Hsa_circ_0009910 knockdown in HeLa cells increases miR‑198 expression levels and decreases c‑Met expression levels and cell viability

Abstract

Cervical cancer (CC) is considered a public health problem. Circular RNAs (circRNAs) serve important roles in different types of cancer, including CC. However, the mechanisms used by circRNAs to facilitate CC progression are currently unclear. The present study analyzed the effects of hsa_circ_0009910 knockdown on microRNA (miRNA/miR)-198 and mesenchymal-epithelial transition factor (c-Met) expression levels and its impact on apoptosis and the viability of HeLa cells. Differentially expressed circRNAs in CC were identified using analysis of circRNA microarray data. Bioinformatics analysis was performed to predict circRNA-microRNA (miRNA) and miRNA-mRNA interactions. The knockdown of hsa_circ_0009910 in HeLa cells was performed using small interfering RNA and the expression levels of hsa_circ_0009910, miR-198 and c-Met were assessed using reverse transcription-quantitative PCR. The viability and apoptosis of HeLa cells were evaluated using MTT, neutral red uptake and ApoLive-Glo™ multiplex assays. Hsa_circ_0009910 was significantly upregulated in HeLa cells and the knockdown of hsa_circ_0009910 increased miRNA-198 expression levels, reduced c-Met expression levels and decreased cellular viability, but not apoptosis, in HeLa cells. Overall, these results indicated that hsa_circ_0009910 could act as a molecular sponge of miRNA-198 and contribute to the upregulation of c-Met expression levels. The hsa_circ_0009910/miRNA-198/c-Met interaction network affects the viability, but not apoptosis, of HeLa cells. Based on this mechanism, the present study suggests that hsa_circ_0009910 may be a promising biomarker for CC.

Keywords: HeLa; Hsa_circ_0009910; c-Met; cervical cancer; miR-198; viability.

Figure 1.

Hsa_circ_0009910 is upregulated in cervical cancer cells. (A) The heat map shows the expression of downregulated and upregulated circRNAs in HeLa cells and HCerEpiC. (B) Hsa_circ_0009910 expression levels in HaCaT and HeLa cells. (C) The hsa_circ_0009910 schematic shows exon 4 (green), exon 5 (pink), miRNA response elements (red triangles), RBP-binding sites (blue crosses) and an open reading frame (green line). (D) Comparison of the MFN2 expression in CESC compared with normal tissue using data retrieved from TCGA. The data are presented as the mean ± standard deviation and from at least three independent experiments where applicable. *P<0.05. circRNA, circular RNA; miRNA, microRNA; RBP, RNA-binding protein; HCerEpiC, human cervical epithelial cells; MFN2, mitofusin 2; nt, nucleotide; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; TMP, transcripts per million.

Figure 2.

Probable target genes for miR-198 and the Gene Ontology biological process. (A) The interaction node with the 12 mRNAs potentially regulated by miR-198 and (B) the pathway enrichment analysis of these 12 mRNAs are shown. FDR <0.05 was considered to indicate a statistically significant difference. miR, microRNA; FDR, false discovery rate.

Figure 3.

Hsa_circ_0009910 knockdown increases miR-198 and decreases c-Met mRNA expression levels. (A) Relative expression of hsa_circ_0009910 in HeLa cells in the HeLa si-circ9910 and HeLa si-NC groups. (B) Binding site of hsa_circ_0009910/miR-198, depicting the sequence of hsa_circ_0009910 (red letters) and the sequence of miR-198 (blue letters). The binding type is 7mer-m8. (C) Expression levels of miR-198 in HeLa si-circ9910 and HeLa si-NC. (D) Basal expression levels of c-Met in HaCaT and HeLa cells. (E) Expression levels of c-Met in HeLa si-circ9910 and HeLa si-NC. Data presented are from at least three independent experiments and expressed as the mean ± standard deviation. *P<0.05. circ, circular; miR, microRNA; si-circ9910, small interfering RNA against hsa_circ_0009910; NC, negative control.

Figure 4.

Knockdown of hsa_circ_0009910 affects HeLa cell viability but not apoptosis. The viability of HeLa cells with hsa_circ_0009910 knockdown was assessed with the (A) MTT, (B) neutral red uptake and (C) ApoLive™ assay. (D) Apoptosis of HeLa cells were assessed using the ApoLive assay. The DMSO 5% and A23187 10 µM groups served as positive controls. All data presented are from at least three independent experiments and are expressed as the mean ± standard deviation. *P<0.05. circ, circular; DMSO, dimethyl sulfoxide.

Figure 5.

Proposed hsa_circ_0009910/miR-198/c-Met regulatory network. Hsa_circ_0009910 promotes cell viability by directly regulating miR-198 and indirectly c-Met mRNA expression levels. circ, circular; MFN2, mitofusin 2; miR, microRNA; circ9910, hsa_circ_0009910.