damage in pancreatic acinar cells and leading to cell apoptosis. Btg2 overexpression reversed the protective effects of Nr4a3 knockdown. Overall, our findings demonstrate that downregulation of Nr4a3 exerts anti-AP effects by regulating Btg2, providing potential therapeutic targets for AP.
研究领域:
分子生物学
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细胞生物学
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生物化学
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医学研究
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遗传学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Nanomaterials
·
2022
·Vol. 12
·期 12
·2072
·
IF 5.719
Tsogzolmaa Ganbold,Qingming Bao,Hai Xiao,Dolgorsuren Zurgaanjin,Caifeng Liu,Shuqin Han,Agula Hasi,Huricha Baigude
Ischemic stroke activates toll-like receptor 4 (TLR4) signaling, resulting in proinflammatory polarization of microglia and secondary neuronal damage. Herein, we report a novel lipid-nanoparticle (LNP)-mediated knockdown of TLR4 in microglia and amelioration of neuroinflammation in a mouse model of transient middle cerebral artery occlusion (tMCAO). siRNA against TLR4 (siTLR4) complexed to the novel LNP (siTLR4/DoGo310), which was based on a dioleoyl-conjugated short peptidomimetic (denote DoGo310), was readily internalized by the oxygen–glucose-deprived (OGD) mouse primary microglia, knocked-down TLR4, and polarized the cell to the anti-inflammatory phenotype in vitro. Systemic administration of siTLR4/DoGo310 LNPs in the tMCAO mice model resulted in the accumulation of siRNA mainly in the Iba1 positive cells in the peri-infarct. Analysis of the peri-infarct brain tissue revealed that a single injection of siTLR4/DoGo310 LNPs led to significant knockdown of TLR4 gene expression, reversing the pattern of cytokines expression, and improving the neurological functions in tMCAO model mice. Our data demonstrate that DoGo310 LNPs could be a promising nanocarrier for CNS-targeted siRNA delivery for the treatment of CNS disorders.Keywords:lipid nanoparticles;siRNA delivery;microglia;neuroinflammation;stroke
研究领域:
神经科学
·
药学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANTI-CANCER DRUGS
·
2023
·Vol. 34
·期 9
·1046
·
IF 2.3
Zhou Qidong,Wu Hao,Miao Fengze,Cao Yirui,Liang Lifei,Huang Jiahua,Qian Zhiyu
Cisplatin-based chemotherapy plays a vital role in the management of muscle-invasive bladder cancer (MIBC); however, off-tumor toxicity and resistance often lead to cancer recurrence and eventual treatment failure. The loss of function of the nucleotide excision repair gene excision repair cross-complementing rodent repair deficiency gene 2 ( ERCC2 ) in cancer cells correlates with sensitivity to cisplatin, while its overexpression causes cisplatin resistance. Small interfering RNA (siRNA) knockdown of ERCC2 combined with cisplatin treatment may improve therapeutic outcomes in patients with bladder cancer. Here, we aimed to develop macrophage-derived mimetic nanovesicles (MNVs) as a nanoplatform for the simultaneous delivery of cisplatin and ERCC2 siRNA for enhancing the efficacy of bladder cancer chemotherapy. The cellular uptake, gene down-regulation, tumor inhibition effects, and biosafety of the synthesized nanodrugs (MNV-Co) as a synergistic therapeutic strategy for MIBC were evaluated in vitro and in vivo . The results indicated high efficacy of MNV-Co against MIBC and low off-tumor toxicity. Furthermore, by down-regulating ERCC2 mRNA and protein levels, MNV-Co improved chemosensitivity, promoted cancer cell apoptosis, and effectively suppressed tumor growth. This study presents a potential approach for delivering cisplatin and ERCC2 siRNA concurrently to treat bladder cancer using a biomimetic nanosystem.
研究领域:
肿瘤学
·
药学
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纳米技术
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分子生物学
✓ 已验证
DOI
PubMed
高影响因子
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