Treatment of cholangiocarcinoma by pGCsiRNA-vascular endothelial growth factor in vivo

Cholangiocarcinoma QBC939 cells were injected subcutaneously into the back of nude mice (1 × 10⁷ cells/mouse). When the tumor grew to about 5 mm in diameter, it was freshly harvested and cut into pieces (1 mm × 1 mm × 1 mm). The nude mice were anesthetized with 2% pentobarbital, and the abdomen was disinfected and cut open along the linea alba. Then the tumor tissue was inoculated closely near the bile duct, and finally the abdomen was closed. After surgery, the mice were fed for 4–6 weeks.

VEGF oligonucleotides synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) were annealed and ligated into the pGCSilencerTMneo2.0-U6/GFP-siRNA expression vector, and positive clones were screened out after transformation (Figure 1). The plasmids were extracted in accordance with the instructions of the kit (Thermo Fisher Scientific, USA) and identified by digestion with HindIII and BamHI. Then the digested products were identified by 2% agarose electrophoresis, and the results showed that 6.3 kb linearized vectors and 63 bp oligonucleotide fragments were obtained. The synthesized plasmids were sent to Shanghai Invitrogen Biotechnology Co., Ltd. (China) for sequencing.

Figure 1.

Twenty-one nude mice with cholangiocarcinoma were randomly divided into 3 groups (mock group, pGCsiRNA-VEGF group, and si-scramble group), with 7 mice in each group. The mice in the mock group were perfused with PBS, those in the pGCsiRNA-VEGF group were infected by an oral perfusion with attenuated Salmonella carrying the plasmid pGCsiRNA-VEGF at 1 × 10⁴ CFU/mL, and those in the si-scramble group were infected by an oral perfusion with attenuated Salmonella carrying plasmid pGCsiRNA. The eating, drinking, and mental status of mice was observed in each group, and the tumor growth status was observed and recorded every other day after treatment.

On Day 28 of treatment, the nude mice were sacrificed in each group. The tumors were harvested, weighed, and photographed. Then the tumor tissues were fixed in 10% formalin solution, embedded in paraffin, and sectioned. After deparaffinization and hematoxylin–eosin (HE) staining, microscopy was performed to observe the effects of different treatments on tissues of mice.

About 1–3 pieces of tumor tissue (about 0.5 g) were harvested from each mouse, washed with normal saline 2–3 times, and prepared into homogenates. The protein was extracted with RIPA lysis buffer, subjected to electrophoresis (30 μg/well), and transferred onto PVDF membranes. Then the membrane was sealed with 5% skim milk at room temperature for 1 h and incubated with anti-VEGF, MMP2, and MMP9 antibodies (1:1,000) at 4°C overnight. After washing, the membrane was incubated again with horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:10,000) at room temperature for 30 min. Finally, color was developed by ECL, images were acquired, and the intensity of protein bands was analyzed with Image Lab software (Bio-Rad, USA).

One piece of tumor tissue (about 1 g) was harvested from each mouse, washed with normal saline 2–3 times, and prepared into homogenates. Total RNA was extracted with 1 mL of TRIzol (Invitrogen, USA) and its concentration was measured. Then, 500 ng of total RNA was reversely transcribed into cDNA according to the instructions of the PrimeScript RT kit (Takara, Japan), and PCR was performed on a fluorescence quantitative PCR instrument (Bio-Rad, USA) using the qRT-PCR kit (Vazyme, Nanjing, China).

Prism software (GraphPad, USA) was used for statistical analysis, and the results were described as mean ± standard deviation. Comparison was conducted between the 2 groups by the Student’s t test, and P < 0.05 was considered statistically significant. All tests were 2-tailed, and all test results had 3 biological replicates.

Tumors, with a diameter of about 5 mm, formed on the right side of nude mice about 2 weeks after inoculation with QBC939 cells, suggesting the successful establishment of the mouse xenograft model of cholangiocarcinoma. After grouping, the treatment was initiated (Day 1), the tumor growth status was observed every other day, and the tumor growth curve of each group was plotted (Figure 2). The tumor volume was measured every other day after treatment. The tumor volume was smaller in the pGCsiRNA-VEGF group than that in the mock group and the si-scramble group on Day 27 of treatment (P < 0.05). On Day 28 of treatment, the nude mice were sacrificed. The tumor weight was smaller in the pGCsiRNA-VEGF group than that in the mock group and the si-scramble group (P < 0.01). Collectively, the tumor growth in nude mice was inhibited (Figures 3 and 4).

Figure 2.

Figure 3.

Figure 4.

The protein expressions of VEGF, MMP2 and MMP9 significantly decreased in pGCsiRNA-VEGF group compared with those in the mock and si-scramble groups (P < 0.01) (Figure 5).

Figure 5.

The mRNA expressions of VEGF, MMP2, and MMP9 were significantly lower in the pGCsiRNA-VEGF group than those in the mock and si-scramble groups (P < 0.05) (Figure 6).

Figure 6.

It was observed by HE staining that in the pGCsiRNA-VEGF group, there was obvious apoptosis of tumor tissues, a large number of cells died, the tissue structures mostly disappeared, karyopyknosis occurred, many apoptotic cells were dispersed around the tissues, and the cells lost their normal morphology (Figure 7).

Figure 7.

The protein expression of VEGF was significantly suppressed in the pGCsiRNA-VEGF group compared with that in the mock and si-scramble groups (Figure 8). Taken together, the expressions of VEGF, MMP2, and MMP9 at the transcriptional and translational levels were inhibited by pGCsiRNA-VEGF. PGCsiRNA-VEGF promoted tissue apoptosis and destroyed the tissue structure.

Figure 8.