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Archive > Volume 29 Issue 6 > 2022,29(6):557-566. DOI:10.3872/j.issn.1007-385X.2022.06.007 Prev Next
Clinical Research
miR-32-5p inhibits the malignant biological behaviors and the growth of nude mice xenograft of pancreatic cancer PANC-1 cells by targeted down-regulation of EZH2 expression
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Abstract:
Objective: To explore the effect of miR-32-5p on the malignant biological behaviors of pancreatic cancer PANC-1 cells via targeting zeste gene enhancer human homolog 2 (EZH2) and its molecular mechanism. Methods: The GEPIA database was used to analyze the expression level of EZH2 in pancreatic cancer tissues and its relationship with the prognostic survival of patients. The association between miR-32-5p expression and clinicopathological features of patients was also analyzed. qPCR was used to detect the expression of miR-32-5p and EZH2 mRNA in pancreatic cancer cells (PANC-1, AsPC-1, SW1990) and normal pancreatic HPDE6-C7 cells. With LipofectamineTM 2000, miR-NC, miR-32-5p mimic, miR-32-5p inhibitor, pcDNA-NC and pcDNA EZH2 plasmids were separately or jointly transfected into pancreatic cancer PANC-1 cells, namely control group (untransfected)，miR-NC group (transfected with miR-NC), miR-32-5p mimic group (transfected with miR-32-5p mimic), anti-miR-32-5p group (transfected with miR-32-5p inhibitor); miR-NC+pcDNA-NC group (transfected with miR-NC+pcDNA-NC), miR-NC+pcDNA EZH2 group (transfected with miR-NC+pcDNA EZH2), miR-32-5p mimic+pcDNA-NC group (transfected with miR-32-5p mimic+pcDNA-NC), and miR-32-5p mimic+pcDNA EZH2 group (transfected with miR-32-5p mimic+pcDNA EZH2). Dual-luciferase reporter gene assay was used to verify the targeting relationship between miR-32-5p and EZH2. MTT method and the clone formation assay were used to detect proliferation ability of the cells in each group; scratch healing test was used to detect cell migration ability; Transwell chamber test was used to detect cell invasion; Western blotting method was adopted to detect the expression of EZH2, epithelial cadherin (E-cadherin)and neural cadherin (N-cadherin) in cells. The tumorigenicity experiment in nude mice was used to detect the development of transplanted tumors; and the expression of Ki67 and metal matrix protease-2 (MMP-2) in tumor tissues was observed by immunohistochemical staining. Results: The GEPIA database showed that the expression level of EZH2 in pancreatic cancer tissues was higher than that in para-cancerous tissues, and the prognostic survival was negatively correlated with the expression level of EZH2 (all P<0.05). The miR-32-5p expression was significantly correlated with the nerve infiltration, tumor differentiation, TNM staging, lymph node metastasis of pancreatic cancer (all P<0.05). Compared with HPDE6-C7 cells, miR-32-5p was up-regulated and EZH2 mRNA was down-regulated in pancreatic cancer cells (PANC-1, AsPC-1, SW1990), and the level of miR-32-5p was negatively correlated with the level of EZH2. miR-32-5p targeted and down-regulated the expression of EZH2 (all P<0.05). Over-expression of miR-32-5p could down-regulate the expression levels of Ki67, MMP-2 and N-cadherin, up-regulate the level of E-cadherin, inhibit proliferation, migration and invasion of PANC-1 cells as well as the volume and weight of transplanted tumors (all P<0.05).Conclusion: miR-32-5p can inhibit the proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) of pancreatic cancer PANC-1 cells as well as the development of nude mice transplanted tumors in vivo through targeting EZH2.
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