HOXD-AS1 Exerts Oncogenic Functions and Promotes Chemoresistance in Cisplatin-Resistant Cervical Cancer Cells

Abstract

Long noncoding RNAs (lncRNAs) are important regulators in various human diseases. The lncRNA HOXD-AS1 is a tumor promoter in ovarian cancer, glioma, and lung cancer, but the specific effects of HOXD-AS1 on cervical cancer (CC) chemoresistance remain unclear. Here, the level of HOXD-AS1 in nonmalignant and CC tissues as well as in CC cells and cisplatin-resistant CC cells was determined. qRT-PCR indicated that HOXD-AS1 was overexpressed in CC tissues and cisplatin-resistant CC cells. Loss-of-function assays showed that downregulation of HOXD-AS1 expression suppressed chemoresistance of cisplatin-resistant CC cells. HOXD-AS1 targeted miR-130a-3p, and in gain-of-function assays miR-130a-3p could reverse cisplatin resistance of CC cells. miR-130a-3p in turn targeted zinc finger E-box homeobox 1 (ZEB1). These results collectively show that HOXD-AS1 can act as a competing endogenous RNA to upregulate ZEB1 expression via miR-130a-3p. The effects of the HOXD-AS1-miR-130a-3p-ZEB1 axis on cisplatin resistance of cisplatin-resistant CC cells were supported by rescue assay results. In summary, HOXD-AS1 enhanced chemoresistance of cisplatin-resistant CC cells by modulating miR-130a-3p/ZEB1 axis expression.

Keywords: HOXD-AS1; ZEB1, cisplatin-resistant; cervical cancer; miR-130a-3p.

Figure 1.

HOXD-AS1 is highly regulated in human cervical cancer (CC) tissues, cell lines, and cisplatin-resistant cell lines. (A) Expression level of HOXD-AS1 in normal tissues and CC tissues determined by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). (B) Histology of nonmalignant and CC tissues. (C) HOXD-AS1 expression in two cisplatin-resistant CC cell lines (CaSki-DDP and HeLa-DDP) and the corresponding parental CC cell lines (CaSki and HeLa). Error bars represent the mean ± SD of at least three independent experiments. **p < 0.01 vs. control group. Color images available online at www.liebertpub.com/hum

Figure 2.

Effects of HOXD-AS1 expression on chemosensitivity of cisplatin-resistant CC cells. (A) CaSki-DDP and HeLa-DDP were stably transfected with HOXD-AS1-specific siRNAs. Control siRNA was used as a negative control (NC). Chemosensitivity was determined 48 h after transfection. (B) MTT assay of effects of HOXD-AS2 silencing on cisplatin IC₅₀ of cisplatin-resistant CC cells. (C) Change in cell proliferation of two chemoresistant CC lines after transfection as measured by colony formation assay. (D) Flow cytometry of HOXD-AS1 effects on CaSki-DDP and HeLa-DDP apoptosis with increasing cisplatin concentrations. (E) Western blotting of apoptosis-related proteins. Error bars represent the mean ± SD of at least three independent experiments. **p < 0.01 vs. control group.

Figure 3.

HOXD-AS1 expression is closely associated with formation of the epithelial-mesenchymal transition (EMT) phenotype in cisplatin-resistant CC cells. (A) Distribution of EMT-related proteins in parental CC cells and cisplatin-resistant cells determined by Western blotting. (B) Transwell assays of two parental CC cell lines and CR cell lines. Scale bar = 200 μm. (C, D) Western blot and immunofluorescence assays were used to examine the level of EMT-related protein in CaSki-DDP and HeLa-DDP cell lines after si-HOXD-AS1 transfection. Scale bar = 50 μm. (E) Transwell assay to measure the metastatic capabilities of CaSki-DDP and HeLa-DDP cells after si-HOXD-AS1 transfection. Scale bar = 200 μm. Error bars represent the mean ± SD of at least three independent experiments. **p < 0.01 vs. control group. Color images available online at www.liebertpub.com/hum

Figure 4.

HOXD-AS1 interaction with miR-130a-3p. (A) qRT-PCR examined the expression level of miR-130a-3p in two tissue types. (B) miR-130a-3p expression levels in two cisplatin-resistant CC cells and corresponding parental cells. (C) Spearman's correlation analysis indicated that the miR-130a-3p expression level negatively correlated with that of HOXD-AS1. (D) Mutation of the HOXD-AS1 in miR-130a-3p abolished the interaction. (E) Dual luciferase reporter assays demonstrating the effects of the combination of HOXD-AS1 and miR-130a-3p. Error bars represent the mean ± SD of at least three independent experiments. **p < 0.01 vs. control group.

Figure 5.

miR-130a-3p in chemoresistance of cisplatin-resistant CC cells. (A) MTT measurement of cisplatin IC₅₀ value for resistant CC cells overexpressing miR-130a-3p. (B) CaSki-DDP and HeLa-DDP cell proliferation was suppressed after transfection with miR-130a-3p mimics as shown by colony formation assay. (C) CaSki-DDP and HeLa-DDP cell apoptosis was increased in the presence of miR-130a-3p mimics as shown by flow cytometry. miR-130a-3p effects on EMT in CaSki-DDP and HeLa-DDP cells as shown by (D) Western blotting and (E) immunofluorescence. Scale bar = 50 μm. Error bars represent the mean ± SD of at least three independent experiments. **p < 0.01 vs. control group. Color images available online at www.liebertpub.com/hum

Figure 6.

HOXD-AS1 positively modulates miR-130a-3p (zinc finger E-box homeobox 1 [ZEB1]) expression in CC tissues and cell lines. (A) Potential binding sites between miR-130a-3p and ZEB1 were deduced using bioinformatics analysis. (B) HOXD-AS1, miR-130a-3p, and ZEB1 interaction examined by dual luciferase assays in HEK-293T cells. (C, D) qRT-PCR and Western blot analyses of two cisplatin-resistant CC cells co-transfected with miR-130a-3p inhibitors and pLent-HOXA-AS1 to test ZEB1 expression. (E) ZEB1 expression levels in nonmalignant tissues and CC tissues by qRT-PCR analysis. (F) Spearman's correlation analysis analyzed the negative relation between miR-130a-3p and ZEB1. Error bars represent the mean ± SD of at least three independent experiments. ^**p < 0.01 vs. control group.

Figure 7.

Regulation of expression of HOXD-AS1, miR-130a-3p, and ZEB1 affects CC cell proliferation, metastasis, and EMT process. (A) Cisplatin IC₅₀ value for indicated cisplatin-resistant CC cells determined by MTT assay. (B) Proliferation of HOXD-AS1-downregulated cells after separate transfection with miR-130a-3p inhibitor and miR-130a-3p inhibitor+si-ZEB1 as assessed by colony formation assay. (C) Reduction in HOXD-AS1 expression induced by miR-130a-3p resulted in increased apoptosis, and the rates were increased again upon addition of si-ZEB1 in the presence of the miR-130a-3p inhibitor as assessed by flow cytometry. (D) Western blotting of EMT-related proteins in CaSki-DDP with suppressed HOXD-AS1 expression. Error bars represent the mean ± SD of at least three independent experiments. **p < 0.01 vs. control group.