Korean J Physiol Pharmacol.  2023 Mar;27(2):131-141. 10.4196/kjpp.2023.27.2.131.

Hsa_circ_0129047 sponges miR-665 to attenuate lung adenocarcinoma progression by upregulating protein tyrosine phosphatase receptor type B

• Xia X
• Fan J
• Fan Z

• KMID: 2539568
• DOI: http://doi.org/10.4196/kjpp.2023.27.2.131

Abstract

Compelling evidence has demonstrated the critical role of circular RNAs (circRNAs) during lung adenocarcinoma (LUAD) progression. Herein, we explored a novel circRNA, circ_0129047, and detailed its mechanism of action. The expression of circ 0129047, microRNA-665 (miR-665), and protein tyrosine phosphatase receptor type B (PTPRB) in LUAD tissues and cells was determined using reverse transcription quantitative polymerase chain reaction and Western blotting. Cell Counting Kit-8 and colony formation assays were conducted to detect LUAD cell proliferation, and western blotting was performed to quantify apoptosis-related proteins (Bcl-2 and Bax). Luciferase reporter and RNA immunoprecipitation assays were used to validate the predicted interaction between miR-665 and circ_0129047 or PTPRB. A xenograft assay was used for the in vivo experiments. Circ_0129047 and PTPRB were downregulated in LUAD tissues and cells, whereas miR-665 expression was upregulated. Overexpression of circ_0129047 suppresses LUAD growth in vivo and in vitro. Circ_0129047 is the target of miR-665, and the miR-665 mimic ablated the antiproliferative and pro-apoptotic phenotypes of LUAD cells by circ_0129047 augmentation. MiR-665 targets the 3ʹUTR of PTPRB and downregulates PTPRB expression. PTPRB overexpression offsets the pro-proliferative potential of miR-665 in LUAD cells. Circ_0129047 sequestered miR-665 and upregulated PTPRB expression, thereby reducing LUAD progression, suggesting a promising approach for preventing LUAD.

Keyword

Adenocarcinoma; circ_0129047; Lung; miR-665; PTPRB

Figure

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  Fig. 1 Hsa_circ_0129047 expression was downregulated in lung adenocarcinoma (LUAD). (A) RT-qPCR analysis results of circ_0129047 in both LUAD and normal tissues. (B) RT-qPCR analysis of circ_0129047 in human bronchial epithelium (BEAS-2B) and LUAD cells (A549, H1975, PC9 and Calu-3). **p < 0.01 vs. BEAS-2B. (C) Loop structures of Circ_012904. (D) Subcellular localization of circ_0129047 in PC9 and Calu-3 cells. (E) Circ_012904 was resistant to RNase R digestion. **p < 0.01 vs. RNAse R. Values are presented as mean ± SD.

• 

  Fig. 2 Augmentation of circ_0129047 expression suppresses LUAD tumors in vitro and in vivo. circ 0129047-overexpressing vectors (OE-circ) and empty vectors were transfected into PC9 and Calu-3 cells (OE-NC). (A) RT-qPCR analysis of circ_0129047 expression 48 h later. (B) Cell proliferation assay using the CCK-8 assay. (C) Expression of apoptosis-related proteins (Bcl-2 and Bax) determined using Western blotting. (D) Colony formation was assessed using colony formation assay (×200). (E) Xenograft tumor growth assay. Values are presented as mean ± SD. LUAD, lung adenocarcinoma; OD, optical density. **p < 0.01 vs. OE-NC.

• 

  Fig. 3 Circ_0129047 targets miR-665. (A) MiR-665 is predicted as a target of circ_0129047 through CircInteractome. (B) RIP assay was performed using Ago2 antibody in LUAD cells, and the enrichment of circ_0129047 and miR-665 was detected. **p < 0.01 vs. Anti-IgG. (C) Luciferase activity of Circ_0129047 in LUAD cells transfected with miR-665 mimics, which bind to the circ 0129047 sequence. **p < 0.01 vs. mimic-NC. (D) RT-qPCR analysis of miR-665 in LUAD tissues and normal tissues. (E) RT-qPCR analysis of miR-665 in LUAD cells (PC9 and Calu-3) and normal cells (BEAS-2B). **p < 0.01 vs. BEAS-2B. (F) Pearson analysis of miR-665 expression and circ_0129047 expression in LUAD tissues. (G) circ 0129047-overexpressing vectors (OE-circ), OE-NC, miR-665 mimic (mimic), mimic-NC, and OE+mimic were transfected into PC9 and Calu-3 cells. The expression of miR-665 was measured using RT-qPCR 48 h after transfection. **p < 0.01 vs. OE-NC; ##p < 0.01 vs. mimic-NC; &&p < 0.01 vs. OE+mimic. Values are presented as mean ± SD. LUAD, lung adenocarcinoma; Ago2, Argonaute 2; WT, wild type; MUT, mutant.

• 

  Fig. 4 MiR-665 is a critical target via which circ_0129047 mediated LUAD tumorigenesis. PC9 and Calu-3 cells were transfected with circ_0129047 overexpression vectors (OE-circ), OE-NC, miR-665 mimic (mimic) or mimic-NC, and OE+mimic. (A) Cell proliferation was analyzed using the CCK-8 assay. (B) Western blot analysis of anti-Bax and anti-Bcl-2 expression. (C) Colony formation determined using colony formation assay (×200). Values are presented as mean ± SD. LUAD, lung adenocarcinoma; OD, optical density; PTPRB, protein tyrosine phosphatase receptor type B. *p < 0.05, **p < 0.01 vs. OE-NC; #p < 0.05, ##p < 0.01 vs. mimic-NC; &p < 0.05, &&p < 0.01 vs. OE+mimic.

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  Fig. 5 MiR-665 targets PTPRB. (A) Predicted miR-665 interactions with PTPRB 3′UTR using miRNA target prediction software based on TargetScan. (B) Luciferase reporter gene assay of PTPRB 3′UTR after miR-665 treatment. **p < 0.01 vs. mimic-NC. (C) PTPRB mRNA expression in LUAD tissues analyzed using RT-qPCR. (D) Pearson correlation analysis was used to analyze the relationship between miR-665 and PTPRB in LUAD tissues. (E) Pearson correlation analysis was used to analyze the relationship between circ_0129047 and PTPRB in LUAD tissues. (F) PTPRB mRNA expression in LUAD cells (PC9 and Calu-3) and normal cells (BEAS-2B) using RT-qPCR. **p < 0.01 vs. BEAS-2B. (G) Western blot analysis determining PTPRB expression in LUAD cells transfected with PTPRB-overexpressing vectors (OE-PTPRB), OE-NC, miR-665 mimic (mimic), mimic-NC, OE-PTPRB+mimic. **p < 0.01 vs. OE-NC; ##p < 0.01 vs. mimic-NC; &&p < 0.01 vs. OE-PTPRB+mimic. Values are presented as mean ± SD. LUAD, lung adenocarcinoma; PTPRB, protein tyrosine phosphatase receptor type B; WT, wild type; MUT, mutant.

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  Fig. 6 The tumorigenic role of miR-665 may be achieved by targeting PTPRB 3ʹUTR. LUAD cells were transfected with PTPRB-overexpressing vectors (OE-PTPRB), OE-NC, miR-665 mimic (mimic), mimic-NC, and OE-PTPRB+mimic. (A) Cell proliferation was analyzed using the CCK-8 assay. (B) Western blot analysis of anti-Bax and anti-Bcl-2 expression in the transfected cells. (C) Colony formation was determined using colony formation assay (×200). *p < 0.05, **p < 0.01 vs. OE-NC; #p < 0.05, ##p < 0.01 vs. mimic-NC; &p < 0.05, &&p < 0.01 vs. OE-PTPRB+mimic. Values are presented as mean ± SD. LUAD, lung adenocarcinoma; OD, optical density; PTPRB, protein tyrosine phosphatase receptor type B.

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