Effect Of RAAV-HIF-1α-siRNA Combined L-ascorbate On Glycometabolism Of Human Pancreatic Cancer MiaPaC2 Cells

ObjectiveThe pancreatic adenocarcinoma lacks blood vessels, which can expressHIF-1αrichly, and HIF-1αcan adjust the glycometabolism of carcinoma cellsduring hypoxia self-adaptation. It will possibly be a effective method of treatingpancreatic adenocarcinoma to target HIF-1α. RNAi can precipitate thedegradation of the target gene mRNA specifically and effectively, and block theexpression of the target gene, achieve the effect of gene knock-out. AAV isconsidered as the ideal carrier in gene therapy for it's having no pathogenicityand immunogenicity, etc. Ascorbate can promote the degradation of HIF-1α.In this study, the MiaPaC2 human pancreatic carcinoma cells were handledwith rAAV-siRNA targeted on HIF-1αand ascorbate in anoxic condition, thehandled cells were harvested and used to observe the expression of Glut-1which is the key enzyme of glycometabolism reaction to investigate the effect oftreating pancreatic adenocarcinoma by pAAV-siRNA targeted on HIF-1αcombined with ascorbate.Methods1.Vector plasmids(pAAV-hrGFP and pAAV-H1-siRNA-hrGFP)and helperplasmids(pAAV-RC and pAAV-Helper) were extracted and amplified;rAAVwas produced by cotransfection of AAV Helper-Free System in HEK293 cell byphosphate-calcium deposit method; rAAV was extracted and purified and it'stiter was calculated;the transfection effect of rAAV into MiaPaC2 humanpancreatic carcinoma cells was judged by observing the fluorescence expressionof GFP.2.The MiaPaC2 cells were cultured and divided into 5 groups:â‘ blankcontrol groupâ‘¡idling virus groupâ‘¢simple ascorbate groupâ‘£simplerAAV-H1-siRNA-hrGFP group⑤rAAV-H1-siRNA-hrGFP combined withascorbate;after cultivated for 24 and 48 hours in anoxic condition(5%CO₂, 1%O₂,94%N₂), the protein of HIF-1αwas determined by Western Blot, themRNA of Glut-1 was detected by RT-PCR, the protein of Glut-1 was determinedby Western Blot.Results1.The rAAV was constructed successfully and it's shape and purity weresatisfactory, the titer of it was 4×10¹³ particles/ml.rAAV-HIF-1α-siRNA wastransfected into MiaPaC2 cells successfully and the fluorescence of GFP wasobserved through the fluorescence microscope.2. From Western Blot, we know that rAAV-HIF-1α-siRNA and L-ascorbatecan successfully suppress the expression of HIF-1αprotein after 24h underhypoxic condition and the expression of HIF-1αprotein after 48h declined moreobviously in the combined group. Applying the RT-PCR method to detect theeffect of each treated factor on the mRNA expression of Glut-1, we discover thatboth rAAV-H1-siRNA-hrGFP and ascorbate can refrain the mRNA expressionof Glut-1, which there is significant difference compared with the control group(P＜0.01), but rAAV-hrGFP have no effect on the mRNA expression ofGlut-1(P＞0.05) and the rAAV-HIF-1α-siRNA repressing the mRNA expressionof Glut-1 can reach the peak in 48 hours which exceeds the role in 24 hoursobviously. Applying the Western blot method to detect the effect of each treatedfactor on the protein expression of Glut-1, we Find the protein expression ofGlut-1 decreased significantly in rAAV-H1-siRNA-hrGFP group compared withthe control group(P＜0.01), as well as ascorbate group andrAAV-H1-siRNA-hrGFP combined with aseorbate group. the protein expressionof Glut-1 in rAAV-hrGFP group have no significant changes compared with thecontrol group(P＞0.05). the protein expression of Glut-1 decreased in 24 hourssignificantly, but the descent of protein expression is more significant in 48hours.Conclusions1. The rAAV was constructed successfully and it's shape, purity and titer were satisfactory; it was transfected into MiaPaC2 cells successfully.2. rAAV-HIF-1α-siRNA and L-ascorbate can both suppress the expressionof HIF-1αprotein and the effect of the combined group is more efficient thanthat seen when rAAV-HIF-1α-siRNA or L-ascorbate was used separately.BothrAAV-H1-siRNA-hrGFP and ascorbate can repress the activity of Glut-1which is the key enzyme in glycometabolism in MiaPaC2 cells,inhibit theproliferation of carcinoma cells and possibly achieve the purpose of treatingpancreatic adenocarcinoma.