miR-552 promotes the proliferation and metastasis of cervical cancer cells through targeting MUC15 pathway - PubMed (original) (raw)

. 2021 Aug 24;12(20):6094-6104.

doi: 10.7150/jca.56098. eCollection 2021.

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miR-552 promotes the proliferation and metastasis of cervical cancer cells through targeting MUC15 pathway

Xinxin Zhang et al. J Cancer. 2021.

Abstract

Accumulating evidence shows that microRNAs (miRNAs) play key roles in tumorigenesis, progression, recurrence and drug resistance of malignant tumors. The tumor-promoting role of miR-552 has been evidenced in multiple tumors. Yet, the relevance of miR-552 in cervical cancer remains undetermined. This study aimed to investigate the role of miR-552 in cervical cancer proliferation and metastasis. Herein, we for first found that miR-552 expression was upregulated in cervical cancer tissues compared with their normal controls. Functional assays revealed that miR-552 promoted the proliferation and metastasis of cervical cancer cells. Mechanically, bioinformatics and luciferase reporter analysis identified MUC15 as a direct target of miR-552. Reduced MUC15 expression was detected in cervical cancer, and MUC15 overexpression exhibited a tumor-suppressive effect. MUC15 restoration partially abolished the discrepancy of growth and metastasis capacity between miR-552 overexpression cervical cancer cells and control cells. Taken together, these data demonstrate that miR-552 acts as a potential oncogene miRNA in cervical cancer, which exerts its function through targeting MUC15.

Keywords: MUC15; cervical cancer; metastasis; miR-552; proliferation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1

Figure 1

Increased miR-552 expression in human cervical cancer. A. Relative expression of miR-552 in human cervical cancer and normal tissues samples was determined by real-time PCR analysis (n=30) (*p < 0.05). B. The expression of miR-552 in cervical cancer cell lines (Ca_Ski, C-33A, HeLa, and SiHa) and normal H8 cervical epithelial cells was investigated via real-time PCR analysis (n=3, *p < 0.05).

Figure 2

Figure 2

miR-552 knockdown suppresses cervical cancer cells proliferation. A. The interference effect of miR-552 in HeLa and SiHa cells was determined by real-time PCR analysis (n=3, *p < 0.05). B. Cell proliferation in HeLa miR-552 sponge or SiHa miR-552 sponge and their control cells was measured by using CCK-8 assays (n=6, *p < 0.05). C. Colony formation assays of HeLa miR-552 sponge or SiHa miR-552 sponge and their control cells (n=4, *p < 0.05). D. Cell cycle in HeLa miR-552 sponge and its control cells was assessed by flow cytometry (n=3, *p < 0.05). E. Cell cycle in SiHa miR-552 sponge and its control cells was assessed by flow cytometry (n=3, *p < 0.05). F. The apoptotic cells of miR-552 sponge and their control cells were assessed by flow cytometry (n=3, *p < 0.05). G. The cleave-PAPR expression in HeLa miR-552 sponge or SiHa miR-552 sponge and their control cells was determined by western bolt analysis.

Figure 3

Figure 3

miR-552 depletion inhibits cervical cancer cells migration and invasion. A. Wound healing assay was performed to compare the migratory properties of HeLa miR-552 sponge and its control cells. Magnification, 100X; *p <0.05 (n=3). B. Wound healing assay was performed to compare the migratory properties of SiHa miR-552 sponge and its control cells. Magnification, 100X; *p <0.05 (n=3). C. The migration ability of HeLa miR-552 sponge and its control cells were performed utilizing polycarbonate membrane inserts in a 24-well plate. Scale bar: 20 µm (n=4, *p < 0.05). D. The migration ability of SiHa miR-552 sponge and its control cells were performed utilizing polycarbonate membrane inserts in a 24-well plate. Scale bar: 20 µm (n=4, *p < 0.05). E. The invasive capacity of HeLa miR-552 sponge and its control cells were analyzed using Matrigel-coated Boyden chamber. Scale bar: 20 µm (n=4, *p < 0.05). F. The invasive ability of SiHa miR-552 sponge and its control cells was analyzed using Matrigel-coated Boyden chamber. Scale bar: 20 µm (n=4, *p < 0.05).

Figure 4

Figure 4

MUC15 was a direct target of miR-552 in cervical cancer cells. A. A potential target site for miR-552 in the 3'-UTR of human MUC15 mRNA, as predicted by the program Targetscan and miRBase. To disrupt the interaction between miR-552 and MUC15 mRNA, the target site was mutated. B. Luciferase reporter assays performed in HeLa miR-552 sponge or SiHa miR-552 sponge and their control cells transfected with wild-type or mutant MUC15 3'-UTR constructs (n=3, *p < 0.05). C. Luciferase reporter assays performed in HeLa miR-552 mimic or SiHa miR-552 mimic and their control cells transfected with wild-type or mutant MUC15 3'-UTR constructs (n=3, *p < 0.05). D. The mRNA expression of MUC15 was checked in HeLa miR-552 sponge or SiHa miR-552 sponge and their control cells by real-time PCR (n=3, *p < 0.05). E. The mRNA expression of MUC15 was checked in HeLa miR-552 mimic or SiHa miR-552 mimic and their control cells by real-time PCR (n=3, *p < 0.05). F. The protein expression of MUC15 was checked in HeLa miR-552 sponge or SiHa miR-552 sponge and their control cells by western blot. G. The protein expression of MUC15 was checked in HeLa miR-552 mimic or SiHa miR-552 mimic and their control cells by western blot. H. Significant correlation was observed between miR-552 and MUC15 expression in human cervical cancer tissues (n=30).

Figure 5

Figure 5

MUC15 overexpression represses cervical cancer cells proliferation and metastasis. A. Relative expression of MUC15 in human cervical cancer and normal tissues samples was determined by real-time PCR analysis (n=30) (p<0.05). B. The expression of MUC15 in cervical cancer cell lines (Ca_Ski, C-33A, HeLa, and SiHa) and normal H8 cervical epithelial cells was investigated via real-time PCR analysis (n=3, *p < 0.05). C. The overexpression effect of MUC15 in HeLa and SiHa cells was determined by real-time PCR analysis (n=3, *p < 0.05). D. The overexpression effect of MUC15 in HeLa and SiHa cells was determined by western blot analysis. E. Cell proliferation in HeLa MUC15 or SiHa MUC15 and their control cells was measured by using CCK-8 assays (n=6, *p < 0.05). F. Colony formation assays of HeLa MUC15 or SiHa MUC15 and their control cells (n=4, *p < 0.05). G. The migration ability of HeLa MUC15 or SiHa MUC15 and their control cells were performed utilizing polycarbonate membrane inserts in a 24-well plate. Scale bar: 20 µm (n=4, *p < 0.05). H. The invasive capacity of HeLa MUC15 or SiHa MUC15 and their control cells were analyzed using Matrigel-coated Boyden chamber. Scale bar: 20 µm (n=4, *p < 0.05).

Figure 6

Figure 6

miR-552 promotes cervical cancer cells progression via targeting MUC15. A. HeLa miR-552 mimic or SiHa miR-552 mimic and their control cells were infected MUC15 overexpression virus and then subjected to CCK8 assay (n=6, *p < 0.05). B. HeLa miR-552 mimic and its control cells were infected MUC15 overexpression virus and then subjected to colony formation assay (n=4, *p < 0.05). C. SiHa miR-552 mimic and its control cells were infected MUC15 overexpression virus and then subjected to colony formation assay (n=4, *p < 0.05). D. HeLa miR-552 mimic and its control cells were infected MUC15 overexpression virus and then subjected to Invasion assay. Scale bar: 20 µm (n=4, *p < 0.05). E. SiHa miR-552 mimic and its control cells were infected MUC15 overexpression virus and then subjected to Invasion assay. Scale bar: 20 µm (n=4, *p < 0.05)

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