A Preliminary Study Of The Effects Of Knockdown Of Endogenous C-Myc Expression By RNAi On Bionomics And Molecular Mechanism In Vitro And In Vivo In 5-8F Nasopharyngeal Carcinoma Cells

【background of nasopharyngeal carcinoma and c-myc】Nasopharyngeal carcinoma is a polygenetic inheritance malignant tumor. The etiology and pathogenesis of NPC are mainly involved in hereditary susceptibility,genomic instability,changes of structure and function of multiple oncogene and anti-oncogene,EB virus infection and chemical carcinogens.c-Myc, a highly conserved proto-oncogene, localizes to human chromosome 8q24. In recent years, many studies have discovered that abnormal activation and overexpression of c-myc oncogene closely correlate with the occurrence, development and prognosis of NPC. Previous experiment by western blot also proved overexpression of endogenous c-myc in 5-8F, 6-10B, HNE1 and CNE1.【construction, identification of siRNA expressing vector targeting c-myc and establishment of stable transfection cell lines】In order to elucidate the effect of c-myc gene on the carcinogenesis in NPC, two targeting sequences namedⅠandⅡwere well designed by siRNA design tool, which were 1357bp-1377bp and 1716bp-1738bp of open reading frame of c-myc respectively. pRNT-U6.1/siRNA-c-myc, a siRNA expressing vector targeting c-myc, was introduced into a c-myc over-expressed 5-8F cells by liposome transfection and a stable cell line 5-8F/siRNA-c-myc was established. Similarly, 5-8F/siRNA-Control was also established. RT-PCR and Western blot methods were used to detect the expression of c-myc gene. The result showed thatⅠagainst c-myc markedly reduced its overexpression by up to 75％, but notⅡ. So a monoclonal cell line, 5-8F/siRNA-c-myc/C3, was selected in subsequent experiments.【changes of ultrastructure of 5-8F cells by knockdown of c-myc】5-8F, 5-8F/siRNA-Control and 5-8F/siRNA-c-mycⅠ/C3 were scanned by transmission electron microscope(TEM). The results indicated that compared with 5-8F and 5-8F/siRNA-Control, super microvilli of 5-SF/siRNA-c-mycⅠ/C3 obviously fell off, its karyoplasmic ratio tended to be normalized and the number of its endoplasmic reticulum and mitochondrion reduced. This suggested that knockdown of c-myc overexpression increased intercellular contact inhibition, inhibited cell growth and reversed malignant biological behavior of 5-8F cells.【effects of knockdown of c-myc on 5-8F cell growth】To investigate the effects of knockdown of c-myc on the malignant phenotype of NPC cells, 200 cells of 5-8F, 5-8F/siRNA-Control and 5-8F/siRNA-c-mycⅠ/C3 were planted in plates and then cultured for 14 days. Colony formation number and colony formation ratio of each group were calculated. In MTT assay, 0.5×10~4 cells of each group were planted in 96 wells and observed for 6 days. While in growth curve assay, 1×10~4 cells of each group were planted in 24 wells and then observed for 7 days. The proliferation rate curves were figured out. The results showed that knockdown of c-myc significantly inhibited colony formation and cell growth of 5-8F compared with 5-8F and 5-8F/siRNA-Control. 1×10~7 cells of each group were injected into nude mice in vivo to study the effect of siRNA-c-myc on xenograft. The nude mice were executed and the xenografts were isolated. We found that the sizes and weights of xenografts causing by 5-8F/siRNA-c-mycⅠ/C3 cells injection were smaller and lighter than that of the control group. Pathological detection showed that xenografts were low differentiated squamous cell carcinoma by HE stained and no difference between groups. To further investigate the function of knockdown of c-myc, flow cytometry was performed. The cells were subjected to FACS analysis. Knockdown of c-myc induced an increased percentage of cells in G1 phase and a corresponding decrease in the percentage of cells in S and G2/M phases. The G0/G1 phase cells of 5-8F/siRNA-c-mycⅠ/C3 (66.2％) markedly increased compared with 5-8F (47.8％) and 5-8F/siRNA-Control(46.9％) cells. But it had no effect on cell apoptosis of 5-8F. These results clearly demonstrated a role for siRNA-c-myc in regulating cell cycle.【down expression of important molecules in Rb/E2F signal pathway by knockdown of c-myc gene】In order to fred the molecular mechanism of G1-S phase inhibition in 5-8F cells by siRNA-c-myc, Western blot methods was used to detect the expression of relevant molecules in Rb/E2F signal transduction pathway. The results indicated that expression of CDK2,CDK4,cyclinD1,pRb,E2F3 and DP2 were down regulated in 5-8F/siRNA-c-mycⅠ/C3 cell compared with 5-8F and 5-8F/siRNA-Control cells. These data suggested that the biological function of c-myc gene correlated to Rb/E2F pathway.【conclusion】In summary, plasmids expressing siRNA against c-myc changed the ultrastructure of 5-8F, decreased cell growth of 5-8F, significantly inhibited colony formation in plates, slowed down xenograft growth in nude mice, effectively restricted the G1-S cell cycle progression and down regulate the expression of important molecules in Rb/E2F pathway. Therefore, c-myc might accelerate G1-S cell cycle, and increase cell proliferation by activating Rb/E2F signal pathway in etiology and pathogenesis of NPC. In brief, our study provided new clues to elucidate the molecular mechanism of NPC carcinogenesis and supplied an efficient system to study gene function in mammalian cells. Furthermore, our experiment provided a theoretical basis for gene therapy of NPC and c-myc is expected to serve as a potential therapeutic target for human NPC.