MLPH regulates EMT in pancreatic adenocarcinoma through the PI3K-AKT signaling pathway

Abstract

Pancreatic adenocarcinoma (PAAD) is an extremely malignant tumor, and most patients develop postoperative metastases. Melanophilin (MLPH) is involved in the progression of various tumors, but its molecular mechanisms and role in pancreatic cancer progression are unknown. In this study, differential MLPH expression in cancer tissues and the adjacent tissues was evaluated using the Gene Expression Profiling Interaction Analysis 2 (GEPIA 2) and Human Protein Atlas (HPA) databases. The role of MLPH in PAAD proliferation, invasion, and migration in vitro was explored via clone formation, Cell Counting Kit-8 assay, Transwell assay, and western blot. The in vivo validation of function was performed using a metastatic nude mouse model. The result showed that the pancreatic cancer tissues had significantly higher MLPH expression levels than the noncancerous pancreatic tissues. MLPH expression changes were related to PAAD cell proliferation, invasion, and migration. The western blotting demonstrated that PAAD cells had reduced Epithelial-mesenchymal transition (EMT)-related marker expression. Furthermore, overexpressing MLPH enhanced cell proliferation, migration, and invasion, and increased EMT-related marker expression. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the molecular mechanism underlying the effect of MLPH on PAAD was significantly related to the PI3K-AKT pathway. LY294002 blocked the MLPH overexpression-mediated enhanced cell invasion and migration and inhibited EMT-associated marker expression. Conversely, 740Y-P reversed the inhibitory effects of MLPH downregulation and led to cell migration, invasion, and EMT. MLPH regulated EMT to mediate PAAD cell invasive migration through the PI3K-AKT pathway. The results indicated that MLPH is a possible target for blocking PAAD metastasis.

Keywords: MLPH; PI3K-AKT signaling pathway; epithelial-mesenchymal transition; pancreatic adenocarcinoma; progression.

Figure 1

Differential expression of MLPH. (A)According to the GEPIA 2 database, MLPH is highly expressed in several cancer tissues including PAAD. (B) MLPH is highly expressed in PAAD tissues. (C) Representative immunohistochemical staining images of MLPH in PAAD and normal tissues from the HPA database. (D) Overall Survival analysis in the Kaplan-Meier Plotter database based on TCGA data. (E) MLPH mRNA expression level in different Histologic Grade of PAAD samples. (The scale bar is 200 μm, the locally enlarged scale bar is 50 μm.*P<0.05).

Figure 2

MLPH enhances the proliferation of PAAD cells. (A, B) Protein levels of MLPH in cells were detected by western blotting after MLPH knockdown and overexpression, respectively. (C, D) The mRNA levels of MLPH in cells were detected by real-time fluorescence quantitative PCR after MLPH knockdown and overexpression, respectively. (E, F) Quantitative statistics of the number of colonies and the number of colonies of cells detected by clone formation assay after MLPH knockdown and overexpression are shown, respectively. (G, H) Proliferative capacity of cells was detected by CCK-8 assay after MLPH knockdown and overexpression, respectively. (si-MLPH group vs. negative control group; oe-MLPH group vs. Vector group, *P < 0.05, ** P < 0.01, *** P < 0.001).

Figure 3

MLPH promotes the migration and invasive capacity of PAAD cells. (A) After the knockdown of MLPH, the migratory ability of the cells was examined through chambers without the addition of matrix gel. (B) After overexpression of MLPH, the migratory capacity of the cells was examined through chambers without the addition of matrix gel. (C) After the knockdown of MLPH, the invasive ability of the cells was tested by the addition of matrix gel to the chambers. (D) After overexpression of MLPH, the invasive capacity of the cells was detected by adding matrix gel to the chambers. (E, F) After overexpression of MLPH, the protein expression of EMT markers (E-cadherin, N-cadherin, and vimentin) were measured by western blotting. (G, H) After the knockdown of MLPH, the protein expression of EMT markers were measured by western blotting. (si-MLPH group vs. negative control group; oe-MLPH group vs. Vector group; The scale bar is 100 μm, *P < 0.05, ** P < 0.01).

Figure 4

MLPH promotes EMT in PAAD cells through the PI3K-AKT signaling pathway. (A, B) KEGG Pathway enrichment analysis by KEGG based on TCGA database. (C, D) Relative protein levels of MLPH, E-cadherin, N-cadherin, Vimentin, p-Akt, and Akt in cells after treatment with or without 740Y-P and the quantitative statistics of protein expression. (*P < 0.05, ** P < 0.01). (E, F) Relative protein levels of MLPH, E-cadherin, N-cadherin, Vimentin, p-Akt, and Akt in cells after treatment with or without LY294002 and the quantitative statistics of protein expression. (*P < 0.05, ** P < 0.01).

Figure 5

MLPH promotes tumor progression in vivo. (A) Subcutaneous tumor formation in nude mice after MLPH knockdown. (B) The tumor growth curves of subcutaneous tumor in nude mice after MLPH knockdown. (C) Subcutaneous tumor weight in nude mice after MLPH knockdown. (D) Subcutaneous tumor formation in nude mice after MLPH overexpression. (E) The tumor growth curves of subcutaneous tumor in nude mice after MLPH overexpression. (F) Subcutaneous tumor weight in nude mice after MLPH overexpression. (G) The general appearance of lung metastasis. (H) The number of lung metastasis nodules. (*P < 0.05, ** P < 0.01, *** P < 0.001).