Cancer Res Treat.  2018 Oct;50(4):1396-1417. 10.4143/crt.2017.537.

Circular RNA-ZFR Inhibited Cell Proliferation and Promoted Apoptosis in Gastric Cancer by Sponging miR-130a/miR-107 and Modulating PTEN

• Liu T
• Liu S
• Xu Y
• Shu R
• Wang F
• Chen C
• Zeng Y
• Luo H

• KMID: 2424811
• DOI: http://doi.org/10.4143/crt.2017.537

Abstract

PURPOSE
This study aimed to probe into the associations among circular RNA ZFR (circ-ZFR), miR-130a/miR-107, and PTEN, and to investigate the regulatory mechanism of circ-ZFR-miR-130a/miR-107-PTEN axis in gastric cancer (GC).
MATERIALS AND METHODS
GSE89143 microarray data used in the study were acquired from publicly available Gene Expression Omnibus database to identify differentially expressed circular RNAs inGC tissues. The expressions of circ-ZFR, miR-130a, miR-107, and PTEN were examined by real-time reverse transcription polymerase chain reaction, while PTEN protein expression was measured by western blot. The variation of GC cell proliferation and apoptosis was confirmed by cell counting kit-8 assay and flow cytometry analysis. The targeted relationships among circZFR, miR-130a/miR-107, and PTEN were predicted via bioinformatics analysis and demonstrated by dual-luciferase reporter assay and RNA immunoprecipitation assay. The impact of ZFR on gastric tumor was further verified in xenograft mice model experiment.
RESULTS
Circ-ZFR and PTEN were low-expressed whereas miR-107 and miR-130a were highexpressed in GC tissues and cells. There existed targeted relationships and interactions between miR-130a/miR-107 and ZFR/PTEN. Circ-ZFR inhibited GC cell propagation, cell cycle and promoted apoptosis by sponging miR-107/miR-130a, while miR-107/miR-130a promoted GC cell propagation and impeded apoptosis through targeting PTEN. Circ-ZFR inhibited cell proliferation and facilitated apoptosis in GC by sponging miR-130a/miR-107 and modulating PTEN. Circ-ZFR curbed GC tumor growth and affected p53 protein expression in vivo.
CONCLUSION
Circ-ZFR restrained GC cell proliferation, induced cell cycle arrest and promoted apoptosis by sponging miR-130a/miR-107 and regulating PTEN.

Keyword

Circ-ZFR; miR-130a; miR-107; PTEN; Stomach neoplasms

MeSH Terms

Animals
Apoptosis*
Blotting, Western
Cell Count
Cell Cycle
Cell Cycle Checkpoints
Cell Proliferation*
Computational Biology
Flow Cytometry
Gene Expression
Heterografts
Immunoprecipitation
Mice
Polymerase Chain Reaction
PTEN Phosphohydrolase
Reverse Transcription
RNA
Stomach Neoplasms*
PTEN Phosphohydrolase
RNA

Figure

• 

  Fig. 1. Circular RNA ZFR (circ-ZFR) was significantly downregulated in gastric cancer (GC) tissues and cells. (A, B) CircZFR was low-expressed in GC tumor tissues compared with adjacent tissues confirmed by microarray analysis and shown in Volcano plot and heat map. (C) Circ-ZFR was significantly downregulated in tumor tissues compared with adjacent tissues. (D) The expression of circ-ZFR was considerably lower in human GC cell lines (AGS, AZ521, and HGC-27) than in gastric epithelial cell line GES-1 determined by real-time reverse transcription polymerase chain reaction. Among three GC cell lines, AGS cell line presented the most significant difference of circ-ZFR expression in comparison with GES-1 cell line. (E) The presence of circ-ZFR in AGS cells showed that there was no less expression afterthe RNase was processed. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. *p < 0.05, **p < 0.01, compared with adjacent tissues or GES-1 or normal control (NC).

• 

  Fig. 2. Overexpression of circular RNA ZFR (circ-ZFR) impeded cell reproduction and cell cycle and promoted apoptosis in gastric cancer (GC). (A) Circ-ZFR expression was remarkably upregulated after transfection with pcDNA3.1-circZFR examined by real-time reverse transcription polymerase chain reaction. (B) Overexpression of circ-ZFR observably suppressed GC cell proliferation, as confirmed in cell counting kit 8 assay. (C) The proportion of cells arrested in G0/G1 phase was significantly higher in pcDNA3.1-circZFR group than in normal control (NC) group, while that of arrested cells in S phase relatively reduced compared with NC group analyzed by flow cytometry assay. (D) The apoptosis rate of GC cells significantly increased after transfection with pcDNA3.1-circZFR detected by flow cytometry assay. PI, propidium iodide. *p < 0.05, **p < 0.01, compared with NC group.

• 

  Fig. 3. Prediction of circRNA-miRNA-mRNA associations in gastric cancer. (A) The binding sites of miR-107/miR-130a on 3′-untranslated regions (3′-UTR) of circular RNA ZFR (circ-ZFR) was confirmed by starbase. (B) The schematic model showing the putative binding sites for miRNAs and 3′-untranslated region of circ-ZFR. (C) The pathways co-affected by miR-107 and miR-130a were displayed by DIANA TOOLS-mirPath v.3. (D) The crossed targeting gene regulated by both p53 pathway and miR-130a/miR-107 via Veen diagram. (E) The binding sites of miR-107/miR-130a on 3′-UTR of PTEN was validated by TargetScan. (F) A biomathematically predicted target network between circ-ZFR, miR-130a/miR-107, and PTEN.

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  Fig. 4. Confirmation of targeted relationships between miR-107/miR-130a and PTEN or circular RNA ZFR (circ-ZFR). (A) MiR-107 mimics and miR-130a mimics led to overexpression of miR-107 and miR-130a, respectively detected by real-time reverse transcription polymerase chain reaction. (B) The expression of circ-ZFR was no significant difference both in miR107‒mimics and miR-130a–mimics group. (C, D) Overexpression of miR-107 and miR-130a both significantly repressed the luciferase activity of the luciferase reporter containing ZFR 3′-untranslated regions (3′-UTR)‒WT but not the reporter containing ZFR 3′-UTR‒Mut confirmed by dual luciferase reporter assay. (E, F) There existed some interactions between ZFR and miR-107/miR-130a verified by RNA immunoprecipitation assay.

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  Fig. 5. Correlations on expression between miR-107/miR-130a and PTEN/circ-ZFR. (A, B) MiR-107 and miR-130a were highly expressed whereas PTEN was lowly expressed in tumor tissues compared with adjacent tissues examined by realtime reverse transcription polymerase chain reaction. (C, D) The expressions of miR-107 and miR-130a were both negatively correlated with ZFR expression determined by Pearson correlation analysis. (E, F) The expressions of miR-107 and miR-130a were both negatively correlated with PTEN expression determined by Pearson correlation analysis. *p < 0.05, compared with adjacent tissues. GC, gastric cancer.

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  Fig. 6. Circ-ZFR suppressed gastric cancer cell reproduction by sponging miR-107/miR-130a. (A, C) MiR-107/miR-130a mimics significantly increased the expression of miR-107/miR-130a, while miR-107/miR-130a inhibitor remarkably decreased miR-107/miR-130a expressions examined by real-time reverse transcription polymerase chain reaction. (B, D) Overexpression of miR-107 and miR-130a both promoted cell proliferation whereas downregulation of miR-107 and miR130a expressions restrained propagation. No significant difference of cell proliferation was found between miR-107/miR130a+pcDNA3.1-ZFR group and normal control (NC) group. However, the proliferation ability of the cells in miR-107/miR-130a+pcDNA3.1-ZFR group was significantly stronger than that in pcDNA3.1-ZFR group, while weaker compared with miR-107/miR-130a–mimics group. *p < 0.05, **p < 0.01, compared with NC group; #p < 0.05, compared with pcDNA3.1-ZFR group; †p < 0.05, compared with miR-107/miR-130a–mimics group.

• 

  Fig. 7. Circular RNA ZFR (circ-ZFR) induced cell cycle arrest by sponging miR-107/miR-130a in gastric cancer. (A, B) The proportion of the cells arrested in G1 phase in pcDNA3.1-ZFR group, miR-107 inhibitor group and miR-130a inhibitor group was considerably larger than in normal control (NC) group. Conversely, the percentage of arrested cell in G1 phase in miR107–mimics group and miR-130a–mimics group was relatively smaller compared with NC group. Nonetheless, the percentage of arrested cell in G1 phase in miR-107/miR-130a+pcDNA3.1-ZFR group was significantly smaller than that in pcDNA3.1-ZFR group, while larger than that in miR-107/miR-130a–mimics group. There was no conspicuous distinction between miR-107/miR-130a+pcDNA3.1-ZFR group and NC group. *p < 0.05, compared with NC group; #p < 0.05, compared with pcDNA3.1-ZFR group; †p < 0.05, compared with miR-107/miR-130a–mimics group.

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  Fig. 8. Circular RNA ZFR (circ-ZFR) facilitated gastric cancer cell apoptosis by sponging miR-107/miR-130a (A, B), The apoptosis of cells was significantly enhanced after transfected with pcDNA3.1-ZFR and miR-107/miR-130a inhibitor, while that of the cells transfected with miR-107/miR-130a–mimics was remarkably inhibited. No significant difference of apoptosis rate was detected between miR-107/miR-130a+pcDNA3.1-ZFR group and normal control (NC) group. However, the cell apoptosis rate in miR-107/miR-130a+pcDNA3.1-ZFR group was considerably lower than that in pcDNA3.1-ZFR group, while significantly higher compared with miR-107/miR-130a‒mimics group. *p < 0.05, ** p < 0.01, compared with NC group; #p < 0.05, compared with pcDNA3.1-ZFR group; †p < 0.05, compared with miR-107/miR-130a–mimics group.

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  Fig. 9. miR-107/miR-130a promoted gastric cancer cell reproduction through targeting PTEN. (A, B) The expression of PTEN was remarkably repressed after transfection with miR-107‒mimics/miR-130a‒mimics, while it was overexpressed followed transfection with pcDNA3.1-ZFR and miR-107 inhibitor/miR-130a inhibitor detected with western blot. (C) The expression of pcDNA3.1-PTEN in gastric cancer cells was dramatically upregulated after transfection with pcDNA3.1-PTEN examined by real-time reverse transcription polymerase chain reaction. (D, E) The proliferation ability of the cells in miR-107/miR130a‒mimics+pcDNA3.1-PTEN group was significantly stronger than that in pcDNA3.1-PTEN group, while weaker compared with miR-107/miR-130a‒mimics group. No significant difference of cell proliferation was observed between miR-107/miR-130a‒mimics+pcDNA3.1-PTEN group and normal control (NC) group. *p < 0.05, compared with NC group; #p < 0.05, compared with pcDNA3.1-PTEN group; † p < 0.05, compared with miR-107/miR-130a‒mimics group.

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  Fig. 10. miR-107/miR-130a influenced gastric cancer cell cycle through targeting PTEN. (A, B) Flow cytometry assay displayed that the proportion of the cells arrested in G1 phase in miR-107/miR-130a–mimics group dramatically reduced compared with normal control (NC) group, while that in S phase increased and there was no significant difference in G2 phase. On the contrary, the percentage of arrested cell in G1 phase in pcDNA3.1-PTEN group conspicuously increased in comparison with NC group whereas that in S phase relatively decreased. Nonetheless, the percentage of arrested cell in G1 phase in miR-107/miR-130a+pcDNA3.1-PTEN group was significantly smaller than that in pcDNA3.1-PTEN group, while larger than that in miR-107/miR-130a–mimics group. There was no significant distinction between miR-107/miR-130a+pcDNA3.1-PTEN group and NC group. *p < 0.05, compared with NC group; #p < 0.05, compared with pcDNA3.1-PTEN group; †p < 0.05, compared with miR-107/miR-130a–mimics group.

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  Fig. 11. miR-107/miR-130a impeded cell apoptosis in gastric cancer through targeting PTEN. (A, B) The apoptotic rate of the cells transfected with miR-107/miR-130a–mimics considerably fell whereas that of the cells transfected with pcDNA3.1-PTEN drastically rose. No significant difference of apoptosis ratio was found between miR-107/miR-130a+pcDNA3.1-PTEN group and normal control (NC) group. Nevertheless, the cell apoptosis rate in miR-107/miR-130a+pcDNA3.1-PTEN group was considerably lower than that in pcDNA3.1-PTEN group, while significantly higher compared with miR-107/miR-130a–mimics group. *p < 0.05, compared with NC group; #p < 0.05, compared with pcDNA3.1-PTEN group; †p < 0.05, compared with miR-107/miR-130a–mimics group.

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  Fig. 12. Circular RNA ZFR curbed gastric cancer (GC) tumor growth and affected p53 protein expression in vivo (A) The tumor volume of the mice in pcDNA3.1-ZFR group was significantly grew more slowly than that in normal control (NC) group. (B, C) The tumor weight of mice in pcDNA3.1-ZFR group was significantly lighter compared with NC group. (D, E) The expression level of miR-107 (D) and miR-130a (E) was decreased after transfected with pcDNA3.1-ZFR. (F) The expression level of p53 protein dramatically increased after transfected with pcDNA3.1-ZFR confirmed by immunohistochemistry (IHC) assay. (G) The expression level of PTEN increased after transfected with pcDNA3.1-ZFR confirmed by IHC assay. *p < 0.05, **p < 0.01, compared with NC group.

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  Fig. 13. The mechanism of circular RNA ZFR (circ-ZFR)–miR-107/miR-130a–PTEN axis. Circ-ZFR was identified as a sponge of miR-130a/miR-107 and indirectly modulated their target PTEN expression. GC, gastric cancer.

Reference

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