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Circular RNA circ_HN1 facilitates gastric cancer progression through modulation of the miR-302b-3p/ROCK2 axis

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Abstract

Gastric cancer (GC) is a malignant tumor with high morbidity and mortality in the world. Circular RNA hsa_circHN1_005 (circ_HN1), also termed as hsa_circ_0045602, is reported as an oncogene in GC. However, the molecular mechanism of circ_HN1 in GC development has not been fully explored. Here, we surveyed the regulatory mechanism of circ_HN1 in GC progression. The levels of circ_HN1, miR-302b-3p, and rho-associated coiled-coil containing protein kinase 2 (ROCK2) mRNA were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, apoptosis, colony formation, cell cycle progresion, migration, and invasion were determined by using cell counting, flow cytometry, colony formation, or transwell assays. Protein levels were detected with Western blotting. The relationship between circ_HN1 or ROCK2 and miR-302b-3p was verified via dual luciferase reporter or RNA immunoprecipitation (RIP) assays. The role of circ_HN1 in vivo was confirmed by xenograft assay. We observed that circ_HN1 and ROCK2 were upregulated while miR-302b-3p was downregulated in GC tissues and cells. Circ_HN1 silencing slowed tumor growth in vivo and impeded cell proliferation migration, invasion, and facilitated cell apoptosis in GC cells in vitro. Circ_HN1 sponged miR-302b-3p to regulate ROCK2 expression. MiR-302b-3p inhibitor reversed circ_HN1 silencing-mediated influence on the malignant behaviors of GC cells. Furthermore, ROCK2 overexpression restored miR-302b-3p mimic-mediated impacts on cell malignant behaviors in GC cells. In conclusion, circ_HN1 exerted an oncogenic role in GC through upregulating ROCK2 via sponging miR-302b-3p, offering evidence that circ_HN1 is a potential target for GC therapy.

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Abbreviations

GC:

Gastric cancer

circ_HN1:

Circular RNA circ_HN1

ROCK2:

Rho-associated coiled-coil containing protein kinase 2

qRT-PCR:

Quantitative real-time polymerase chain reaction

CCK-8:

Cell counting kit-8

E-cad:

E-cadherin

c-caspase3:

Cleaved caspase-3

PCNA:

Proliferating cell nuclear antigen

RIP:

RNA immunoprecipitation

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Authors and Affiliations

  1. Department of General Surgery, The First Affiliated Hospital of Soochow University, Medical College of Soochow University, No. 188, Shizi Street, Suzhou, 215006, Jiangsu, China

    Ding Wang & Yuting Kuang

  2. Department of General Surgery, Affiliated Tumor Hospital of Nantong University, Nantong Tumor Hospital, Nantong, Jiangsu, China

    Ding Wang, Xiaohui Jiang, Guangxin Cao & Xueliang Zhang

  3. Department of Radiotherapy, Affiliated Tumor Hospital of Nantong University, Nantong Tumor Hospital, Nantong, Jiangsu, China

    Yi Liu

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  1. Ding Wang
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Supplementary file1 (TIF 648 kb)

Fig. S1 The association between some miRNAs and circ_HN1. (A) The expression of 6 miRNAs (miR-520a-3p, miR-520d-3p, miR-526b-3p, miR-628-5p, miR-302b-3p, and miR-760) in GC tissues (10 random samples) was assessed by qRT-PCR. (B and C) Influence of circ_HN1 knockdown on the expression of these miRNAs in MKN7 and HGC-27 cells was determined by qRT-PCR. *P < 0.05.

Supplementary file2 (TIF 797 kb)

Fig. S2 The association between some mRNAs and miR-302b-3p. (A) The levels of 6 proteins (ROCK2, MAP3K7, HMGA2, COL5A1, ADAM9, and EZH2) in GC tissues (3 random samples) were assessed by Western blotting. (B and C) Effect of circ_HN1 silencing on the levels of these proteins in MKN7 and HGC-27 cells was determined by Western blotting. *P < 0.05.

Supplementary file3 (TIF 4467 kb)

Fig. S3 Influence of circ_HN1 overexpression on migration and invasion of GES-1 cells. (A) The overexpression efficiency of circ_HN1 in GES-1 cells was revealed by qRT-PCR. (B and C) Effect of circ_HN1 overexpression on migration and invasion of GES-1 cells was determined by transwell assay. *P < 0.05.

Supplementary file4 (TIF 3084 kb)

Fig. S4. A schematic diagram presenting the regulatory mechanism of circ_HN1 in GC progression.

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Wang, D., Jiang, X., Liu, Y. et al. Circular RNA circ_HN1 facilitates gastric cancer progression through modulation of the miR-302b-3p/ROCK2 axis. Mol Cell Biochem 476, 199–212 (2021). https://doi.org/10.1007/s11010-020-03897-2

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