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. 2022 Jul 25:12:903484.
doi: 10.3389/fonc.2022.903484. eCollection 2022.

MiR-520a-5p/PPP5C regulation pattern is identified as the key to gemcitabine resistance in pancreatic cancer

Ruibiao Fu  1 , Qian Shao  1 , Bin Yang  1 , Yan Chen  1 , Qinghuang Ye  1 , Xi Chen  1 , Jinhui Zhu  1
Affiliations

MiR-520a-5p/PPP5C regulation pattern is identified as the key to gemcitabine resistance in pancreatic cancer

Ruibiao Fu et al. Front Oncol. .

Abstract

Objective: To explore the effects of the expression level of miR-520-5p/PPP5C in pancreatic cancer cells and exosomes on cell viability, angiogenesis, autophagy, which involved in the mechanism of gemcitabine resistance in pancreatic cancer.

Methods: APSC-1 cell line was treated with gemcitabine, after which its exosomes were extracted for NTA assay. Subsequently, the drug resistance of APSC-1 cells was assayed using CCK8, as well as the activity of HUVEC cells treated with exosomes from each group of APSC-1 cells after drug resistance treatment as well as overexpression treatment. Five groups of HUVEC cells treated with exosomes were subjected to in vitro tubule formation assay. levels of PPP5C in each group of ASPC-1 cells and their exosomes, levels of overexpressed PPP5C, and related exosomal proteins were examined by WB. mRNA expression levels of PPP5C and levels of miR-520a were examined by qPCR The relationship between miR-520a-5p and PPP5C was investigated. After that, the autophagy of PPP5C was detected. Finally, it was analyzed by TCGA database for survival prognosis analysis.

Results: APSC-1 cells had an IC50 value of 227.1 μM for gemcitabine, elevated PPP5C expression, drug resistance, and enhanced HUVEC cell activity; exosomes CD9, CD63, and CD81 were significantly expressed in all groups; meanwhile, enhanced PPP5C expression not only promoted in vitro tubule formation but also increased autophagy levels; meanwhile, its relationship with miR-520-5p and There was a targeted inhibitory relationship between its level and miR-520-5p and PPP5C, and its elevated level also led to a decrease in the survival level of patients over 3-5 years.

Conclusion: PPP5C has a prognostic role in pancreatic cancer by promoting the value-added and invasion of pancreatic cancer cells, and a targeted inhibitory relationship between miR-520-5p and PPP5C was found.

Keywords: PPP5C; Pancreatic cancer; autophagy; gemcitabine; miR-520-5p.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Drug resistance and cellular activity: (A) IC50 values of gemcitabine in ASPC-1 cells; (B) CCK8 detection of cell proliferation; (C) CCK8 detection of cell proliferation after overexpression treatment.
Figure 2
Figure 2
Tubule formation capacity assay (A) in vitro tubule formation capacity assay for each group (experimental plots); (B) in vitro tubule formation capacity assay for each group (bar graph); (C) in vitro tubule formation capacity assay for each group after overexpression treatment (experimental plots); (D) in vitro tubule formation capacity assay for each group after overexpression treatment (bar graph). Note: *p < 0.05, **p < 0.01.
Figure 3
Figure 3
Autophagy levels: (A) WB autophagy level assay in each group of cells; (B) immunofluorescence autophagy level assay in each group of cells. .
Figure 4
Figure 4
Expression of PPP5C as well as exosomal proteins: (A) serine/threonine protein phosphatase expression assay; (B) PP5C mRNA content in each group of ASPC-1 cells; (C) exosome markers in each group; (D) exosome diameter distribution in each group of cells; (E) PPP5C expression in each group of ASPC-1 exosomes; (F) PPP5C in each group after overexpression.
Figure 5
Figure 5
Mechanism of PPP5C expression imbalance: (A) hsa-miR-520a-5p expression in each group of ASPC-1 cells; (B) hsa-miR-520a-5p expression in each group of ASPC-1 exosomes; (C) luciferase activity assay; (D) hsa-miR-520a-5p overexpression versus hsa-miR-520a-5p in the exosomes of drug-resistant cells. miR-520a-5p expression assay; (E) expression level of PPP5C after hsa-miR-520a-5p overexpression. Note: * indicates p < 0.05, *p < 0.05, **p < 0.01.
Figure 6
Figure 6
Prognostic analysis of PPP5C in pancreatic cancer patients from TCGA database: (A) Distribution of high and low risk samples. (B) Survival curves. (C) ROC curves.
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