Abstract
BackgroundOur previous study showed that the ribosomal protein L21 (RPL21) may play an important role in the development and survival of pancreatic cancer. In this article, RNA interference (RNAi) experiments were performed with RPL21-specific small interfering RNA (siRNA) to elucidate the mechanism by which RPL21 controls PC PANC-1 and BxPC-3 cell proliferation.MethodsIn the present study, PANC-1, BxPC-3 cells, and BALB/c nude mice were used to investigate antitumor effect and mechanism by which RPL21 controls cell proliferation and apoptosis in vitro and in vivo. The effects of RPL21 knockdown on PANC-1 and BxPC-3 cell proliferation, cell cycle and cell apoptosis in vitro were determined using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays and flow cytometry assay. The mechanism of RPL21 regulating cell proliferation was investigated using transcriptome sequencing analysis and luciferase reporter assay. The effects of RPL21 knockdown on PANC-1 and BxPC-3 cell proliferation in vivo were determined using BALB/c nude mice tumor model.ResultsIn PANC-1 and BxPC-3 cells, the knockdown of RPL21 expression with corresponding siRNA suppressed cell proliferation in vitro and in vivo, inhibited DNA replication, and induced arrests in the G1 phase of the cell cycle. Further results showed that the mini-chromosome maintenance (MCM) protein family (MCM2-7), CCND1 and CCNE1 were down-regulated significantly in PANC-1 and BxPC-3 cells after transfected with RPL21 siRNA, which suggests that the suppression of DNA replication is due to the reduced expression of MCM2-7 family, and the induction of G1 arrest is correlated with the inhibition of CCND1 and CCNE1. Luciferase reporter assay showed that RPL21 controls the DNA replication and G1-S phase progression possibly through the regulation of E2F1 transcription factor in PC cells. Moreover, RPL21 siRNA showed an apoptosis-inducing effect only in BxPC-3 and PANC-1 cells but not in normal HPDE6-C7 cells. The increase of caspase-8 activities and the loss of mitochondrial membrane potential after RPL21 silencing indicates that the RPL21 gene may be involved in caspase-8-related mitochondrial apoptosis.ConclusionOur findings suggest that siRNA against the RPL21 gene possesses a potential anti-cancer activity for PC cells by inhibiting their proliferation and DNA replication, as well as inducing cell cycle G1 arrest and cell apoptosis.
Highlights
These results indicate that siL21-1 and siL21-2 dramatically reduce the ribosomal protein L21 (RPL21) expression at mRNA and protein levels in BxPC-3 and PANC-1 cells
The number of clones in PANC-1 transfected with siL21-1 and siL21-2 were significantly decreased by 84 and 88%, respectively, compared with control groups (P < 0.05), while in BxPC-3 cells, the inhibiting values were 79 and 81%. These findings indicate that ribosomal protein gene RPL21 is closely related to proliferation of Pancreatic cancer (PC) PANC-1 and BxPC-3 cells
SiL21-1 and siL21-2 did not induce apoptosis in the normal pancreatic duct epithelial HPDE6-C7 cells, compared with control groups and NC (Supplementary Figure S2). These results suggest that silencing RPL21 markedly induces apoptosis only in PC cells but not in pancreatic normal cells, which indicates that RPL21 gene may be a therapeutic target for PC treatment
Summary
Its occurrence was a process with multi-factors and multi-steps, including activation of protooncogenes, inactivation of tumor-suppressor genes, alterations in cell cycle related proteins and so on. Many cancer-related genes interact with each other and are involved in the progression of PC [4]. Some proto-oncogenes (EGFR, KRAS, etc.) have been characterized as therapeutic targets for clinic treatment to improve survival rate [5]. The current strategies haven’t demonstrated significant improvement on the survival rates yet [6]. The molecular mechanism of pancreatic cancer remains unclear, and genes associated with PC progression still need to be identified. Our previous study showed that the ribosomal protein L21 (RPL21) may play an important role in the development and survival of pancreatic cancer. RNA interference (RNAi) experiments were performed with RPL21-specific small interfering RNA (siRNA) to elucidate the mechanism by which RPL21 controls PC PANC1 and BxPC-3 cell proliferation
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