Effect Of MUC4Gene Silencing By RNAi On Cell Proliferation And Migration In Human Pancreatic Carcinoma BxPC-3Cells

Objective: To construct MUC4-ShRNA lentivirus expression vector andinvestigate the effect of MUC4gene silencing by RNA interference (RNAi)on cell proliferation and migration in human pancreatic carcinoma BxPC-3cells in vitro and in vivo.Methods:（1）Eight sequence of ShRNA were designed based on MUC4gene toconstruct MUC4-ShRNA lentivirus expression vector. Quantitative real-timePCR (qPCR) was used to test the effect of MUC4gene silencing.（2）In vitro study：Transfection of BxPC-3cells with MUC4-ShRNAlentivirus expression vector to construct the lentiviral expression clones(BxPC-3-MUC-ShRNA) for MUC4gene silencing; application of qPCR andWestern blot to determine transcription levels of MUC4gene and proteinexpression; using CCK8to test cell proliferation and apoptosis; Transwellmigration assay was used to test the invasive ability of BxPC-3-MUC-ShRNAcells.（3）In vivo study: Flank xenograft tumors of BxPC-3cells wereprepared by subcutaneous injection of5×10~6cells (including blank controlempty plasmid transfection (NC group), MUC4gene silencing (KD group)and BxPC-3cell control group) suspended in serum free media into nudemice (16–22g). Tumor size and metastasis were measured after tumorinoculation.（4）MRI was used to observe tumor signal changes and tumor volume changes at different time points (every week for8weeks after tumorinoculation)Results:（1）Eight sequences of SiRNA were successfully constructed forconfirming the gene silencing effect.（2） MUC4-ShRNA lentivirus expression clones (BxPC-3-MUC-ShRNA) were successfully obtained and qPCR test demonstrated the genesilencing effect. It was found that the sequence of ShRNA of A1000141induced significant MUC4sequence-specific silencing in BxPC-3cells.（3）qPCR and Western blot results showed that MUC4gene and proteinexpression in BxPC-3-MUC-ShRNA cells obviously reduced; CCK-8assayand Transwell assay showed that cell proliferation, migration and invasionwere markedly inhibited in shRNA MUC4-transfected BxPC-3pancreaticcancer cells.（4）Human ectopic pancreatic tumor xenograft model in nude mice wassuccessfully constructed and the incidence of tumor xenograft was100%(36/36). Tumor volume and weight in KD group are lower than the controlgroup. Liver or distant metastasis lesions were not found in KD group. Therewas statistically difference between control group and KD group (P<0.05).（5）MRI study on the human ectopic pancreatic tumor xenograft model:1week after inoculating, xenograft tumors of BxPC-3cells in nude mice wereisointensity on T1WI sequence, homogeneously high signal on the FIESTAand T2WI.2-3weeks later, the signal was similar to the first weekperformance. From4to10weeks, low signal was found in center of tumorson T1WI (liquefaction necrosis area), and its surrounding showed slightly lowsignal or isointensity on T1WI, and high signal on T2WI. With the growth of the tumor volume, the necrotic area increased. Among the three groups oftumor xenograft MRI signal showed no obvious difference.Conclusion:（1）ShRNA against MUC4reduced the expression of MUC4. Cellproliferation, migration and invasion were markedly inhibited and apoptosiswas increased in shRNA MUC4-transfected BxPC-3pancreatic cancer cells.MUC4genes play an important role in the development of pancreatic cancer.Inhibition of MUC4gene expression shows potential for therapeuticintervention in human pancreatic cancer.（2）MRI can be used to non-invasively observe the growth process ofectopic human pancreatic tumor xenograft in nude mice.