Inhibition of C35 gene expression by small interfering RNA induces apoptosis of breast cancer cells.
C35 was reported to be a new biomarker and therapeutic target for breast cancer. To explore the functional importance of C35, we constructed small interfering RNA (siRNA) targeting C35 and investigated the effects of the siRNAs on C35 expression and apoptosis of T47D cells. C35 siRNAs were constructed and named psiRNA-C35-1 and psiRNA-C35-2. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blots were used to detect the effects of the siRNAs on mRNA and protein expression of C35 in T47D cells. The effects of the two siRNAs on apoptosis of T47D cells were detected by flow cytometry and terminal dUTP nicked-end labelling assays. Also, the apoptosis related molecule caspase-3 was detected using Western blots. The psiRNA-C35-1 and psiRNA-C35-2 siRNAs were verified by both EcoR I/Hind III digestion analysis and automated DNA sequencing. RT-PCRs and Western blots showed that C35 mRNA and protein expression in T47D cells were obviously inhibited after psiRNA-C35-1 and psiRNA-C35-2 transfection. Flow cytometry and terminal dUTP nicked-end labelling assays showed that apoptosis of T47D cells was significantly induced after transfection with psiRNA-C35-1 and psiRNA-C35-2 (p < 0.05). Also, caspase-3 expression in the psiRNA-C35-1 and psiRNA-C35-2 transfected cells was obviously higher than that of the Lipofectamine and pTZU6+1 transfected cells. This study showed that apoptosis of T47D cells can be significantly induced by inhibiting C35 expression using siRNAs, which may be caused by activating caspase-3. C35 might play an important role in apoptosis of breast cancer cells, and therapeutic strategies targeting C35 may be useful for breast cancer treatment.
Shandong Provincial Institute of Parasitic Diseases, Ji'ning, Shandong, China.
Qiaoqiao Q
QQ
Liu
Kun
K
Yin
Song
S
Zhu
Ling
L
Zhang
Peie E
PE
Wen
Cuiling L
CL
Li
Dianbo B
DB
Zhang
Miao
M
Liu
Ge
G
Yan
D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics / Q000502:physiology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; C517054:MIEN1 protein, human; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D053148:Caspase 3