LncRNA AFAP1-AS1 exhibits oncogenic characteristics and promotes gemcitabine-resistance of cervical cancer cells through miR-7-5p/EGFR axis

Abstract

Background: Drug resistance is the main factor contributing to cancer recurrence and poor prognosis. Exploration of drug resistance-related mechanisms and effective therapeutic targets are the aim of molecular targeted therapy. In our study, the role of long non-coding RNA (lncRNA) AFAP1-AS1 in gemcitabine resistance and related mechanisms were explored in cervical cancer cells.

Methods: Gemcitabine-resistant cervical cancer cell lines HT-3-Gem and SW756-Gem were constructed using the gemcitabine concentration gradient method. The overall survival rates and recurrence-free survival rates were evaluated by Kaplan-Meier analysis. The interaction was verified through a Dual-luciferase reporter gene assay and a Biotinylated RNA pull-down assay. Cell proliferation ability was assessed through methyl-thiazolyl-tetrazolium (MTT), soft agar, and colony formation experiments. Cell cycle and apoptosis were detected by flow cytometry.

Results: Up-regulation of AFAP1-AS1 in cervical cancer predicted a poor prognosis. Besides, patients in the gemcitabine-resistance group had higher levels of AFAP1-AS1 than the gemcitabine-sensitive group. AFAP1-AS1 promoted tumor growth and induced gemcitabine tolerance of cervical cancer cells. In addition, AFAP1-AS1 mediated epidermal growth factor receptor (EGFR) expression by serving as a molecular sponge for microRNA-7a-5p (miR-7-5p). This present study also proved that the knockdown of EGFR or overexpression of miR-7a-5p abolished the accelerative role of AFAP1-AS1 overexpression in cancer progression and gemcitabine tolerance.

Conclusions: In general, the AFAP1-AS1/miR-7-5p/EGFR axis was tightly related to the progression and gemcitabine tolerance of cervical cancer, providing potential targets for the management of cervical cancer.

Keywords: Cervical cancer; Epidermal growth factor receptor (EGFR); Gemcitabine-resistance; Long non-coding RNA (lncRNA) AFAP1-AS1; miR-7-5p.

Figure 1. Up-regulated lncRNA AFAP1-AS1 was associated with chemo-resistance of cervical cancer cells. (A) Comparative expression of AFAP1-AS1 was assessed using qRT-PCR in 60 pairs of cervical cancer samples and control normal tissues. (B and C) Overall survival rates and recurrence-free survival rates of selected patients were shown via Kaplan-Meier survival analysis. (D) AFAP1-AS1 expression in the chemotherapy-resistance group (n = 43) or chemotherapy-sensitive (n = 17) group was examined through qRT-PCR. (E) qRT-PCR was used to detect the relative expression of AFAP1-AS1 in cervical cancer cell lines. ***p < 0.001.

Figure 2. LncRNA AFAP1-AS1 boosted the progression of cervical cancer cells. Empty vectors (EV) and AFAP1-AS1 overexpression vectors were used to regulate the level of AFAP1-AS1. (A) The relative level of AFAP1-AS1 in HT-3 or SW756 cells was evaluated by qRT-PCR. (B) Cell growth was detected by MTT assay every two days in HT-3 or SW756 cells. (C) Representing images of cell growth conditions were shown. Scale bar = 100 µm. (D and E) Anchorage-independent growth ability was determined by soft agar assay. Representing pictures (D) and statistics of average colony number were shown (E). (F and G) Colony formation ability (F) of cells in different groups and relative cell confluence (G) were shown. (H–J) A nude mice model of cervical cancer was built by injecting with EV or AFAP1-AS1 expression vector pretreated HT-3 cells (2 × 10⁶). Tumor volume curve (H), representing tumor images (I) and tumor weight (J) were shown. (K and L) Expressions of PCNA, pMAPK, MAPK, pAKT, and AKT in HT-3 and SW756 cells were examined by western blot. Corresponding statistical results were also shown (L). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3. Higher expression of AFAP1-AS1 was interrelated with gemcitabine resistance in cervical cancer cells. (A) Gemcitabine-resistant cervical cancer cell lines HT-3-Gem and SW756-Gem were constructed and the cell viability was examined. (B and C) The detection of the effects of gemcitabine on HT-3/SW756 cells and HT-3-Gem/SW756-Gem cells by flow cytometry assay. (D and E) Soft agar assay (D) and statistics of average colony number (E) were conducted to compare anchorage-independent growth ability between HT-3-Gem and SW756-Gem and parent cells. (F) qRT-PCR was used to examine the expression of AFAP1-AS1 in resistant cells and parent cells. (G) The silencing effect of sh-AFAP1-AS1 was evaluated by qRT-PCR. (H) The function of sh-AFAP1-AS1 on gemcitabine resistance was assessed by MTT. (I and J) Flow cytometry was used to detect the cell apoptosis rate and was shown in the form of a bar chart. (K and L) Results of the western blot showed the expressions of cleaved-caspase-3 and cleaved-PARP. Corresponding statistical results were also shown (L). **p < 0.01; ***p < 0.001.

Figure 4. Enforced AFAP1-AS1 expression increased gemcitabine tolerance in gemcitabine-sensitive cervical cancer cells (A) Gemcitabine tolerance in each group was determined via MTT. (B) The function of enforced AFAP1-AS1 on cell growth was examined by colony formation assay. (C) The relative cell confluence of the AFAP1-AS1 group was analyzed. (D and E) The HT-3 cells were treated with 10 nM gemcitabine and SW756-Gem cells were incubated with 20 nM gemcitabine. Flow cytometry was conducted to assess the role of AFAP1-AS1 in gemcitabine for the induction of cell apoptosis. (F and G) Detection of expression of cell apoptosis and MAPK signal pathway-related proteins through western blot. Corresponding statistical results were also shown **p < 0.01; ***p < 0.001.

Figure 5. LncRNA AFAP1-AS1 mediated the EGFR expression by serving as a molecular sponge for miR-7-5p. (A) Bioinformatics analysis identified putative miR-7-5p binding sites with AFAP1-AS1. (B and C) Effects of AFAP1-AS1 on the expression of miR-7-5p were assessed through qRT-PCR. (D and E) To confirm the interaction between miR-7-5p and AFAP1-AS1, luciferase reporter assay and pull-down assay were conducted. (F) The heatmap was drawn to exhibit the expression of EGFR in HT-3-Gem cells and HT-3 cells. (G) Using bioinformatics prediction, potential binding sites between miR-7-5p and EGFR were identified. (H) Effects of miR-7-5p on the EGFR expression were evaluated through qRT-PCR. (I) The luciferase reporter examination evidenced the interaction between EGFR and miR-7-5p. (J) Pull-down assay showed an interaction between miR-7-5p and EGFR. (K and L) qRT-PCR and western blot were executed to verify the effects of AFAP1-AS1 and miR-7-5p on the expression of EGFR. n.s. p > 0.05; ***p < 0.001.

Figure 6. Effects of the AFAP1-AS1/miR-7-5p/EGFR axis on cervical cancer cells. (A) The cell growth rate was measured through an MTT assay. (B and C) Soft agar assay (B) and corresponding statistics of average colony number (C) showed effects of sh-EGFR or miR-7-5p mimic on tumor-promoting activity of AFAP1-AS1. (D) Drug resistance in each group was determined through MTT assay. (E and F) Expression of EGFR, pMAPK, MAPK, pAKT, and AKT was detected by western blot assay. Corresponding statistical results were also shown. **p < 0.01; ***p < 0.001.