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. 2024 Feb 27;9(3):720-731.
doi: 10.1016/j.ncrna.2024.02.016. eCollection 2024 Sep.

miR-218-5p, miR-124-3p and miR-23b-3p act synergistically to modulate the expression of NACC1, proliferation, and apoptosis in C-33A and CaSki cells

Manuel Joaquín Romero-López  1 , Hilda Jiménez-Wences  1   2 , Merlin Itsel Cruz-De La Rosa  1 , Judit Alarcón-Millán  1   2 , Miguel Ángel Mendoza-Catalán  2 , Elizabeth Ortiz-Sánchez  3 , José Manuel Tinajero-Rodríguez  3   4 , Daniel Hernández-Sotelo  4 , Gladys Wendy Valente-Niño  1 , Dinorah Nashely Martínez-Carrillo  1   2 , Gloria Fernández-Tilapa  1   2
Affiliations

miR-218-5p, miR-124-3p and miR-23b-3p act synergistically to modulate the expression of NACC1, proliferation, and apoptosis in C-33A and CaSki cells

Manuel Joaquín Romero-López et al. Noncoding RNA Res. .

Abstract

Background: In cervical cancer (CC), miR-218-5p, -124-3p, and -23b-3p act as tumor suppressors. These miRNAs have specific and common target genes that modulate apoptosis, proliferation, invasion, and migration; biological processes involved in cancer.

Methods: miR-218-5p, -124-3p, and -23b-3p mimics were transfected into C-33A and CaSki cells, and RT-qPCR was used to quantify the level of each miRNA and NACC1. Proliferation was assessed by BrdU and apoptosis by Annexin V/PI. In the TCGA and The Human Protein Atlas databases, the level of NACC1 mRNA and protein (putative target of the three miRNAs) was analyzed in CC and normal tissue. The relationship of NACC1 with the overall survival in CC was analyzed in GEPIA2. NACC1 mRNA and protein levels were higher in CC tissues compared with cervical tissue without injury.

Results: An increased expression of NACC1 was associated with lower overall survival in CC patients. The levels of miR-218-5p, -124-3p, and -23b-3p were lower, and NACC1 was higher in C-33A and CaSki cells compared to HaCaT cells. The increase of miR-218-5p, -124-3p, and -23b-3p induced a significant decrease in NACC1 mRNA. The transfection of the three miRNAs together caused more drastic changes in the level of NACC1, in the proliferation, and in the apoptosis with respect to the individual transfections of each miRNA.

Conclusion: The results indicate that miR-218-5p, -124-3p, and -23b-3p act synergistically to decrease NACC1 expression and proliferation while promoting apoptosis in C-33A and CaSki cells. The levels of NACC1, miR-218-5p, -124-3p, and -23b-3p may be a potential prognostic indicator in CC.

Keywords: Apoptosis; NACC1; Proliferation; miR-124-3p; miR-218-5p; miR-23b-3p.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Expression of genes predicted to be targeted by miR-218-5p, -124-3p, and −23b-3p in CC tissues. (A–H) Levels of NAA15, GSK3B, RFFL, SERPINB10, CCDC6, MARK1, TCF4, NACC1 were analyzed. The expression data from 306 CC tissue samples and 13 normal cervical epithelial tissues recorded in TCGA were assessed. The GEPIA2 platform was used to process the data. P < 0.05. *P < 0.05.
Fig. 2
Fig. 2
Expression of NAA15, GSK3B, RFFL, SERPINB10, CCDC6, MARK1, TCF4, and NACC by immunohistochemistry in the normal cervical epithelium and CC tissue (A–H). Images available at https://www.proteinatlas.org, accessed 29 October 2022.
Fig. 3
Fig. 3
Relationship between the NACC1 expression level and overall survival of patients with CC at different stages of evolution. Kaplan-Meier survival curve. The red line implies the high expression group and the black line represents the low expression group of NACC1. (A) In patients with CC at different stages of evolution, the NACC1 expression level is not related to OS. (B) High level of NACC1 is significantly related to lower OS in CC patients who present EMT, activation of the PI3K-AKT pathway, or hormonal disturbances.
Fig. 4
Fig. 4
NACC1 is a putative target of miR-218-5p, -124-3p, and −23b-3p. (A) Position and recognition sequences for miR-218-5p, -124-3p, and −23b-3pin the 3′-UTR region of NACC1. (B) Basal expression of miR-23b-3p, -124-3p, -218-5p, and (C) NACC1 in C-33A and CaSki cells compared to HaCaT cells. Statistical analysis was performed using GraphPad Prism v.8.0. A P < 0.05 value was considered statistically significant *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 5
Fig. 5
NACC1 mRNA level in response to overexpression of miR-218-5p, -124-3p, and −23b-3p. (A–F) Levels of miR-23b-3p, -124-3p and -218-5p in C-33A and CaSki cells transfected with each mimic. (G) NACC1 mRNA level in response to overexpression of miR-23b-3p, -124-3p, -218-5p or to the co-expression of the three microRNAs, compared to scrambled, in C-33A cells and (H) CaSki cells. The levels of miRNAs and NACC1 were normalized with RNU6 and GAPDH, respectively. Statistical analysis was performed using GraphPad Prism v.8.0. When the value was p < 0.05, differences were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 6
Fig. 6
Effect of miR-218-5p, -124-3p, and −23b-3p overexpression on proliferation in CaSki and C-33A cells. Cell proliferation decreased at 48 h, 72 h, and 96 h after transfection with miR-23b-3p (B and G), miR-124-3p (C and H), or miR-218-5p (D and I) mimics in both cell lines. The percentage of inhibition of proliferation was higher when the three miRNAs were overexpressed in C-33A (A and E) and CaSki cells (F and J). When the value was p < 0.05, the differences were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 7
Fig. 7
miR-218-5p, -124-3p, and −23b-3p modulate apoptosis in C-33A and CaSki cells. Changes in apoptosis were determined by flow cytometry. C-33A (A) and CaSki (B) cells were transfected with miR-218-5p + miR-124-3p + miR-23b-3p mimics or with each individual miRNA. When the value was p < 0.05, the differences were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001.
None
Supplementary Fig. 1Expression of 24 genes, predicted to be targeted by miR-218-5p, -124-3p, and, −23b-3p in CC samples. (A-X) Levels of LHX4, NAA15, GABRB3, GSK3B, PRLR, RFFL, SERPINB10, CACUL1, NACC1, PURA, RUNX2, ABL2, GNA12, RAPGEF2, MITF, MARK1, PTPN11, JAG1, TCF4, CCDC6, FZD4, ZBTB16, SP1, AFF1 were analyzed. The expression data from 306 CC tissue samples and 13 normal cervical epithelial tissues recorded in TCGA were assessed. The GEPIA2 platform was used to process the data. P < 0.05. *P < 0.05.
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