ABSTRACT
MicroRNAs (miRNAs) regulate cancer cell proliferation by binding directly to the untranslated regions of messenger RNA (mRNA). MicroRNA-148a (miR-148a) is expressed at low levels in breast cancer (BC). However, little attention has been paid to the sequestration of miR-148a. Here, we performed a knockdown of miR-148a using anti-miRNA oligonucleotides (AMOs) and investigated the effect on BC cell proliferation. BC cell proliferation was significantly suppressed by AMO flanked by interstrand cross-linked duplexes (CL-AMO), whereas single-stranded and commercially available AMOs had no effect. The suppression was caused by sequestering specifically miR-148a. Indeed, miR-148b, another member of the miR-148 family, was not affected. Importantly, the downregulation of miR-148a induced a greater and longer-lasting inhibition of BC cell proliferation than the targeting of oncogenic microRNA-21 (miR-21) did. We identified thioredoxin-interacting protein (TXNIP), a tumor suppressor gene, as a target of miR-148a and showed that CL-AMO provoked an increase in TXNIP mRNA expression. This study provide evidence that lowly expressed miRNAs such as miR-148a have an oncogenic function and might be a promising target for cancer treatment.
Subject(s)
Breast Neoplasms , Cell Proliferation/drug effects , MicroRNAs , Oligonucleotides, Antisense , RNA, Neoplasm , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Female , Humans , MCF-7 Cells , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , MicroRNAs/metabolism , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , RNA, Neoplasm/antagonists & inhibitors , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolismABSTRACT
Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4-1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs.
Subject(s)
Antibodies, Monoclonal/biosynthesis , CRISPR-Cas Systems , Chickens/genetics , Egg White/chemistry , Receptor, ErbB-2/antagonists & inhibitors , Animals , Animals, Genetically Modified , Antibodies, Monoclonal/isolation & purification , Bioreactors , Female , Gene Editing/methods , Humans , Plasmids/chemistry , Plasmids/metabolism , Receptor, ErbB-2/genetics , Receptor, ErbB-2/immunology , Trastuzumab/biosynthesis , Trastuzumab/isolation & purification , Zygote/chemistry , Zygote/metabolismABSTRACT
Breast cancer is the most frequent cancer affecting women worldwide. Traditional chemotherapy, hormone therapy, and targeted therapy are used for breast cancer treatment. However, breast cancer is a heterogeneous disease, and patients often develop drug resistance. Therefore, various new therapeutic strategies have been investigated, including microRNA regulation. Anti-microRNA oligonucleotides (AMOs) are one of the most potent agents in oligonucleotide therapy. The inhibition activity of an AMO can be increased by flanking its single-stranded antisense sequence (the widely used structure for AMOs) with interstrand cross-linked duplexes (CLDs). An extrastable CLD improves nuclease resistance and stabilizes hybridization with a target. This study investigated the effects of anti-microRNA-21 (miR-21) AMO modified with CLDs on breast cancer cells without using reporter assay. The CLD-modified AMO suppressed breast cancer cell proliferation for a long duration compared to other types of AMOs. In addition, it expectedly up-regulated the miR-21-controlled expression of tumor suppressor genes. Therefore, an AMO flanked by CLDs can be a promising strategy for breast cancer treatment.
Subject(s)
Base Pairing , MicroRNAs/chemistry , MicroRNAs/genetics , Nucleic Acid Conformation , Oligonucleotides/chemistry , Oligonucleotides/genetics , Base Sequence , Breast Neoplasms , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Gene Expression , Humans , Molecular Structure , PTEN Phosphohydrolase/genetics , TransfectionABSTRACT
We previously reported the kinetics analysis of cardiomyocyte beating using scanning electrochemical microscopy (SECM). In this study, a stage-top incubator and a capillary micropipette (MP) for delivering drugs were assembled with an SECM instrument, and the responses of rat cardiomyocytes were analyzed under a culture environment after drug stimulation. When adenosine triphosphate (ATP) was delivered to synchronously beating cardiomyocytes, the beating acceleration effect of ATP was counteracted by the synchronously beating network in the culture dish. In contrast, cardiomyocytes cultured on a pattern of islands in a culture dish showed fluctuations in the duration of beating upon the addition of ATP. We also examined the effect of the cardiotoxic agent astemizole on cardiomyocytes and successfully detected motion fluctuations. Therefore, drug stimulation via MPs and beating measurement by SECM are effective routes for the evaluation of drug candidates through the analysis of time-course beating motion fluctuations of the cardiomyocytes.
