PDX1, a key factor in pancreatic embryogenesis, can exhibit antimetastatic activity in pancreatic ductal adenocarcinoma

Figures & data

Table 1 Real-time PCR primers used in gene expression analysis

Gene	Primer sequence (5’ → 3’)		PCR product (bp)
18S	Fw	CGCGGTTCTATTTTGTTGGT	501
Rv	ATGCCAGAGTCTCGTTCGTT
EEF1a	Fw	GACACGTAGATTCGGGCAAG	173
Rv	GATACCACGTTCACGCTCA
PDX1	Fw	GTCCTGGAGGAGCCCAAC	272
Rv	CGGCGGTTTTGGAACCAGAT
CDH1	Fw	AGTGCCTGCTTTTGATGATG	338
Rv	AGCTTGAACTGCCGAAAAATC
KRT8	Fw	ATGTTGTCCATGTTGCTTCG	125
Rv	ACCCTCAACAAGTTTGCC
MUC1	Fw	CTGGTCTGTGTTCTGGTTGC	250
Rv	CCACTGCTGGGTTTGTGTAAG
KLF5	Fw	ACAAATCAGACAGCAGCAATGGACA	312
Rv	GGTGGTGGGTAAATTTGGATTGTGA
VIM	Fw	GCAGAAGAATGGTACAAATCCA	144
Rv	TTTAAGGGCATCCACTTCACA
SNAIL	Fw	CCAATCGGAAGCCTAACTAC	124
Rv	GCGGTGGGGTTGAGGATCTC
SLUG	Fw	AGAAGGTTTTGGAGCAGTTTTTG	160
Rv	TGGTTGCTTCAAGGACACAT
ZEB1	Fw	GAACAGTGTTCCATGCTTAAGAGCG	217
Rv	GGGCGGTGTAGAATCAGAGTCATTC

Abbreviations: Fw, forward primer; Rw, reverse primer; bp, base pair.

Figure 1 Construction of PDX1 overexpression in PANC-1 cells. (A) Scheme of the lentiviral construct used to obtain GFP-labeled PANC-1^PDX1 (1) and PANC-1^Control (2) cells. (B) Western blot analysis of the expression of the PDX1 protein in PANC-1^PDX1 cells and in the PANC-1^Control group, and the expression of PDX1 in PANC-1^PDX1 after treatment with anti-PDX1 siRNA and negative siRNA. Abbreviations: PCNA - proliferating cell nuclear antigen gene promoter; PDX1 -Pancreatic And Duodenal Homeobox 1 gene; PGK - promoter of 3-phosphoglycerate kinase gene; puro - puromycin-resistance gene encoding N-acetyl-transferase; CMV - cytomegalovirus promoter; GFP  - green fluorescent protein gene, 5'-LTR, 3'- LTR - 5' and 3' long terminal repeat; GAPDH - Glyceraldehyde 3-phosphate dehydrogenase; kDa - kilodalton.

Figure 2 Effects of PDX1 overexpression on PANC-1 cell proliferation. (A) Growth curves of cells were plotted after transfection with indicated vectors by MTT assays. The OD value of cells in the control group was lower than that of PANC-1^PDX1+ cells. *P<0.05, **P<0.01 compared with control group (B) Flow cytometry results showed that the number of cells in the S stage of PANC-1^PDX1+ was significantly lower than of PANC1^Control, whereas the number of cells in the G₁ and G₂ stages of PANC-1^PDX1+ cells was higher than that of PANC1.^Control

Figure 3 Expression of PDX1 significantly reduces both migration and invasion of human pancreatic cancer cells. (А) Effect of PDX1 overexpression on pancreatic cancer cell wound recovery. Nearly confluent cell monolayers of PANC-1^Control and PANC-1^PDX1 cells were scratched, and wound recovery was monitored after 24 hrs. The rate of wound closure ± SEM is shown. The experiment was performed in triplicate with a similar trend. (B) The transwell cell invasion assay of PANC-1^Control and PANC-1^PDX1 cells. Representative fields of invasive cells on a membrane are captured. The average number of invasive cells per field from three independent experiments ± SEM is shown. *P<0.05; **P<0.01.

Figure 4 Relative expression of pro-epithelial and pro-mesenchymal genes in PANC1^Control and PANC-1^PDX1 cells. TFs are the transcription factors. Data are presented as the mean ± SEM for three to five independent experiments; *P<0.05, **P<0.01 compared with the control group.

Table 2 Quantitation of embryos with migrating cells^a

Cells	PANC-1^Control			PANC-1^PDX1
siRNA	No siRNA	siPDX1	siNeg	No siRNA	siPDX1	siNeg
% of embryos with migration	50%	30%	23%	12,5%	40%	6%

Note: ^aResults of 8 independent experiments are summarized in the table. The variation between the experiments was 10%.

Abbreviation: PDX1, pancreatic and duodenal homeobox 1.

Figure 5 Representative images of migration of PANC-1^PDX1/PANC-1^Control cells within injected embryos. Fluorescent (left) and bright/fluorescent fields (right) images are presented 20× magnification. hpt is hour post-transplantation, dpt is days post-transplantation.