To investigate the action mechanism of lncRNA CDKN2B-AS1/hsa-miR-134-5p/CCND1 axis in cervical cancer based on bioinformatics

Subjects

We recruited 173 CC patients from Bethune International Peace Hospital, including 55 patients at stage I, 52 patients at stage II, 32 patients at stage III, and 34 patients at stage IV. Their clinical characteristics were shown in Supplementary Table 1. CC patients at stage I and II underwent surgery (no preoperative treatment), who at stage III and IV were treated with cisplatin. We investigated their survival time within 5 years after surgery or cisplatin treatment, and collected their blood samples and tumor tissues (from CC patients underwent surgery treatment) for experiments. We had obtained the informed consents from volunteers and the approval from the Ethics Committee of Bethune International Peace Hospital before the study began.

Cell culture

Human CC cells (HeLa and SiHa) and human normal cervical epithelial cells (Ect1/E6E7) were provided by ATCC (USA), and were grown in RPMI 1,640 medium (MeilunBio, China) with 10 % FBS (Tianhang Biotechnology Co., LTD, Every Green, China) and 1 % penicillin/streptomycin (Solarbio, China) in a humidified 37 °C incubator with 5 % CO₂.

Bioinformatics analysis

The datasets of GSE228568, GSE150227, GSE167362, and GSE241703 were taken from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). GSE228568 was obtained by array of non-coding RNA profiling in 6 HPV-positive cervical cancer tissues (including 3 HPV16 and 3 HPV18 positive patients) and 3 HPV-negative normal cervical tissues. GSE150227 is a lncRNA dataset obtained by high throughput sequencing of non-coding RNA profiling in cervical normal and tumor tissues. GSE167362 is an RNA dataset obtained by high throughput sequencing of RNA profiling in three cervical cancer tissues and three para-carcinoma tissues. GSE241703 was obtained by high throughput sequencing of expression profiling in human immortalized keratinocytes (HaCaT) and HeLa cells. For prediction of CDKN2B-AS1 downstream miRNAs and hsa-miR-134-5p target genes, the ENCORI/starBase (Encyclopedia of RNA Interactomes) database (https://rnasysu.com/encori/index.php) was used.

RT-qPCR

Total RNA, extracted by the TRIzol™Plus RNA purification kit (ThermoFisher, America), was used to synthesize cDNA by RevertAid RT reverse transcription kit (ThermoFisher) for RT-qPCR. CDKN2B-AS1 level and mRNA levels of CCND1 and PCNA were normalized by GAPDH. U6 was the internal reference gene of hsa-miR-134-5p. Formula 2^−ΔΔCt was used to calculate the relative expression. Primer sequences were shown in Supplementary Table 2.

Western blotting

Total protein, extracted from cells, was isolated by 10 % SDS-PAGE and transferred to PVDF membrane (Merck Milipore, Germany) which was then incubated with 5 % skim milk at room temperature for 90 min. After washing with TBST buffer, PVDF membrane was incubated with primary antibody (anti-CCND1/GAPDH, Abcam, Britain) at 4 °C overnight and incubated with the secondary antibody (Sigma-Aldrich, America) at room temperature for 60 min. Protein signals were detected using chemiluminescent reagents (Absin, China).

Cell proliferation assay

CC cells were seeded in a 96-well plate at 3×10³ cells per well, transfected with or without si-CDKN2B-AS1. 10 μL of CCK-8 solution (Solarbio, China) was added to each well after 48 h to incubate for 2 h at 37 °C. The OD values at 450 nm were measured using a microplate reader.

Transwell migration assay

CC cells with or without si-CDKN2B-AS1 transfection were diluted to a concentration of 1×10⁵ using FBS-free medium with 0.1 % BSA. Transwell chambers (Corning, America) containing 200 μL of cell solution were placed in a 24-well plate containing complete medium to incubate for 12 h. Finally, after fixation and staining with 4 % paraformaldehyde and 0.1 % crystal violet, migratory cells were quantified.

Transwell invasion assay

60 µL of Matrigel with a concentration of 1 mg/mL was added in transwell chambers to incubate for 1–3 h at 37 °C. Then 100 µL of FBS-free medium was added and incubated for 30 min at 37 °C. 200 μL of cell solution with a concentration of 1×10⁵ was added into transwell chambers after removing the unbound Matrigel carefully. Then the transwell chambers with cell solution were placed in a 24-well plate containing complete medium to incubate for 24 h. The next steps were referred to transwell migration assay.

Cell apoptosis assay

The apoptosis of CC cells was evaluated using an Annexin V-FITC apoptosis detection kit (Beyotime, China). Simply put, the treated HeLa and SiHa cells were digested and centrifuged (1000 g, 5 min). After the cells were suspended with 195 μL binding solution, 5 μL Annexin V-FITC was added first, then 10 μL propyl iodide (PI) staining solution was added, mixed and incubated at room temperature for 10–20 min away from light. Then the apoptotic cells were analyzed by the Beckman Cytomics FC 500 flow cytometry.

Dual-luciferase reporter assay

CC cells were pre-inoculated into a 6-well plate (1×10⁶ cells/well). Next, cells were transfected with wt-CDKN2B-AS1, mut-CDKN2B-AS1, wt-CCND1 or mut-CCND1 luciferase vectors and hsa-miR-134-5p mimic. After 48 h, the cells were harvested for luciferase activity by means of the dual luciferase reporter gene assay system (Promega, America).

Statistical analysis

Associations between CDKN2B-AS1 levels and several clinical parameters were assessed by Chi-squared. The likelihood ratio test was used for multiple Cox regression analysis. The log-rank test was used for Kaplan-Meier survival analysis. The statistical differences between the experimental data of two groups were evaluated by means of Student’s t-test. This study used SPSS Statistics 19.0 for statistical analysis. p<0.05 was considered statistically significant.

CDKN2B-AS1 was abnormally expressed in CC

The GEO2R online tool in GEO database was used for the gene expression analysis of the GSE228568, GSE150227, and GSE167362 datasets. The differentially expressed lncRNAs were screened by setting conditions p<0.05 and |logFC|>1. We found that CDKN2B-AS1 expressed highly (p<0.001, logFC>1) in HPV-positive cervical tissues (vs. normal cervical tissues) and CC tumor tissues (vs. para-carcinoma tissues) (Supplementary Figure 1A). In our research, CDKN2B-AS1 showed high expression levels in HeLa and SiHa cells (vs. Ect1/E6E7 cells) and CC tumor tissues (vs. para-carcinoma tissues) of CC patients at stage I and II (Figure 1A–C). The expression of CDKN2B-AS1 was significantly increased in CC patients with elevated grading (Figure 1D). Moreover, the results of the Chi-square test analysis showed that CDKN2B-AS1 was strongly correlated with FIGO classification of CC (p<0.05) (Tables 1 and 2).

Figure 1:

CDKN2B-AS1 was abnormally expressed in CC. (A) CDKN2B-AS1 highly expressed in HeLa and SiHa cells compared with Ect1/E6E7 cells. (B–C) CDKN2B-AS1 highly expressed in the tumor tissues compared with para-carcinoma tissues of CC patients at stage Ⅰ and Ⅱ. (D) The expression of CDKN2B-AS1 in the serum from CC patients with different grades. ***p<0.001.

CDKN2B-AS1 promoted CC cell functions

Transfection of the small interfering RNA of CDKN2B-AS1 (si-CDKN2B-AS1) markedly inhibiting its expression, and the overexpressed plasmid of CDKN2B-AS1 (ov-CDKN2B-AS1) significantly increased its expression (Figure 2A). Knocking down CDKN2B-AS1 weakened the cell viability of HeLa and SiHa cells (Figure 2B). The expression of PCNA, a marker of cell proliferation, was suppressed by CDKN2B-AS1 silencing, and was enhanced by CDKN2B-AS1 overexpression (Figure 2C), suggesting that CDKN2B-AS1 helped CC cells to proliferate. Knocking down CDKN2B-AS1 decreased the number of migratory and invasive cells, which were enhanced by CDKN2B-AS1 up-regulation (Figure 2D and E), suggesting that CDKN2B-AS1 promoted CC cells to transfer. Moreover, the apoptotic rate of CC cells was enhanced by CDKN2B-AS1 silencing and was reduced by CDKN2B-AS1 overexpression (Figure 2F), indicating that CDKN2B-AS1 mediated the anti-apoptosis in CC cells. These results demonstrate that CDKN2B-AS1 played a pro-carcinogenic role in CC, and its knockdown suppressed the malignant phenotypes of CC cells.

Figure 2:

Effects of CDKN2B-AS1 on the functions of CC cells. (A) The efficiency of si-CDKN2B-AS1 and ov-CDKN2B-AS1. (B) CDKN2B-AS1 knockdown weakened the viability of CC cells. (C) Effects of knockdown and overexpression of CDKN2B-AS1 on PCNA expression. (D–E) Effects of knockdown and overexpression of CDKN2B-AS1 on migration and invasion of CC cells. (F) Effects of knockdown and overexpression of CDKN2B-AS1 on the apoptosis of CC cells. NC, negative control. **p<0.01, ***p<0.001.

High level of CDKN2B-AS1 represented poor prognosis and outcome in CC

We constructed the Kaplan–Meier survival curves of CDKN2B-AS1 to predict the outcome of CC patients. We found that the overall survival rate in the high CDKN2B-AS1 group was lower than that in the low group at any given time in the CC patients who underwent surgical treatment (log-rank p=0.041) (Figure 3A). After cisplatin treatment, the overall survival time of CC patients with high CDKN2B-AS1 level was shorter than that of patients with low level (log-rank p=0.041) (Figure 3B). These results suggest that high CDKN2B-AS1 level represented worse outcome.

Figure 3:

Prognostic value of CDKN2B-AS1 in CC. (A) Kaplan-Meier survival curve of CC patients at stage I and II who underwent surgical treatment. (B) Kaplan-Meier survival curve of CC patients at stage Ⅲ and Ⅳ who underwent cisplatin treatment.

A multifactorial COX proportional risk model was constructed by incorporating variables such as CDKN2B-AS1 level, age, FIGO stage, histologic subtype, and so on. The effects of CDKN2B-AS1 level on survival time of CC patients treated with surgery (HR=3.871, 95 % CI 1.185–12.650, p=0.025) and cisplatin (HR=1.951, 95 % CI 1.080–3.524, p=0.027) were found to be statistically significant (Table 3 and 4).

CDKN2B-AS1 targeted and negatively regulated hsa-miR-134-5p

In the ENCORI/starBase database, hsa-miR-134-5p and hsa-miR-3118 were predicted to be the downstream miRNAs of CDKN2B-AS1. Since CDKN2B-AS1 showed a higher TDMDScore for hsa-miR-134-5p binding, hsa-miR-134-5p was selected as our interesting molecule for this study. Hsa-miR-134-5p was low-expressed in CC cells and tumors (Figure 4A and B). The luciferase vectors of wild-type/mutant CDKN2B-AS1 (wt-/mut-CDKN2B-AS1) were constructed based on target sequences predicted by the starbase database (Supplementary Figure 2). Transfection of the mimic significantly increased cellular level of hsa-miR-134-5p (Figure 4C), which attenuated the luciferase activity in CC cells transfected with wt-CDKN2B-AS1, but hardly affected the cells with mut-CDKN2B-AS1 transfection (Figure 4D and E). Furthermore, CDKN2B-AS1 silencing promoted hsa-miR-134-5p expression, and overexpressing CDKN2B-AS1 reduced hsa-miR-134-5p level (Figure 4F). These results manifested the negatively targeted regulation of CDKN2B-AS1 on hsa-miR-134-5p.

Figure 4:

CDKN2B-AS1 targeted and negatively regulated hsa-miR-134-5p. (A–B) the expression of hsa-miR-134-5p in CC cells and tumors. (C) The working efficiency of hsa-miR-134-5p mimic. (D–E) Effects of hsa-miR-134-5p mimic on the luciferase activity of CC cells transfected with wt-CDKN2B-AS1 or mut-CDKN2B-AS1. (F) Effects of knock-down or overexpression of CDKN2B-AS1 on hsa-miR-134-5p expression. ***p<0.001; ns, no significant difference.

CDKN2B-AS1 promoted CCND1 expression through sponging hsa-miR-134-5p

We predicted the downstream genes of hsa-miR-134-5p in the ENCORI/starBase database. Then we made the Venn diagram of these genes against the genes differentially expressed in the cervical tumor and para-carcinoma tissues (from GSE167362) and in HaCaT and HeLa cells (from GSE241703), yielding 43 target genes (Supplementary Figure 1B). KEGG enrichment analysis displayed that these genes are enriched in the oxytocin signaling pathway, tight junction, pancreatic cancer, AMPK/FoxO/Apelin/cAMP and other signaling pathways. CCND1 was widely involved in cancer-related signaling pathways (Supplementary Figure 3 and Supplementary Table 3). In CC cells and tumors, CCND1 expression was high (Figure 5A–D). Hsa-miR-134-5p mimic decreased the luciferase activity of CC cells transfected with wild-type CCND1 vector (wt-CCND1), but hardly affect which in mutant CCND1 vector (mut-CCND1) transfection group (Figure 5E and F). The inhibitor of hsa-miR-134-5p (miR-inhibitor) resulted in an increased expression level of CCND1 (Figure 5G–I). Moreover, knocking down CDKN2B-AS1 decreased CCND1 expression, which was saved by inhibiting hsa-miR-134-5p (Figure 5J–L). These results demonstrate that CDKN2B-AS1 promoted CCND1 expression by inhibiting hsa-miR-134-5p, which may be an important molecular mechanism for CDKN2B-AS1 in promoting CC development.

Figure 5:

CDKN2B-AS1 promoted CCND1 expression through sponging hsa-miR-134-5p. (A–D) The expression of CCND1 in CC cells and tumors. (E–F) Effects of hsa-miR-134-5p mimic on the luciferase activity of CC cells transfected with wt-CDKN2B-AS1 or mut-CDKN2B-AS1. (G–I) Effect of hsa-miR-134-5p inhibition on the expression of CCND1. (J–L) Effect of si-CDKN2B-AS1 transfection alone or combined with miR-inhibitor on CCND1 expression. ***p<0.001; ns, no significant difference.