The lentiviral expression vector was purchased from the Zimmer Medical International Trading Co., Ltd. (Shanghai, China). DH5α Escherichia coli (E. coli) was obtained from the Chinese Academy of Sciences (Shanghai, China). The restriction endonucleases BbsI and BamHI, T4 ligase and Taq enzyme were purchased from Takara (Dalian, China). Roswell Park Memorial Institute (RPMI)-1640 culture medium, fetal bovine serum (FBS), trypsin, Lipofectamine 2000 and TRIzol reagent were purchased from Gibco (Invitrogen Life Technologies, Carlsbad, CA, USA). A plasmid DNA extraction kit was obtained from Qiagen (Shanghai, China). A reverse transcription kit was purchased from Promega (Fitchburg, WI, USA). All oligonucleotides, including PCR primers, were purchased from Shanghai YingJun Biotechnology Co., Ltd. (Shanghai, China). A Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). A Boyden chamber for the migration assay was obtained from Corning Costar (Rochester, NY, USA). Matrigel (0.3 mg/ml) was purchased from BD Biosciences (Bedford, MA, USA).

BxPC-3 cells, a human PC cell line, was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. These cells were grown in RPMI-1640 media supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 95% air and 5% CO₂.

The eight MUC4-siRNAs, and a negative control (with a scrambled sequence that did not match any known gene) were selected based on the full-length cDNA of human MUC4 mRNA (gene bank no. NM_004532) using Ambion siRNA design software. A siRNA against GAPDH was included as a positive control to verify transfection reliability, RNA extraction and gene expression quantification. The TTCAAGAGA sequence was used in the loop of all constructs to avoid premature termination. The sequences of eight shRNAs coresponding to MUC4-siRNA are listed in Table I. PCR products were annealed using the following conditions: 95°C for 5 min, 85°C for 4 min, 75°C for 5 min, 70°C for 5 min and preservation at 4°C. After the annealing process, 20 µl of shRNA template (10 µl of forward primer and 10 µl of reverse primer), 10 µl of annealing buffer (10X) and 70 µl of double distilled water (ddH₂O) were mixed at 100°C for ligation.

After agarose electrophoresis purification, shRNA was inserted into the pUETP/td-tomoto/puro vector at the BamHI and BbsI sites. The resulting vector was inoculated into competent DH5α E. coli, selected using kanamycin resistance, and verified with sequencing using Shanghai 3D Biopharm Bio-technology. A total of eight constructs, referred to as pUETP/td-tomoto/puro-shRNA-1-8 (A139-A146) were evaluated in cultured BxPC-3 cells. The experiment also included pUETP/td-tomoto/puro-shNC (shNC) as a negative control and pUETP/td-tomoto/puro-shPC (shPC) as a positive control.

Two microliters of total RNA were reverse transcribed with Premix Ex Taq™ (Takara) using random primers. MUC4 first-strand cDNA was amplified with specific primers as follows: forward, 5-GGAG AGGTATCGCCCTGATAGATT-3 and reverse, 5-ACGGTA GTTGGGCCTTTCTTC-3. The primers used for housekeeping gene, glyceraldehyde-phosphate dehydrogenase (GAPDH), were as follows: forward, 5-CATGAGAAGTATG ACAACAGCCT-3 and reverse, 5-AGTCCTTCCACGATAC CAAAGT-3. We evaluated the specificity of the primers using GenBank. The PCR products were separated on a 1.5% agarose gel and analyzed using an imaging system from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

Whole cell proteins were prepared from 5×10⁶ cells after transfection for 96 h using WB lysis buffer. For WB, we used an anti-MUC4 mouse monoclonal antibody (8G7) and an HRP-linked anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). We used the BCA protein assay kit to detect the protein concentration in the supernatant (Takara, Otsu, Japan). The protein bands were visualized using an enhanced chemiluminescence system (Bio-Rad Laboratories).

After seeding in 6-well plates at a density of 1×10⁵ cells/well, BxPC-3 cells were transfected with A139-A146, sh-NC, or sh-PC. After incubation for 96 h, MUC4 mRNA was examined using qPCR. Subsequently, we counted the number of cells at 48, 72 and 96 h after transfection using a colorimetric CCK-8 assay at 450 nm. Migration was examined using a 24-well, 8-µm pore transparent insert containing a gelatin-coated polycarbonate membrane filter. Cell migration was quantified by counting the cells that migrated to the lower chamber using crystal violet staining and an optical microscope. Tumor formation and growth were examined in nude mice.

The quantitative results are expressed as the mean value ± SD. All statistical analyses were carried out by one-way analysis of variance (ANOVA) using SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA). A P-value of <0.05 was considered to indicate a statistically significant difference.

Eight pairs of MUC4-shRNA were ligated into the lentiviral vector using T4 DNA ligase. A KpnI single enzyme digestion was performed, and the fragments were examined by agarose gel electrophoresis. A 2.95+7.5 kb fragment confirmed that the shRNA size conformed to the design (Fig. 2B). A transfection experiment on 293T cells showed that more than 80% of the cells produced red fluorescent protein, as observed with fluorescence microscopy (Fig. 2D).

The BxPC-3 cell lines were transfected with eight pairs of lentiviral vectors based on MUC4-shRNA. The qPCR results showed that expression of MUC4 mRNA in the BxPC-3 cells was significantly decreased by all eight shRNAs (P<0.05 vs. the NC; Fig. 3A) at 96 h after transfection. A141 had the strongest suppressive effect on MUC4 mRNA expression among the eight shRNAs and was used for further experiments. To obtain MUC4 knockdown in BxPC-3 cells, the BxPC-3 cell line was transfected with shRNA-A141, and cultured for nearly 20 days until the cell fluorescence ratio reached 100% (Fig. 3B).

After stable transfection with shRNA-A141 (KD group), qPCR and western blot analysis were performed. The results showed that the efficiency of MUC4 mRNA interference was ~91% (Fig. 4A). At the same time, MUC4 knockdown in the BXPC-3 cells (KD) showed reduced growth compared with cells transfected with empty or scrambled RNAi vectors (NC and CON groups), and the number of floating cells in the cell culture increased (Fig. 4B). Transwell migration assays showed that the number of cells in the polycarbonate membrane for the KD group were significantly lower than that of the NC group (P<0.01) (Fig. 4C). In fact, the expression level of MUC4 in the KD group was also significantly reduced, compared with the NC group (P<0.01) (Fig. 4D). The results showed that MUC4 protein knockdown in the BxPC-3 cells was noteworthy.

BxPC-3 cells of the KD, NC and CON groups were transplanted into nude mice. Tumors formed within 7–10 days. Lower tumor growth rates were observed for tumors derived from the KD group, whereas the transplanted tumors derived from the other groups grew rapidly (Fig. 5A). At 8 weeks after cell inoculation, the tumor volume reached 195.02±14.24 mm² in the KD group, which was less than a tenth of that in the NC and CON groups (Fig. 5C). Throughout the experiment, no significant differences were observed in the activity and eating behavior of the KD cell-transplanted nude mice. In the NC and CON groups, the activity of the nude mice gradually reduced with tumor enlargement.