Construction of a BAC Vector and Its Application for Haplotype Analysis of CYP2A6 / 医学研究杂志

Objective To construct one high-copy BAC vector with cloning capacity for large DNA fragment insertion and use it for the haplotype analysis of CYP2A6 gene.Methods Oligos including multiple cloning sites were annealed and ligated into the Hind Ⅲ/BamH Ⅰ site of pGEM-3Z and pBeloBACll,separately.Then,the intermediate vectors were digested by Hind Ⅲ and ligated together,so as to get the head-to-tail oriented high-copy BAC vector pBAC-BJH after the blue-white spotting test.STI PCR method was used for the amplification of whole CYP2A6 gene,and purified amplicon was double digested by BstB Ⅰ/Mlu Ⅰ and ligated into the same site of newly constructed vector pBAC-BJH.Several clones were then picked up and sequenced for the haplotype analysis of carriers with newly discovered CYP2A6 variant 355A＞T.Results One high-copy BAC vector pBAC-BJH with 7 newly added multiple cloning sites was successfully constructed.High copy number and multiple cloning sites were the advantages of this plasmid.With this vector,haplotype analysis result for 22C＞T,51A＞G,355A＞T in carrier with newly detected CYP2A6mutation was CGT.Conclusion One vector pBAC-BJH convenient for cloning large DNA fragment was successfully developed,and it can be used for the haplotype analysis of cytochrome p450 gene and cloning or sequencing of other genes with large genomic DNA inserts.

Formononetin regulates dilated cardiomyopathy-mediated heart failure in rats via HSP90/AKT cardiomyocyte apoptosis and mechanism / 西安交通大学学报(医学版)

【Objective】 To investigate the effects of formononetin (FMN) on cardiomyocyte apoptosis and HSP90/AKT in rats with dilated cardiomyopathy-mediated heart failure. 【Methods】 Echocardiography, ELISA, histological staining, and TUNEL staining were used to observe the protective effect of different doses of FMN on dilated cardiomyopathy-mediated heart failure in rats and the apoptosis of cardiomyocytes. The potential targets of formononetin on dilated cardiomyopathy-mediated heart failure were obtained from TCMSP, DisGeNet, GeneCards, and other databases, the key targets were obtained according to the protein-protein interaction (PPI) network, and the key targets were verified by molecular docking. Western blotting was used to further verify the regulatory role of key targets in the treatment of dilated cardiomyopathy-mediated heart failure with formononetin. 【Results】 Formononetin could reduce the levels of LVIDS, LVIDD, NT-pro BNP, cTn-T, CK, CK-MB, and LDH in rats with dilated cardiomyopathy-mediated heart failure, increase the levels of EF and FS, and reduce the apoptosis of cardiomyocytes. FMN had a strong binding effect on 10 key targets (AKT1, HSP90AA1, CASP3, MAPK1, MMP9, SRC, ALB, HRAS, IGF1, and EGFR) screened by network pharmacology, with HSP90AA1 and AKT1 having the strongest binding effect. Formononetin decreased the expression of HSP90, AKT and downstream CASP3 protein, but increased the expression of p-AKT in myocardial tissue. 【Conclusion】 Formononetin may inhibit the expression of HSP90, promote phosphorylation of AKT to p-AKT, and inhibit the expression of CASP3, thereby reducing the apoptosis of cardiomyocytes and improving myocardial tissue damage, so as to achieve the purpose of treating dilated cardiomyopathy-mediated heart failure.

Retraction note to: Lentivirus-Mediated Short-Hairpin RNA Targeting Protein Phosphatase 4 Regulatory Subunit 1 Inhibits Growth in Breast Cancer / 한국유방암학회지

The investigator raised the possibility that the authors hadn't conducted the research. Therefore, the entire article has been retracted in accordance with this journal's policy and Editorial decision.

Erratum: Lentivirus-Mediated Short-Hairpin RNA Targeting Protein Phosphatase 4 Regulatory Subunit 1 Inhibits Growth in Breast Cancer / 한국유방암학회지

This article was initially published on the Journal of Breast Cancer with a misspelled name of the second author.

Lentivirus-Mediated Short-Hairpin RNA Targeting Protein Phosphatase 4 Regulatory Subunit 1 Inhibits Growth in Breast Cancer / 한국유방암학회지

PURPOSE: Protein phosphatase 4 regulatory subunit 1 (PP4R1), as an interaction partner of the catalytic serine/threonine-protein phosphatase 4 catalytic subunit has been shown to involve in cellular processes and nuclear factor kappaB signaling. However, the functions of PP4R1 in human breast cancers remain unclear. This study is designed to explore the effect of PP4R1 knockdown on the biological characteristics of breast cancer cells. METHODS: A lentivirus-mediated short hairpin RNA (shRNA) was designed to knockdown the expression of PP4R1 in ZR-75-30 breast cancer cells. The efficiency of lentivirus-mediated shRNA infection was determined using fluorescence microscopy to observe lentivirus-mediated green fluorescent protein expression and confirmed to be over 80%. PP4R1 expression in infected ZR-75-30 cells was detected by quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation and colony formation ability were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and colony formation assay, respectively. Flow cytometry was used to measure cell cycle progression and cell apoptosis. In addition, apoptosis makers, including poly-ADP-ribose polymerase (PARP) and caspase-3, were investigated in PP4R1-silenced ZR-75-30 cells by western blot assay. RESULTS: We successfully constructed lentivirus-mediated shRNA to target PP4R1 in ZR-75-30 cells. MTT assay and colony formation assay showed the loss of PP4R1 suppressed the proliferation of ZR-75-30 cells. Flow cytometry analysis indicated cell cycle arrest and increased cell apoptosis in PP4R1 knockdown cells. Further, the apoptosis response in cells depleted of PP4R1 was illustrated by downregulation of PARP and upregulation of caspase-3. CONCLUSION: Our results suggest that PP4R1 could promote breast cancer cell proliferation and might play a vital role in breast cancer occurrence.