Korean J Physiol Pharmacol.  2023 May;27(3):197-208. 10.4196/kjpp.2023.27.3.197.

LINC00562 drives gastric cancer development by regulating miR-4636–AP1S3 axis

• Xu L
• Liu D
• Wang X

• KMID: 2541632
• DOI: http://doi.org/10.4196/kjpp.2023.27.3.197

Abstract

Dysregulation of certain long non-coding RNAs may facilitate tumor initiation and progression. However, numerous carcinogenesis-related long non-coding RNAs have not been characterized. The goal of this study was to elucidate the role of LINC00562 in gastric cancer (GC). The expression of LINC00562 was analyzed using real-time quantitative PCR and Western blotting. The proliferative capacity of GC cells was determined using Cell Counting Kit-8 and colony-formation assays. The migration of GC cells were evaluated using wound-healing assays. The apoptosis of GC cells was assessed by measuring the expression levels of apoptosis-related proteins (Bax and Bcl-2). Xenograft models in nude mice were constructed for in vivo functional analysis of LINC00562. The binding relationship between miR-4636 and LINC00562 or adaptor protein complex 1 sigma 3 (AP1S3), obtained from public databases, was confirmed using dual-luciferase and RNA-binding protein immunoprecipitation experiments. LINC00562 was expressed in GC cells at high levels. Knockdown of LINC00562 repressed GC cell growth and migration, promoted apoptosis in vitro, and inhibited tumor growth in nude mouse models. LINC00562 directly targeted miR-4636, and miR-4636 depletion restored the GC cell behavior inhibited by LINC00562 absence. AP1S3, an oncogene, binds to miR-4636. MiR-4636 downregulation increased AP1S3 level, restoring GC cell malignant behaviors inhibited by AP1S3 downregulation. Thus, LINC00562 exerts carcinogenic effects on GC development by targeting miR-4636-mediated AP1S3 signaling.

Keyword

Competitive endogenous RNA; Long noncoding RNA; MicroRNA; Stomach neoplasms

Figure

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  Fig. 1 LINC00562 expression was enhanced in GC. (A) LINC00562 expression levels in tumor and normal specimens. (B) LINC00562 expression levels in GSE-1, RGM-1, HGC-27, AGS and MKN45 cells, **p < 0.01 in contrast to GES-1. (C) Distribution of LINC00562 in cytoplasm and nucleus of AGS and HGC-27 cells. Values are presented as mean ± SD. GC, gastric cancer.

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  Fig. 2 Silencing LINC00562 restrained GC cell growth and migration but enhanced apoptosis. (A) LINC00562 expression in HGC-27 and AGS cells transfected with si-lnc or si-NC. (B) Effect of LINC00562 silencing on cell proliferation was assessed by CCK-8 assay in HGC-27 and AGS cells. (C) Protein levels of Bax and Bcl-2 were affected by LINC00562 silencing in HGC-27 and AGS cells. (D) Effect of LINC00562 silencing in colony formation by HGC-27 and AGS cells. (E) Impact of LINC00562 silencing on cell migration was assessed using wound-healing assay in HGC-27 and AGS cells. Values are presented as mean ± SD. ×200. GC, gastric cancer. **p < 0.01 in contrast to si-NC.

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  Fig. 3 Depletion of LINC00562 restrained GC tumor growth in vivo. Nude mice (N = 6) were subcutaneously injected with HGC-27 cells that were infected with sh-lnc or sh-NC to induce tumorigenesis. (A) Representative images of tumor tissues. (B) Tumor volumes were measured once a week. (C) Tumor weight was measured after tumor growth for 5 weeks. Values are presented as mean ± SD. GC, gastric cancer. **p < 0.01 in contrast to sh-NC.

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  Fig. 4 LINC00562 targeted miR-4636. (A) LncBase (https://diana.e-ce.uth.gr/lncbasev3) predicted LINC00562–miR-4636 binding sites. (B) Putative binding sites were validated using dual-luciferase assays, **p < 0.01 in contrast to miR-NC. (C) Binding of LINC00562 to miR-4636 was confirmed by RIP assay, **p < 0.01 in contrast to Anti-IgG. (D) MiR-4636 expression levels in tumor and normal specimens. (E) MiR-4636 expression levels in GES-1, RGM-1, and GC cells (HGC-27 and AGS), **p < 0.01 in contrast to GES-1. (F) Correlation between miR-4636 expression and LINC00562 expression in tumor samples was determined using Pearson’s analysis. Values are presented as mean ± SD. GC, gastric cancer.

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  Fig. 5 MiR-4636 inhibition reversed the inhibitory effects of LINC00562 silencing on GC cell survival. HGC-27 and AGS cells were transfected with si-lnc, si-NC, inhibitor, inhibitor-NC, or si-lnc+inhibitor. (A) RT-qPCR was used to assess the expression level of miR-4636. (B) CCK-8 assay tested the proliferative capacity of cells. (C) Western blotting confirmed the expression of Bax and Bcl-2. (D) Wound-healing assay showed cell-migration ability. (E) Colony-formation assay was used for proliferation analysis. Values are presented as mean ± SD. ×200. GC, gastric cancer. *p < 0.05, **p < 0.01 in contrast to si-NC; #p < 0.05, ##p < 0.01 in contrast to inhibitor-NC; &p < 0.05, &&p < 0.01 in contrast to si-lnc+inhibitor.

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  Fig. 6 MiR-4636 targeted AP1S3. (A) TargetScan (http://www.targetscan.org/vert_72/) predicted miR-4636–AP1S3 binding site. (B) Predicted binding site was validated using dual-luciferase assays, **p < 0.01 in contrast to miR-NC. (C) Binding of AP1S3 to miR-4636 was confirmed using RIP assay, **p < 0.01 in contrast to Anti-IgG. (D) AP1S3 mRNA levels in tumor and normal specimens. (E) AP1S3 mRNA levels in GES-1, RGM-1, and GC cells (HGC-27 and AGS), **p < 0.01 in contrast to GES-1. (F) Correlation between miR-4636 expression and AP1S3 expression in tumor samples was determined using Pearson’s analysis. Values are presented as mean ± SD. AP1S3, adaptor protein complex 1 sigma 3; GC, gastric cancer.

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  Fig. 7 MiR-4636 depletion inhibited the anti-cancer effects of AP1S3 knockdown in GC. HGC-27 and AGS cells were transfected with si-AP1S3, si-NC, inhibitor, inhibitor-NC, or si-AP1S3+inhibitor. (A) Western blotting revealed the expression of AP1S3 protein. (B) CCK-8 assay was used to analyze the cell proliferative capacity. (C) Western blotting confirmed the expression of Bax and Bcl-2. (D) Wound-healing assay showed cell migration ability. (E) Colony-formation assay was used for proliferation analysis. Values are presented as mean ± SD. ×200. AP1S3, adaptor protein complex 1 sigma 3; GC, gastric cancer. *p < 0.05, **p < 0.01 in contrast to si-NC; #p < 0.05, ##p < 0.01 in contrast to inhibitor-NC; &p < 0.05, &&p < 0.01 in contrast to si-AP1S3+inhibitor.

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