OBJECTIVETo study the role of Lon gene in tumor cell proliferation, apoptosis and cell stress response.

METHODSSmall interfering RNAs (smRNAs) for Lon gene were designed using Ambion software and synthesized. The recombinant plasmid pSilencer U6 2.1/Lon was constructed with the smRNAs and pSilencer U6 2.1, followed by transfection into MCF7 cells via Lipofectamine(TM) 2000. The positive cLones were detected by RT-PCR 24 h after cell transfection. The transfected MCF7 cells were then subjected to cisplatin treatment, ultraviolet (UV) exposure and heat stress, respectively, after which the cells growth was tested with MTT assay and the measurements were plotted against time or concentration depending on the treatment administered. Apoptosis of MCF7 cells following the treatments was measured with flow cytometry.

RESULTSThe mRNA of Lon gene was downregulated in cells transfected with the recombinant plasmid pSilencer U6 2.1-Lon, and RT-PCR fail to detect the specific band of Lon as could be detected in untransfected and mock-transfected MCF7 cells. MTT assay showed that pSilencer U6 2.1-Lon transfection resulted in reduced cell proliferation capacity. Stress response test revealed that MCF7 cells with Lon gene down-regulation enhanced cell sensitivity for UV and cisplatin, which was not observed for non-transfected or mock transfection group. The same changes were also observed for heat stress exposure at 41 degrees Celsius;, but not at 43 degrees Celsius; or 45 degrees Celsius;. Increased cell apoptosis rate from (1.14-/+0.79)% to (22.47-/+3.15)% occurred following pSilencer U6 2.1-Lon transfection of the cells.

CONCLUSIONSLon gene can be significantly downregulated by introduction of siRNA in MCF7 cells to result in enhanced sensitivity of MCF7 cells to UV, cisplatin and heat stress.

Antineoplastic Agents ; pharmacology ; Apoptosis ; Breast Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; drug effects ; radiation effects ; Cisplatin ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Humans ; Protease La ; genetics ; RNA Interference ; Reverse Transcriptase Polymerase Chain Reaction ; Temperature ; Time Factors ; Ultraviolet Rays