Bioengineered miR-34a modulates mitochondrial inner membrane protein 17 like 2 (MPV17L2) expression toward the control of cancer cell mitochondrial functions

Abstract

Genome-derived microRNAs (miRNAs or miRs) control post-transcriptional gene expression critical for various cellular processes. Recently, we have invented a novel platform technology to achieve high-yield production of fully humanized, bioengineered miRNA agents (hBERAs) for research and development. This study is aimed to produce and utilize a new biologic miR-34a-5p (or miR-34a) molecule, namely, hBERA/miR-34a, to delineate the role of miR-34a-5p in the regulation of mitochondrial functions in human carcinoma cells. Bioengineered hBERA/miR-34a was produced through in vivo fermentation production and purified by anion exchange fast protein liquid chromatography. hEBRA/miR-34a was processed to target miR-34a-5p in human osteosarcoma and lung cancer cells, as determined by selective stem-loop reverse transcription quantitative polymerase chain reaction analysis. The mitochondrial inner membrane protein MPV17 like 2 ( MPV17L2 ) was validated as a direct target for miR-34a-5p by dual luciferase reporter assay. Western blot analysis revealed that bioengineered miR-34a-5p effectively reduced MPV17L2 protein outcomes, leading to much lower levels of respiratory chain Complex I activities and intracellular ATP that were determined with specific assay kits. Moreover, Seahorse Mito Stress Test assay was conducted, and the results showed that biologic miR-34a-5p sharply reduced cancer cell mitochondrial respiration capacity, accompanied by a remarkable increase of oxidative stress and elevated apoptotic cell death, which are manifested by greater levels of reactive oxygen species and selective apoptosis biomarkers, respectively. These results demonstrate the presence and involvement of the miR-34a-5p-MPV17L2 pathway in the control of mitochondrial functions in human carcinoma cells and support the utility of novel bioengineered miRNA molecules for functional studies.

MiRNAs Action and Impact on Mitochondria Function, Metabolic Reprogramming and Chemoresistance of Cancer Cells: A Systematic Review

“…The main aim of these 12 studies [ 38 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 ] was to comprehensively analyze the metabolic balance and/or chemoresistance associated with miRNAs in different cancers. In nine studies [ 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 ], the aim was to investigate the effect of miRNAs on mitochondrial function in cancer tissues and cell lines. Five studies [ 64 , 65 , 66 , 67 , 68 ] reported an the association between miRNAs and the regulation of glucose metabolism.…”

“…In total, 51 miRNAs were differentially expressed in 12 cancers: bladder (1 study), breast (24 studies), cervical (2 studies), colon (3 studies), colorectal (1 study), liver (1 study), lung (1 study), melanoma (2 studies), osteosarcoma (1 study), pancreatic (1 study), prostate (2 studies), and tongue (2 studies). High expression of miRNAs (n = 37) was observed in 17 studies [ 25 , 38 , 50 , 51 , 54 , 57 , 59 , 61 , 62 , 63 , 66 , 67 , 68 , 69 , 70 , 72 , 76 ] and lower expression (n = 12) in 9 studies [ 44 , 56 , 58 , 64 , 65 , 67 , 73 , 74 , 76 ]. Of the studies describing the overexpression of miRNAs, four involved the miR-181 family, reported in three tumor types: cervical [ 69 ], colon [ 54 , 62 ], and liver [ 68 ].…”

“…Of the studies describing the overexpression of miRNAs, four involved the miR-181 family, reported in three tumor types: cervical [ 69 ], colon [ 54 , 62 ], and liver [ 68 ]. Additionally, four miRNAs, miR-21 [ 54 , 69 ], miR-34a-5p [ 63 ], miR-195 [ 57 , 70 ], and miR-210 [ 52 , 67 ], were analyzed in seven independent studies in several types of cancer, including cervical, lung, osteosarcoma, breast, and colon cancer. In studies with lower miRNA expression, miR-17 [ 73 , 74 ] and miR-125b [ 46 , 47 ] were observed in two independent studies, on prostate cancer and breast cancer, respectively.…”

“…Twenty-six studies conducted during the search period reported 21 miRNAs with tumor suppressive actions: miR-let-7a [ 44 , 55 ], miR-1 [ 56 ], miR-17* [ 73 ], miR-17-3p [ 74 ], miR-24-2 [ 77 ], miR-27a [ 45 ], miR-34a-5p [ 63 ], miR-125b [ 46 , 47 ], miR-128 [ 64 ], miR-133a [ 48 ], miR-140-5p [ 65 ], miR-195 [ 57 , 70 , 77 ], miR-200a [ 58 ], miR-223 [ 75 ], miR-340 [ 71 ], miR-342-3p [ 49 ], miR-365-2 [ 77 ], miR-519d [ 76 ], miR-663 [ 59 ], miR-1291 [ 72 ], and miR-4485 [ 60 ]. Of these studies, 20 were in breast cancer, 1 was in lung cancer and osteosarcoma, 2 were in melanoma, 2 were in prostate cancer, and 1 was in pancreatic cancer ( Table 2 ).…”

Comprehensive and Quantitative Analysis of the Changes in Proteomic and Phosphoproteomic Profiles during Stimulation and Repression of Steroidogenesis in MA-10 Leydig Cells

“…The MPV17L2 protein levels were also modulated by the treatments. MPV17L2 is an integral mitochondrial inner membrane protein [ 20 ], and as such, it is implicated in mitochondrial ribosome assembly [ 20 ] and in reactive oxygen species (ROS) production [ 31 ], two processes known to influence steroidogenesis in Leydig cells [ 32 ]. We found that the MPV17L2 protein levels increased 5-fold in the presence of AICAR, while it was reduced by 5-fold in the Fsk-treated cells.…”