Gastric Carcinoma Therapy By RNAi-Mediated Silencing Of HIF-1α

Hypoxia inducible factor-1alpha (HIF-1α) is the oxygen-regulated subunit of hypoxia inducible factor-1( HIF- l) transcription factor. It is a kind of hypoxia dependence protein and decide the activity of HIF-1. At normal oxygen press, it is degradated quickly by ubiquitin-proteasome pathway, but under low oxygen level, it is quick stable, and the expression level of HIF-1αincreasing. HIF-1αand HIF-1βcompose heterodimer, and then HIF-1 over express, and its activity increase, then, promote the expression of a series of gene products that are involved in glycolysis velocity exaltation and angiogenesis. The tumor cell take place a series of adaptation reaction to the low oxygen environment. All these play a importante role in tumour energy metabolism, Angiogenesisrise, cell proliferation and metastasis, increase the resistance to chemotherapy and radiation therapy at the same time.RNA interference is a kind of gene blocking technology about Post-transcriptional gene silence(PTGS), which has been extensively applicated for the inhibition of gene expression in the mammal cells in recent years. Compt short interference RNA (21-23nt) is obtained by the incision enzyme RNaseⅢ(Dicer), which mediate specific degredation of its homologous and complimentary mRNA, to silence the expression of target genes.In this research, siRNA of HIF-1αwas synthesizd and transferred into the gastric adenocarcinoma cell line of BGc-823, then injected into the subcutaneous transplanted tumor of nude mice. The changes of HIF-1αgene were detected by RT-PCR and Western Blot, the cell growth and angiogenesis were examined.Our studies might offer a new way for the gene therapy of gastric carcinoma.PARTⅠINHIBITING OF GENE HIF-1αBY RNA INTERFERENCE IN VITROObjective: The plasmid containing short hairpin RNA(shRNA)of HIF-1αis constructed so as to suppress the expression of HIF-1αand to observe the effects of the proliferation and apoptosis in gastric cancer cell line BGC823 by RNA interference.Methods: The recombinant plasmid pshRNA-HIF-1αwere constructed and transfected into gastric cancer cells of BGC-823 by LipofectamineTM2000, the expression of HIF-1αin BGC-823 were observed by RT-PCR and Western Blot, the inhibition of the cell proliferation were examined by MTT. TUNEL and flow cytometry were selected to detect apoptosis. BGC823 cells were inoculated into subcutaneous tissue of nud mice, the inhibitory effects of shRNA targeting HIF-1αon gastric cancer were observed after intratumoral injection of shRNA expression vector.Results: The restriction enzyme digestion and sequencing result showed that the recombinant plasmid of pshRNA-HIF-1αhad been constructed successfully. After it transfecting into the BGC-823, the genetic transcription and protein expression of HIF-1αwere inhibited significantily, compared with controls, its inhibition ratio were 97.4% and 55.7% respectively. The inhibition ratio of the cell proliferation was higher than that of the control ones by MTT, the cell ratio was increased in G0-G1, and decreased in S and G2-M. The apoptosis ratio was also higher by the flow cytometry and TUNEL.Conclusion: It has been initially proved that the recombinant plasmid pshRNA-HIF-1αcan inhibit the genetic transcription and protein expression of HIF-1α, and inhibit the proliferation and induce the apoptosis of gastric cancer cell of BGC-823 in vivo and in vitro, which is a basic foundation for the oncogene therapy in future. PARTⅡEXPERIMENTAL RESEARCH IN GASTRIC RCINOMA OF NUDE MICE BY RNA INTERFERENCE TARGETING HIF-1αObjective: To constructe BGC-823 transplanted tumor modle in mude mice, evaluate the efficacy of inhibiting tumor growth and antiangiogenic effect by RNA interference targeting HIF-1αin nude mice .Methods: BGC-823 cell was inoculated into subcutaneous tissue of nud mice and BGC-823 transplanted tumor modle was constructed. Small interference RNA targeting HIF-1αwas designed, shRNA- HIF-1αvector was constructed and injected into the tumor, the volum and weight of tumor were measured. HIF-1α,vascular endothelial growth factor(VEGF) and tumor microvessel density(MVD) were detected by immunohistochemistry.Results: Compared with control group, tumor sizes in group of injecting of shRNA-HIF-1αdecreased significantly. The protein expression level of HIF-1αand VEGF also reduced obviously, the MVD was much lower too.Conclusion: The expression of HIF-1αin subcutaneous tumor have been obviously down-regulated by pshRNA-HIF-1αRNA interference targeting HIF-1αand pshRNA-HIF-1αcan inhibit the angiogenesis and the growth of transplanted tumor.