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Archive > Volume 30 Issue 12 > 2023,30(12):1051-1060. DOI:10.3872/j.issn.1007-385X.2023.12.003 Prev Next
Basic Research
METTL3-modified RNASEH1-AS1 regulates the malignant biological behaviors of colorectal cancer SW480 cells through the BUD13/ANXA2/Wnt/β-catenin axis
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Abstract:
Objective: To explore the molecular mechanism by which methyltransferase-like protein 3 (METTL3) -modified RNASEH1-AS1 regulates the malignant biological behaviors of colorectal cancer cells SW480 through the BUD13/membrane associated protein A2/Wnt/β-catenin (BUD13/ANXA2/Wnt/β-catenin) axis. Methods: Twenty-four CRC patients who were surgically treated in our hospital from June 2022 to November 2022 were selected as the study subjects, and the patients' CRC tissues and corresponding paracancerous tissues were collected, and the qPCR method was used to detect the METTL3, RNASEH1-AS1, BUD13,ANXA2, β -catenin, GSK-3β mRNA in CRC tissues and paracancerous tissues expression, and the correlation of RNASEH1-AS1 expression with METTL3 expression and BUD13 expression in CRC tissues was analyzed by Pearson's method. Colorectal cancer cells SW480 were routinely cultured, and the experiments were divided into sh-NC group, sh-RNASEH1-AS1 group, NC group, sh-METTL group, si-NC group, si-BUD13 group, sh-RNASEH1-AS1+pc-NC group, sh-RNASEH1-AS1+pc-ANXA2 group, sh-METTL+pc-NC group, sh-METTL+pc-ASEH1-AS1 group, and sh-NC, sh-RNASEH1-AS1, sh-NC, sh-METTL, si-NC, si-BUD13, sh-RNASEH1-AS1 and pc-NC, sh-RNASEH1-AS1 and pc- ANXA2, sh-METTL and pc-NC, sh-METTL and pc-ASEH1-AS1 were transfected in SW480 cells. The expression of METTL3, RNASEH1-AS1, BUD13, and ANXA2 in SW480 cells was detected by qPCR. The cell clone formation assay detected the proliferative ability of SW480 cells in the transfected groups; FCM assay detected the apoptosis of SW480 cells in the transfected groups; cell scratch assay detected the migratory ability of SW480 cells in the transfected groups; and WB assay detected the migration of SW480 cells in the transfected groups. ANXA2, β-catenin, p-β-catenin, GSK-3β, p-GSK-3β, c-Myc, cyclinD1 protein expression in each group of SW480 cells; RNA immunoprecipitation (RIP) assay to detect the targeting relationship between RNASEH1-AS1 and BUD1, and BUD1 and ANXA2. Results: Both database analysis and qPCR assay results of CRC tissues and cancerous tissues of nationals showed that METTL3, RNASEH1-AS1, BUD13, ANXA2, β -catenin, and GSK-3β were significantly highly expressed in CRC tissues compared with paracancerous tissues (all P<0.01), and RNASEH1-AS1 expression was positively correlated with the METTL3 (r=0.698, P<0.01) and BUD13 (r=0.784, P<0.01) expression were positively correlated. METTL3, RNASEH1-AS1, BUD13, and ANXA2 mRNA were highly expressed in colon cancer cells (all P<0.05), and the expression of RNASEH1-AS1, BUD13, and ANXA2 was significantly decreased in SW480 cells after knockdown of RNASEH1-AS1 or METTL3 (all P<0.05), while overexpression of RNASEH1-AS1 significantly upregulated the expression of the above molecules (all P<0.05).Knockdown of RNASEH1-AS1 or METTL3 inhibited the proliferation, migration and expression of p-β-catenin, p-GSK-3β, c-Myc,cyclinD1 proteins, and promoted apoptosis of SW480 (all P<0.05), whereas over-expression of RNASEH1-AS1 promoted the proliferation, migration and expression of p-β-catenin p-β-catenin, p-GSK-3β, c-Myc, cyclinD1 protein expression and inhibited its apoptosis (all P<0.05). RNASEH1-AS1 promoted the expression of ANXA2 by recruiting BUD13 targeting (all P<0.05); over-expression of ANXA2 partially reversed the knockdown of the effect of RNASEH1-AS1 on SW480 cells (all P<0.05). Conclusion: METTL3-modified RNASEH1-AS1 regulates the malignant biological behavior of SW480 cells through the BUD13/ANXA2/Wnt/β-catenin axis.
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