ABSTRACT
Development of anticancer treatments based on microRNA (miRNA/miR) such as miR-34a replacement therapy is limited to the use of synthetic RNAs with artificial modifications. Herein, we present a new approach to a high-yield and large-scale biosynthesis, in Escherichia coli using transfer RNA (tRNA) scaffold, of chimeric miR-34a agent, which may act as a prodrug for anticancer therapy. The recombinant tRNA fusion pre-miR-34a (tRNA/mir-34a) was quickly purified to a high degree of homogeneity (>98%) using anion-exchange fast protein liquid chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-34a showed a favorable cellular stability while it was degradable by several ribonucleases. Deep sequencing and quantitative real-time polymerase chain reaction studies revealed that tRNA-carried pre-miR-34a was precisely processed to mature miR-34a within human carcinoma cells, and the same tRNA fragments were produced from tRNA/mir-34a and the control tRNA scaffold (tRNA/MSA). Consequently, tRNA/mir-34a inhibited the proliferation of various types of human carcinoma cells in a dose-dependent manner and to a much greater degree than the control tRNA/MSA, which was mechanistically attributable to the reduction of miR-34a target genes. Furthermore, tRNA/mir-34a significantly suppressed the growth of human non-small-cell lung cancer A549 and hepatocarcinoma HepG2 xenograft tumors in mice, compared with the same dose of tRNA/MSA. In addition, recombinant tRNA/mir-34a had no or minimal effect on blood chemistry and interleukin-6 level in mouse models, suggesting that recombinant RNAs were well tolerated. These findings provoke a conversation on producing biologic miRNAs to perform miRNA actions, and point toward a new direction in developing miRNA-based therapies.
Subject(s)
Antineoplastic Agents/chemical synthesis , Bioengineering/methods , Cell Survival/drug effects , MicroRNAs/chemical synthesis , Prodrugs/chemical synthesis , Animals , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival/physiology , Dose-Response Relationship, Drug , Hep G2 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/isolation & purification , MicroRNAs/pharmacology , Prodrugs/isolation & purification , Prodrugs/pharmacology , Structure-Activity Relationship , Xenograft Model Antitumor Assays/methodsABSTRACT
Near-infrared autofluorescence imaging of tissues under long-wavelength laser excitation in the green and red spectral region complemented by cross-polarized elastic light scattering was explored for cancer detection. Various types of normal and malignant human tissue samples were utilized in this investigation. A set of images for each tissue sample was recorded that consisted of two autofluorescence images obtained under 532- and 632.8-nm excitation and light-scattering images obtained under linearly polarized illumination at 700, 850, and 1000 nm. These images were compared with the histopathology of the tissue sample. The experimental results indicated that for various tissue types, the intensity of the autofluorescence integrated over the 700 to 1000-nm spectral region was considerably different in cancer tissues than in that of the contiguous non-neoplastic tissues. This difference provided the basis for the detection of cancer and delineation of the tumor margins. Variations on the relative intensity were observed among different tissue types and excitation wavelengths.
