ABSTRACT
To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected TrxR1 knockout sites were obtained. Next, we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector. After transfection of the plasmid into HCT-116 cells, TrxR1 knockout HCT-116 cells were selected using puromycin resistance. The TrxR1 knockout efficiency was identified and verified by DNA sequencing, immunoblotting, TRFS-green fluorescent probe, and cellular TrxR1 enzyme activity detection. Finally, the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay. The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116 (HCT116-TrxR1-KO) cells. The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation. Based on these results, we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.
Subject(s)
Humans , CRISPR-Cas Systems/genetics , Gene Editing , Gene Knockout Techniques , HCT116 Cells , /metabolismABSTRACT
Thioredoxin reductase (TrxR),a family of antioxidant family member,is widely distributed in the body.Its main function is to regulate the redox status of enzymes and transcription factors at the cellular level,and to participate in cell growth,pro-liferation and apoptosis.Meanwhile,it also provides favorable conditions for the occurrence and deterioration of malignant tumors.TrxR has three kinds of isoenzymes.TrxR2 distributed in mitochondria is also up-regulated in most malignant tumors and its expression is much higher than that in paracancerous tissues and normal tissues in recent studies.In addition,there is a lot of correlation between the up-regulated expression of TrxR2 and clinicopathological features as well as prognosis of many malignant tumors.It is speculated that TrxR2 may be involved in the occurrence, deterioration and metastasis in malignant tumors.This article reviews the progress of TrxR2 in several malignancies to explore whether or not TrxR2 can be a biomarker of malignancy and serve as a novel target for oncol-ogy treatment.
ABSTRACT
Objective To investigate the effect of baicalein on mouse ovarian injury induced by triptolide. Methods Female healthy BALB/C mice were randomized into four groups. The mice in control group were given water contained 5% DMSO. The mice in model group were given 25 μg/kg triptolide orally. While the mice in baicalein treated groups were given 25 μg/kg triptolide plus 100 mg/kg and 500 mg/kg baicalein, respectively. On the 41st d of experiment, continuous vaginal smear were made to measure the sexual cycle of mice. On the 47th d, the superovulation experiment was carried out. On the 50th d, the mice were sacrificed to collect the ovaries. Ovaries were examined by hematoxylin and eosin (HE) staining, and the levels of follicles were counted. The levels of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in the ovarian tissue of each group were measured to evaluate the level of oxidative stress. Western blotting assay was performed to measure the expression levels of thioredoxin reductase (TrxR), thioredoxin (Trx), glutathione reductase (GR), and GSH-Px. Results Triptolide significantly induced ovarian damage, reduced the number of follicles, induced ovarian interstitial connective tissue regional osteoporosis, and decreased the activities of SOD and GSH-Px, while increased the content of MDA. On the contrary, when adding baicalein, the mice ovary development was obviously improved. The follicle cell number increased, and the connective tissue in interstitial region of ovary was well-stacked. The activity of SOD and GSH-Px in ovarian tissue was increased and the content of MDA was decreased. Western Blotting results showed that the expression levels of TrxR in model group mice were significantly down-regulated compared with those of control group. However, compared with control group, the expression levels of TrxR were significantly up-regulated by baicalein treatment. Conclusion Baicalein can enhance the anti-oxidative ability of mouse ovarian tissue by up-regulating TrxR expression, and may play an important role in the protection of ovarian injury induced by triptolide.
ABSTRACT
Objective:To study the effect of sulforaphane(SFN) on the growth of human bladder cancer cell and its mechanism in vitro. Method:Morphological characteristics of T24 cell nucleus induced by SFN were observed by AO/EB fluorescein staining .The effect of SFN on the T24 cell cycle was determined by flow cytometry. The expression of TrxR at the transcriptional and translational levels were measured by RT-PCR and Western blot methods respectively. Results:(1) After the cells were treated with 10 ?mol/L and 20 ?mol/L SFN for 24 h and 48 h, nuclear fragmentation and apoptotic body were seen under fluorescence microscope. (2) SFN could block the cell cycle at the G0/G1 phase showed by flow cytometry. Moreover, the treatment of 20 ?mol/L SFN for 48h could cause the appearance of sub-G1 before G0/G1 phase. (3) The expression of TrxR mRNA were increased by the treatment of 10 ?mol/L SFN for 4 h, 10 h,24 h ,compared with the control group. Furthermore ,the treatment with high dose SFN (20 ?mol/L ) for 10h or 24 h could induce the expression of TrxR mRNA more significantly . (4)The expression of TrxR protein in the 10 ?mol/L SFN for 24 h group was augmented compared with the control group , and aftertreatment with 20 ?mol/L SFN for 8 h and 24 h, its expression was significantly higher than that in the control group . Conclusion:SFN can inhibit the growth of T24 cell ,induce apoptosis and arrest T24 cell in G0/G1 phase. Its mechanism is associated with the induction of TrxR both at the transcriptional and translational levels.