Molecular Beacon Imaging to Visualize Ki67 mRNA for Cell Proliferation Ability

Abstract

The objective of this study is to visualize the ability of cell proliferation based on molecular beacons (MB). Two types of MB to detect messenger RNA (mRNA) were used. One is a Ki67 MB of a target for cell proliferation ability. The other one is a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MB as a control of stable fluorescence in cells. To enhance the MB internalization into cells, the MB were incorporated into cationized gelatin nanospheres (cGNS). There was no difference in the physicochemical properties and the cell internalization between the cGNS_{Ki67 MB} and cGNS_{GAP MB}. When basic fibroblast growth factor (bFGF) was added to KUM6 cells of a mouse bone marrow-derived mesenchymal stem cell line, the expression of Ki67 and the cell proliferation increased with the bFGF concentration. After the incubation for the cell internalization of cGNS incorporating MB (cGNS_MB), the cells were further incubated for 24 h with or without different concentrations of bFGF. The fluorescence of cGNS_{Ki67 MB} significantly increased with the increase of bFGF concentration, whereas that of cGNS_{GAP MB} was constant, irrespective of the bFGF concentration. A time-lapse imaging assay revealed a fast enhancement of cGNS_{Ki67 MB} fluorescence after the bFGF addition compared with no bFGF addition. On the other hand, for cGNS_{GAP MB}, a constant fluorescence was observed even at any time point after the bFGF addition. It is concluded that the cGNS_MB system is promising for the chronological visualization of proliferation ability in living cells.

Impact statement

In the present study, molecular beacon (MB) to detect the mRNA of Ki67 as a target for cell proliferation ability was designed. The MB incorporated in cationized gelatin nanospheres was readily internalized into the cells. When basic fibroblast growth factor was added, the increase of Ki67 expression, and the consequent cell proliferation ability was chronologically visualized by the MB fluorescence. The MB imaging system is believed to be useful in the wide fields of basic cell research, cancer diagnosis, drug discovery, and regenerative medicine.