[Effects of RNAi-silenced id1 gene on the biology behavior of esophageal cancer cell lines Eca-109].
OBJECTIVE
To study the effect of shRNA expressing vector targeting to id1 gene on the biological behavior of the esophageal cancer cell.


METHODS
The specific shRNA sequence was designed, synthesized and cloned into pGFU6/neo vector. Then, the resulting recombinants were transfected into Eca-109 cells using Lipofetamine 2000 following the instruction of the manufactures. The expression levels of id1 mRNA were analyzed by RT-PCR and the levels of Id1, p16 and PCNA protein were detected by Western blot assay. The growth of Eca-109 cells was analyzed using clone formation assay and trypan blue exclusion assay. Distribution of cell cycle and proliferation of Eca-109 cells were assessed by flow cytometry.


RESULTS
The sequence of recombinant plasmid was confirmed by sequence analysis. The expression levels of id1 mRNA and Id1 protein significantly decreased in Eca-109 cells transfected with pGFU6/neo-Id1-1 vector compared to the negative control group (P < 0.05). The recombinant plasmid expressing id1 shRNA could effectively inhibit cell proliferation and induce cell cycle arrest in G0/G1 phase with a remarkably decrease of cells in S phase (P < 0.05).


CONCLUSIONS
All the above data suggested that the expression of id1 gene was significantly inhibited in Eca-109 cells transfected with pGFU6/neo-id1-1 recombinant. Furthermore, the proliferation of id1 knockdown cells was depressed.
Pei
Fei
F
The Medicine-Biology Laboratory of Life Science College, Sichuan University, Chengdu 610041, China.
Zhang
Tang-Tong
TT
Zhang
Na-Juan
NJ
Yang
Chao
C
Yang
Chun-Lei
CL
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004938:Esophageal Neoplasms / Q000235:genetics* / Q000473:pathology*; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1 / Q000235:genetics* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; C497453:ID1 protein, human; D051798:Inhibitor of Differentiation Protein 1; D009363:Neoplasm Proteins; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins