Anti Pancreatic Cancer Targeting Silencing Of Glutamine Transporter ASCT2 Based On RNA Interference

Objective: To evaluate the blocking effect of RNA interference on the expression of glutamine transporter ASCT2 in human pancreatic cancer ASCP1,BXPC3 and PAN-C cell lines,the biological characteristics of the proliferation and apoptosis of pancreatic cancer cells with ASCT2 gene silencing were detected at two levels,respectively in vitro and in vivo in nude mice,and the related molecular mechanisms were explored.Methods:1.Pancreatic cancer cell lines ASPC1,BXPC3 and PAN-C were used as cell models in vitro.Plotting the growth curve of glutamine deficient cells: When the cell growth density was 70%~80%,6 orifice plates were digested and 10~5 cells were spread in each hole.Each cell was paved with two six orifice plates,respectively,group Gln+(including 2 mM Gln)and Gln-group,respectively.The number of cells in third,fifth,seventh and ninth days were counted respectively,and the growth curves of each group were drawn.2.Cell cloning experiment after glutamine deletion: when the cell growth density was 70%~80%,the cells were spread in the cell culture dish with diameter 3.5mm respectively.Each dish was paved with 800 cells,which were set as Gln+ group(including 2 mM Gln)and Gln-group respectively.The cells were cultured for 1 time after 1 week of cell culture,and cultured to two weeks to carry out crystal violet staining,and the digital cameras were photographed.3.A short hairpin RNA(shRNA)lentivirus expression vector targeting ASCT2 gene was constructed,and 293 T cells were transfected with pMDLg/p RRE,p RSV-Rev,pMD2.G in the three plasmid system of lentivirus packaging and packaged out of the lentivirus.After infection of pancreatic cancer cell line 24 hr by using lentivirus,puromycin was screened for 72 hr.The expression of ASCT2 was detected by real-time fluorescence quantitative PCR and Western blotting.The content of ATP in pancreatic cancer cell lines with ASCT2 gene silencing and the content of glutamine in culture medium supernatant were detected by the cell metabolism detection kit of Biovision company.4.The ASCT2 gene silenced and stable pancreatic cancer cell line was constructed,and the negative NC sh RNA was used as the control group.Cell count was used to detect the number of cell cultures for first,third,fifth and 7 days.The growth curve of the two groups was plotted.5.The cell apoptosis rate was detected by flow cytometry and the distribution of cell cycle was analyzed.Protein immunoblotting was used to determine the cell cycle distribution.The changes in the expression of p AKT,p70S6K,p4EBP1,Bcl-2,Bax and Cleaved-Caspase3 after ASCT2 gene silencing.6.The tumor model was used in nude mice to measure the tumor weight change and the tumor volume growth curve was compared.The changes of the expression of ASCT2 and Cleaved-Caspase3 in tumor tissues were detected by protein immunoblotting.Results:1.The cell growth curve showed that after 7 days of continuous culture,the cell growth of three kinds of pancreatic cancer cell lines was significantly slower,and the cells in the 3 control groups were all exponentially increased after the culture of 48 hr.2.The results of cell cloning experiment showed that the growth inhibition of ASPC1 and BXPC3 cells in the glutamine deficient group was obvious,the number and volume of cloned groups were significantly lower than that of the normal medium group.3.ASCT2 gene silencing efficiency: after purinamycin screening 72 hr,the RT PCR method and Western blot method were used to analyze the silencing efficiency of ASCT2 m RNA and protein transcription.The results showed that the inhibition rate of gene and protein was more than 90%(p< 0.001).4.After silencing the ASCT2 gene of three cells,the uptake rate of glutamine decreased by more than 50%,and the levels of ATP and ?-KG in cells decreased significantly.5.The effect of ASCT2 gene silencing on growth curve: After continuous culture of 168 hr,the growth of 3 groups of cells with ASCT gene silenced was obviously inhibited,and the apoptosis of PAN-C cells was the most obvious after the inhibition of ASCT2,and the 3 control groups increased exponentially after the culture of 48 hr.6.The effect of ASCT2 gene silencing on cell cycle and apoptosis: The flow cytometry showed that compared with the control group,BXPC3 cells appeared "subdiploid" apoptotic peak(subG2 peak)before G1 phase,G2 stage cells increased significantly,G1 phase cells decreased(all p<0.05),and the comparison of S phase cell ratio was not significant(p>0.05).It was suggested that the cell cycle was obviously blocked in G2/M phase,PAN-C cells decreased in S phase and increased in G1 and G2 phase(all p<0.05),suggesting that cell cycle was blocked in G1/S phase,Before G1 phase,ASPC-1 cells appeared sub G2 peak,cells in S stage decreased obviously,G2 stage cells increased significantly(all p<0.05),and the comparison of proportion of G1 phase cells was not significant(p>0.05),suggesting that cell cycle was blocked in G2 phase.Apoptosis results showed that the apoptotic rate of ASCT2 gene silencing group increased significantly(all p<0.05).The results of protein immunoblotting showed that after the silencing of ASCT2 gene,the proportion of Cleaved-Caspase3 and Bax of apoptosis related proteins increased obviously,while the proportion of anti apoptosis related protein Bcl-2 decreased significantly(all p<0.05).7.The effect of ASCT2 gene silencing on the Akt/m TOR pathway: The relative expression of p-Akt,p-S6k and p-4EBP1 increased significantly in the control group after adding insulin(all p<0.05),and the level of insulin induced p-Akt,p-S6 k and p-4EBP1 protein expression increased significantly after the ASCT2 gene silencing(all p<0.05),indicating that ASCT2 played an important role in the activation of the Akt/m TOR pathway signal pathway.8.The effect of ASCT2 gene silencing on the proliferation of BXPC3 nude mice:The growth volume rate of the sh ASCT2 group was significantly smaller than that in the Control group(P<0.01),and the tumor mass results showed that the mass of the sh ASCT2 group was less than the Control group(P<0.01).The results of protein immunoblotting showed that compared with the control group,the expression of ASCT2 protein in the tumor tissues of the ASCT2 gene silencing group decreased obviously(p<0.05),and the expression of Cleaved-Caspase3 increased obviously(p<0.05),indicating that the ASCT2 silencing could inhibit the tumor cell formation ability by inducing apoptosis.Conclusion:1.The growth and survival of pancreatic cancer cells ASPC1,BXPC3 and PAN-C depend on glutamine metabolism.ASCT2 mediates the glutamine metabolism of these three cells.2.ASCT2 gene silencing effectively inhibited the growth of ASPC1 and BXPC3 pancreatic cancer cells.3.The inhibitory effect of ASCT2 silencing on ASPC1,BXPC3 and PAN-C in pancreatic cancer cells may be related to cell cycle and apoptosis.4.The inhibitory effect of ASCT2 silencing on ASPC1,BXPC3 and PAN-C in pancreatic cancer cells may be related to the involvement of ASCT2 in the activation of Akt/m TOR pathway.5.ASCT2 silence significantly inhibited the proliferation of pancreatic cancer xenografts in nude mice,indicating that ASCT2 deficiency could inhibit tumor growth in vivo.Our findings suggest that ASCT2 can be a potential target for pancreatic cancer treatment.