Use of recombinant microRNAs as antimetabolites to inhibit human non-small cell lung cancer

During the development of therapeutic microRNAs (miRNAs or miRs), it is essential to define their pharmacological actions. Rather, miRNA research and therapy mainly use miRNA mimics synthesized in vitro. After experimental screening of unique recombinant miRNAs produced in vivo, three lead antiproliferative miRNAs against human NSCLC cells, miR-22-3p, miR-9-5p, and miR-218-5p, were revealed to target folate metabolism by bioinformatic analyses. Recombinant miR-22-3p, miR-9-5p, and miR-218-5p were shown to regulate key folate metabolic enzymes to inhibit folate metabolism and subsequently alter amino acid metabolome in NSCLC A549 and H1975 cells. Isotope tracing studies further confirmed the disruption of one-carbon transfer from serine to folate metabolites by all three miRNAs, inhibition of glucose uptake by miR-22-3p, and reduction of serine biosynthesis from glucose by miR-9-5p and -218-5p in NSCLC cells. With greater activities to interrupt NSCLC cell respiration, glycolysis, and colony formation than miR-9-5p and -218-5p, recombinant miR-22-3p was effective to reduce tumor growth in two NSCLC patient-derived xenograft mouse models without causing any toxicity. These results establish a common antifolate mechanism and differential actions on glucose uptake and metabolism for three lead anticancer miRNAs as well as antitumor efficacy for miR-22-3p nanomedicine, which shall provide insight into developing antimetabolite RNA therapies.

Erratum: Author correction to 'Bioengineered miR-124-3p prodrug selectively alters the proteome of human carcinoma cells to control multiple cellular components and lung metastasis in vivo' Acta Pharmaceutica Sinica B 11 (2021) 3950-3965

[This corrects the article DOI: 10.1016/j.apsb.2021.07.027.].

Bioengineered miR-124-3p prodrug selectively alters the proteome of human carcinoma cells to control multiple cellular components and lung metastasis

With the understanding of microRNA (miRNA or miR) functions in tumor initiation, progression, and metastasis, efforts are underway to develop new miRNA-based therapies. Very recently, we demonstrated effectiveness of a novel humanized bioengineered miR-124-3p prodrug in controlling spontaneous lung metastasis in mouse models. This study was to investigate the molecular and cellular mechanisms by which miR-124-3p controls tumor metastasis. Proteomics study identified a set of proteins selectively and significantly downregulated by bioengineered miR-124-3p in A549 cells, which were assembled into multiple cellular components critical for metastatic potential. Among them, plectin (PLEC) was verified as a new direct target for miR-124-3p that links cytoskeleton components and junctions. In miR-124-3p-treated lung cancer and osteosarcoma cells, protein levels of vimentin, talin 1 (TLN1), integrin beta-1 (ITGB1), IQ motif containing GTPase activating protein 1 (IQGAP1), cadherin 2 or N-cadherin (CDH2), and junctional adhesion molecule A (F11R or JAMA or JAM1) decreased, causing remodeling of cytoskeletons and disruption of cell-cell junctions. Furthermore, miR-124-3p sharply suppressed the formation of focal adhesion plaques, leading to reduced cell adhesion capacity. Additionally, efficacy and safety of biologic miR-124-3p therapy was established in an aggressive experimental metastasis mouse model

Bioengineered miR-328-3p modulates GLUT1-mediated glucose uptake and metabolism to exert synergistic antiproliferative effects with chemotherapeutics

MicroRNAs (miRNAs or miRs) are small noncoding RNAs derived from genome to control target gene expression. Recently we have developed a novel platform permitting high-yield production of bioengineered miRNA agents (BERA). This study is to produce and utilize novel fully-humanized BERA/miR-328-3p molecule (hBERA/miR-328) to delineate the role of miR-328-3p in controlling nutrient uptake essential for cell metabolism. We first demonstrated successful high-level expression of hBERA/miR-328 in bacteria and purification to high degree of homogeneity (>98%). Biologic miR-328-3p prodrug was selectively processed to miR-328-3p to suppress the growth of highly-proliferative human osteosarcoma (OS) cells. Besides glucose transporter protein type 1, gene symbol solute carrier family 2 member 1 (GLUT1/), we identified and verified large neutral amino acid transporter 1, gene symbol solute carrier family 7 member 5 (LAT1/) as a direct target for miR-328-3p. While reduction of LAT1 protein levels by miR-328-3p did not alter homeostasis of amino acids within OS cells, suppression of GLUT1 led to a significantly lower glucose uptake and decline in intracellular levels of glucose and glycolytic metabolite lactate. Moreover, combination treatment with hBERA/miR-328 and cisplatin or doxorubicin exerted a strong synergism in the inhibition of OS cell proliferation. These findings support the utility of novel bioengineered RNA molecules and establish an important role of miR-328-3p in the control of nutrient transport and homeostasis behind cancer metabolism.

Bioengineered miR-27b-3p and miR-328-3p modulate drug metabolism and disposition the regulation of target ADME gene expression

Drug-metabolizing enzymes, transporters, and nuclear receptors are essential for the absorption, distribution, metabolism, and excretion (ADME) of drugs and xenobiotics. MicroRNAs participate in the regulation of ADME gene expression imperfect complementary Watson-Crick base pairings with target transcripts. We have previously reported that Cytochrome P450 3A4 (CYP3A4) and ATP-binding cassette sub-family G member 2 (ABCG2) are regulated by miR-27b-3p and miR-328-3p, respectively. Here we employed our newly established RNA bioengineering technology to produce bioengineered RNA agents (BERA), namely BERA/miR-27b-3p and BERA/miR-328-3p, fermentation. When introduced into human cells, BERA/miR-27b-3p and BERA/miR-328-3p were selectively processed to target miRNAs and thus knock down and mRNA and their protein levels, respectively, as compared to cells treated with vehicle or control RNA. Consequently, BERA/miR-27b-3p led to a lower midazolam 1'-hydroxylase activity, indicating the reduction of CYP3A4 activity. Likewise, BERA/miR-328-3p treatment elevated the intracellular accumulation of anticancer drug mitoxantrone, a classic substrate of ABCG2, hence sensitized the cells to chemotherapy. The results indicate that biologic miRNA agents made by RNA biotechnology may be applied to research on miRNA functions in the regulation of drug metabolism and disposition that could provide insights into the development of more effective therapies.