[RNA interference targeting c-Met inhibits proliferation of human laryngeal carcinoma Hep-2 cell line in vitro and in its xenografts in nude mice].
OBJECTIVE
The proto-oncogene c-Met was found to express on human laryngeal carcinoma Hep-2 cell line in previous research. In the present study, the author further examined whether inhibition of c-Met by RNA interference (RNAi) might inhibit biologic activity of Hep-2 cell line in vitro and proliferation using a murine laryngeal carcinoma model.


METHODS
RNAi plasmid that can express small interfering RNA targeting c-Met or siRNA that did not match any known human coding mRNA(control siRNA plasmid)was designed, constructed, and transfected into Hep-2 cell line by using cationic liposome Lipofectamine2000 as transfecting agent. In vitro, the transfection efficacy was tested by RT-PCR and Western Blot method, then elected the most inhibitive c-Met-siRNA sequence. Cell proliferation, movement and invasion were studied using MTT, cell migration assay and cell invasion assay, respectively. The Hep-2 cells were transplanted into nude mice, then the time of tumor formation and growth were observed. After tumor formation, c-Met-siRNA was given as the anti-tumor therapy. Expression of c-Met, MMP-9 and VEGF were detected by Western Blot method.


RESULTS
After the pSilencer2.0/c-Met-shRNA recombinant plasmid transfection into laryngeal carcinoma Hep-2 cells, the expression of mRNA and protein of c-Met decreased significantly in Hep-2 cells. On the 35th day after tumor vaccination, the tumor volume was (138 ± 27) mm³ in c-Met-siRNA transfection group, Which was diminished significantly in contrast with control group (P < 0.01). The expression of c-Met, MMP-9 and VEGF in the tumor of experiment group was decreased significantly, respectively (P < 0.05).


CONCLUSIONS
The results indicated that c-Met-siRNA can down-regulate the expression of c-Met and markedly inhibit laryngeal carcinoma Hep-2 cell proliferation, movement and invasion and the growth of transplantation tumor of nude mice. The siRNA expressing plasmid mediated gene therapy might be a new strategy in targeting molecular therapy of cancer of larynx.
Ji
Chang-you
CY
Department of Otorhinolaryngology Head and Neck Surgery, Daping Hospital and the Field Surgery Reseearch Institute, Third Military Medical University, Chongqing 400042, China.
Xie
Zhi-nian
ZN
Chen
Ji-chuan
JC
Wang
Yi-nan
YN
Guan
Li-lian
LL
Li
Hong-tao
HT
Zhang
Min
M
Liu
Rong-rong
RR
D000818:Animals; D017209:Apoptosis; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D015316:Genetic Therapy; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D000090063:Proto-Oncogene Mas; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection; D023041:Xenograft Model Antitumor Assays
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D034741:RNA, Small Interfering; D019859:Proto-Oncogene Proteins c-met