Effects of upregulated expression of microRNA-16 on biological properties of culture-activated hepatic stellate cells.
In our previous studies, we identified miR-16 as being downregulated during activation of hepatic stellate cells (HSCs) by microarray hybridization. However, the roles and related mechanisms of miR-16 in HSCs are not understood. In this study, The miRNA RNAi technique was used to analyze the effects of miR-16 on biological properties of HSCs in vitro. The lentiviral vector encoding miR-16 was constructed and transfected. Furthermore, the expression level of miR-16 was measured by real-time PCR. Cellular growth and proliferation capacity were assayed using the cell counting kit-8 (CCK-8). The apoptosis rate and cell-cycle distribution were measured by flow cytometry. Cell morphological characteristics were identified by phase-contrast microscopy, fluorescence microscopy and electron microscopy. The underlying mechanisms related to the changes in biological properties were assessed. The identity of the recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. Virus titer was 10(8) > ifu/m. Restoring the intracellular miRNAs by miR-16 administration greatly reduced the expression levels of cyclin D1 (CD1). Cell-cycle arrest and typical features of apoptosis were detected in activated HSCs treated with pLV-miR-16. Our results indicate that transduction of miR-16 offers a feasible approach to significantly inhibit HSC proliferation and increase the apoptosis index. Thus, targeted transfer of miR-16 into HSC may be useful for the treatment of hepatic fibrosis.
Guo
Can-Jie
CJ
Department of Gastroenterology, Xinhua Hospital, Shanghai Jiaotong University, China.
Pan
Qin
Q
Jiang
Bo
B
Chen
Guang-Yu
GY
Li
Ding-Guo
DG
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D049109:Cell Proliferation / Q000187:drug effects; D019938:Cyclin D1 / Q000096:biosynthesis / Q000235:genetics; D003893:Desmin / Q000096:biosynthesis; D015536:Down-Regulation; D005822:Genetic Vectors; D055166:Hepatic Stellate Cells / Q000473:pathology / Q000502:physiology*; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015854:Up-Regulation
D020413:3' Untranslated Regions; C530106:Ccnd1 protein, rat; D003893:Desmin; C547573:MIRN16 microRNA, rat; D035683:MicroRNAs; D019938:Cyclin D1