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Objective To investigate the effect of Smad3 on the apoptosis and autophagy of pancreatic cancer cells Panc-1 and BxPC-3, in order to preliminarily verify the effect of Smad3 expression in the progression of pancreatic cancer. Methods Bioinformatics analysis was performed based on GEPIA2 database to explore the correlation of Smad3 with the occurrence, development, and survival of pancreatic cancer. The CRISPR/Cas9 system and short hairpin RNA (sh-RNA) were used respectively to knock out or reduce the expression of Smad3. Real-time quantitative PCR (qPCR) and Western blotting were subsequently adopted to detect the changes of Smad3 at mRNA and protein levels. The sh-RNA experimental groups were transferred with ShSmad3-1 and ShSmad3-2 sequences, and the CRISPR/Cas9 experimental group were transferred with Smad3 guide RNA (gRNA) sequence, using Sh-Scramble sequence (Scr) and empty plasmid as the corresponding control groups, respectively. Western blotting was applyed to determine the protein changes of LC3, p62, p38 and p-p38 in the cells. Meanwhile, Smad3 knockdown pancreatic cancer cells were stained with PI-Annexin V kit and then analyzed by flow cytometry. To further prove the effect of Smad3 on pancreatic cancer cells, the pLVX-Smad3 plasmid was used to perform a rescue experiment, and in another assay, the cells were treated with p38 inhibitor SB203580 to observe the changes of related proteins. Results The results of GEPIA2 database and clinical specimen analysis showed that Smad3 was highly expressed in pancreatic cancer tissues (P < 0.05). After transfection of ShSmad3-1 and ShSmad3-2 into pancreatic cancer cells BxPC-3, both the mRNA and protein levels of Smad3 were significantly decreased (P < 0.05). Meanwhile, 2 Smad3 knockout cell lines were screened out after transferring gRNA into Panc-1. As compared with the control group, Smad3 knockdown remarkably increased the apoptosis of pancreatic cancer cells (P < 0.05), and Western blotting also indicated that after Smad3 knockdown or knockout, the protein level of p-p38 was up-regulated (P < 0.05), while those of LC3 and p62 were down-regulated (P < 0.05). Further study found that the protein levels of p62 and LC3 were restored by the rescue assay as well as by the treatment of pancreatic cancer cells with SB203580 (P < 0.01). Conclusion Interference in Smad3 promotes excessive autophagy in pancreatic cancer cells and leads to increased apoptosis, possibly through activation of p38/MAPK signaling pathway. |
