Gastric Carcinoma Therapy By RNAi-Mediated Silencing Of Hsp70

Hsp is a kind of guard protein, which educes important pathology and physiology function when disadvantageous factors attack the organism. High expression of Hsp is induced to lessen the contamination when it sufferes stimulation as environmental temperature changes , inflammation, heat, infection of microorganism, tumor, and radiation, while low expression in normal condition. It has been discovered that Hsp70 plays multiplicitas roles in the development of tumors. On one hand, as a molecular companion ,it has something to do with the functional metabolism of tumor cells to protect them against the changes of harmful factors. On the other hand, as the heterogeneity between the tumor cells and the normal ones in the growing, metabolism and differentiation ,Hsp70 has become a target carrier of tumor antigen polypeptide to play a part in the immune reaction as an immunologic adjuvant.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 RNaseâ…¢(Dicer), which mediate specific degredation of its homologous and complimentary mRNA, to silence the expression of target genes.In this research ,siRNA of Hsp70 was synthesizd and transferred into the gastric adenocarcinoma cell line of SGC7901, then injected into the hypodermal transplanted tumor of nude mice. The changes of Hsp70 gene were detected by RT-PCR and Western Blot, and the size of tumors were compared in order to find a new way for the gene therapy of gastric carcinoma.PARTâ… INHIBITION OF GFP EXPRESSION IN GASTRIC CARCINOMA CELLS BY SHRNAObjective The plasmid containing short hairpin RNA(sh-RNA)of green fluorescent protein(GFP)is constructed and the expression of GFP is investigated.Methods A 19bp reverse repeated motif of GFP target sequence with 8bp spacer was synthesized, and inserted into pTZU6+1 to generate the pshRNA-GFP plasmid. Recombinant plasmid pshRNA-GFP and pEGFP- -C1 or pTZU6+1 and pEGFP-C1 were cotransfected into gastric carcinoma cells to detect the expression of GFP respectively. Results After recombinant plasmid pshRNA-GFP was transfected after 48h, cells of GFP expression decreased significantly, and expression of GFP protein reduced in SGC-7901 and BGC-823 cells. Conclusion The results show that the short hairpin RNA of GFP can efficiently suppress expression of GFP in SGC-7901 and BGC-823 cells.PARTâ…¡INHIBITING EXPRESSION OF Hsp70 IN GASTRIC CARCINOMA CELL SGC-7901 BY RNA INTERFERENCEObjective The plasmid containing short hairpin RNA(shRNA)of Hsp70 is constructed so as to suppress the expression of Hsp70 in gastric carcinoma cell SGC-7901.Methods Three pairs of reversed duplicate sequence were designed, which were inserted into contrivance promotor U6 of pTZU6+1 to generate the pshRNA-Hsp70 plasmid and pshRNA-Hsp70'. Recombinant plasmids were transfected into human gastric carcinoma cell SGC-7901 to detect Hsp70 expression respectively.Results Electrophoresis analysis and sequencing showed that the recombinate plasmid of pshRNA-Hsp70 and pshRNA-Hsp70'were successfully constructed .After rcombinant plasmid pshRNA- Hsp70 was transfected, the level of Hsp70 mRNA decreased obviously, and expression of Hsp70 protein also reduced in SGC-7901 cells. Conclusion The results show that the short hairpin RNA of Hsp70 can efficiently suppress expression of Hsp70 in SGC-7901 cells.PARTâ…¢EXPERIMENTAL RESEARCH IN GASTRIC CARCINOMA OF NUDE MICE BY RNA INTERFERENCE TARGETING HSP70Objective Hypodermically gastric carcinoma of mude mice is const- -ructed, the effect of pshRNA-Hsp70 applying for gene therapy in gastric carcinoma by inhibiting Hsp70 in nude mice is investigated.Methods SGC-7901 was injected into nude mice hypodermically to form a gastric carcinoma. Small interference RNAs targeting Hsp70 were designed, shRNA- Hsp70 vector was constructed and injected into nude mice to induce RNAi. The changes of Hsp70 gene were detected with RT-PCR and Western blot. The size of tumors were compared between experimental group and the control one.Results Compared with control group,tumor sizes in group injection of shRNA- Hsp70 decreased significantly. The mRNA level of Hsp70 and its protein expression also reduced obviously.Conclusion The expression of Hsp70 in nude mouse have been obviously down-regulated by pshRNA-Hsp70, which shows that it can depress the growing of tumors.