FIGURE 2.

Exogenous overexpress LDHA enhances proliferation, migration, and invasion in vitro and promotes tumorigenesis and liver metastasis in vivo. (A) The protein expression of LDHA of various pancreatic cancer cell lines was confirmed by immunoblotting. Beta‐actin was used as loading control. The mRNA expression of LDHA and invasion capability of various pancreatic cancer cell lines. Invasion capability through Matrigel and ordered from strongest to lowest, from Panc02 cells to Capan‐2 cells (n = 3 per group). (B) The overexpression and knockdown of LDHA‐transfected Panc02 and Panc‐1 cells was confirmed by immunoblotting and RT‐PCR. Beta‐actin was used as loading control (n = 3 per group). (C) LDHA overexpression (OE) promoted the colony formation capabilities in Panc02 and Panc‐1 cells (n = 6 per group, **P < 0.01, ***P < 0.001) compared with controlled cells (NC). LDHA knockdown (SH) suppressed colony formation in Panc‐1 and Panc02 cells (n = 6 per group, *P < 0.05, **P < 0.01) compared to controlled cells (NC). (D) The invasive properties were analyzed by Matrigel‐coated Boyden chamber assay and scored under a light microscope (200×). LDHA knockdown (SH) suppressed the invasive properties in Panc02 cells (**P < 0.01) and Panc‐1 cells (*P < 0.05) compared to the respective controlled cells (NC) (n = 6 per group). Overexpression of LDHA (OE) promoted cell invasion in both Panc02 and Panc‐1 cells (n = 6 per group, ***P < 0.01) compared to the respective controlled cells (NC). (E) A wound was produced and monitored at 0, 24, and 48 h as the cells moved and filled the damaged area (200×). The data were plotted as the percentage area healed (n = 3 per group, *P < 0.05, **P < 0.01). (F) The tumor size and average weight of primary xenografts of orthotopic implantation model (n = 6 per group, **P < 0.01). H&E staining confirmed the tumorigenesis of LDHA‐OE cells in orthotopic model (400×). The Ki‐67 and LDHA expression in the histological sections was detected by immunohistochemical (IHC) staining (n = 12 per group, ***P < 0.001, 400×). (G) Macroscopic liver metastasis lesion was visible on the surface of the liver tissue (white arrowed) in LDHA‐OE group and the number of liver metastatic lesions in mice (n = 6 per group) was analyzed by counting the macroscopic lesion from each hepatic lobe (***P < 0.001). H&E staining identified adenocarcinoma in the liver section of the LDHA‐OE group, while no liver metastasis lesion was identified with the control group (400×). The Ki‐67 and LDHA expression in the histological sections was detected by immunohistochemical (IHC) staining (n = 12 per group, 400×). NC: controlled cells; SH: LDHA knockdown cells; OE: LDHA overexpressed cells