LncRNA AATBC indicates development and facilitates cell growth and metastasis of cervical cancer as a sponge of miR-1245b-5p

Abstract

With the increasing incidence and mortality rate, cervical cancer has been considered one of the most frequent malignant tumors in females. Exploration of tumor progression-related biomarkers could facilitate the identification of novel and targeted therapy strategies. To assess the significance of lncRNA AATBC (AATBC) and its potential regulatory mechanism in cervical cancer, and to identify a potential biomarker, this study enrolled 123 patients with cervical cancer. Paired tissue samples were collected. The expression levels of AATBC and miR-1245b-5p were analyzed by RT-qPCR and their significance in the development and prognosis of cervical cancer was evaluated using chi-square and Cox analyses. In vitro, the regulatory effect of AATBC on the cellular processes of cervical cancer was estimated by CCK8 and Transwell assay. The interaction between ATTBC and miR-1245b-5p was assessed by luciferase reporter assay. Significant upregulation of AATBC and reduced miR-1245b-5p level in cervical cancer were observed, which showed a negative correlation between their expression levels. Close relationships of AATBC and miR-1245b-5p with the FIGO stage and lymph node metastasis were revealed. AATBC showed a significant prognostic value and miR-1245b-5p was found to mediate the tumor inhibitory effect of AATBC knockdown, which is speculated to be the underlying molecular mechanism of AATBC in cervical cancer development. Upregulation of AATBC indicted the malignant development and adverse prognosis of cervical cancer. AATBC served as a tumor promoter of cervical cancer by modulating miR-1245b-5p.

INTRODUCTION

Cervical cancer is one of the most frequent malignant tumors of the female reproductive system. Recently, the incidence and mortality rate of cervical cancer have increased gradually, and the patients tend to be younger. It has been widely accepted that the continuous infection of the human papillomavirus (HPV) induces cervical intraepithelial neoplasia, which is considered the major pathogen involved in cervical cancer.^1–3 Great progress has been made in the screening of HPV and the development of correlated vaccines, which do benefit the early detection and prevention of cervical cancer.⁴ However, HPV infection is not the only cause of cervical cancer, and its specific occurrence mechanism remains unclear. Currently, radical surgery and adjuvant chemoradiotherapy are used as regular treatments for early or locally advanced cervical cancer, but due to metastasis, chemotherapy resistance, and recurrence, the survival rate of advanced and recurrent cervical cancer patients has not improved.^5–7 Therefore, there is a need to explore novel therapeutic strategies to guide the prevention of cervical cancer and improve therapeutic sensitivity, which could reduce recurrence and, therefore, improve patients' outcomes.

In the human genome, most transcripts lack the function of coding protein, referred to as noncoding RNAs (ncRNAs), including microRNAs (miRNAs), with a length of 22–23 nt, and long noncoding RNAs (lncRNAs), with a length over 200 nt according to the molecular mass.⁸ It has been widely reported that ncRNAs are involved in a variety of biological processes and regulate the expression of RNA, DNA, and proteins, and thereby effecting the development of malignant tumors.⁹ Nowadays, numerous studies have revealed that the abnormal expression of lncRNAs in cervical cancer could indicate patients' prognosis and regulate cellular processes. For example, lncRNA TP73-AS1 serves as a regulator of cervical cancer, competitively regulating the miR-329-3p/ARF1 axis and further modulating the development of cervical cancer.¹⁰ In a recent study, Li et al. screened 130 dysregulated lncRNAs in cervical cancer using the lncRNA microarray analysis, wherein the alerting expression of lncRNA AATBC (AATBC) was observed.¹¹ AATBC was suggested to regulate the progression of various human cancers, such as nasopharyngeal carcinoma, prostate cancer, breast cancer, and bladder cancer; AATBC was also found to be dysregulated in each of these conditions.^12–15

Hence, the present study examined whether AATBC is differentially expressed in cervical cancer and whether it participates in tumor progression. This study aimed to (1) clarify the expression of AATBC in cervical cancer; (2) explore the significance of AATBC in the development of cervical cancer and patients' prognosis; and (3) assess the function of AATBC in regulating cellular processes and disclose the potential molecular mechanism.

MATERIALS AND METHODS Sample collection

Normal and tumor tissues were collected from 123 patients enrolled at our hospital from 2015 to 2018. Patients were diagnosed with colposcopy and histopathology. The collected samples were confirmed by at least two pathologists. All patients had never received chemoradiotherapy or immunotherapy before sample collection. Samples were stored in liquid nitrogen and immediately transferred to the −80°C refrigerator for following analyses. This study had been approved by the Ethics Committee of Beijing Luhe Hospital, Capital Medical University. Written informed consent was obtained from all participants included in the study. The clinicopathological features of the patients are summarized in Table 1 and analyzed using the chi-square test.

TABLE 1 Association of AATBC and miR-1245b-5p with patients' clinical features.

[Table omitted. See PDF]

Patients were grouped according to the average expression levels of AATBC and miR-1245b-5p in tumor tissues, respectively.

Follow-up survey

All patients were followed up on telephone or outpatient review for 3–60 months. The endpoints were defined as cervical cancer-related deaths, malignant progression, and recurrence. Kaplan–Meier analysis was conducted to summarize the outcomes of patients, and Cox regression analysis was used to evaluate the prognostic significance of AATBC adjusted for age, histological type, differentiation, tumor size, FIGO stage, and lymph node metastasis.

Real-time quantitative PCR

Samples were mixed with Trizol reagent to lyse the tissues and cells. Then, the mixture was moved to a sterilized tube with 200 μl trichloromethane and stood still for 3 min. After centrifugation at 12,000 rpm for 10 min, the precipitation was obtained and the value of OD260/OD280 was detected using a microplate reader to assess the purity of the extracted RNA.

Reverse transcription was conducted with the extracted RNA, GoScript Reverse Transcription Mix (Promega, USA, for AATBC), or Tap Man microRNA Reverse Transcription Kit (Applied Biosystems, USA, for miR-1245b-5p) to generate cDNA. The expression of AATBC (forward 5′- AAGGCCGGTTATCAACGT-3′, reverse 5′-GCCAGTCCCTCACTGCTCT-3′) and miR-1245b-5p (forward 5′-TCGTTAGGCCTAGCTGCATTAAC-3′, reverse 5′-CCGTAGTTAGGCATCGTGTTAGGCTTTTGCCAG-3′) was evaluated by PCR with GADPH (forward 5′-GTCATCCCTGAGCTAGACGG-3′, reverse 5′-GGGTCTTACTCCTTGGAGGC-3′) and U6 (forward 5′-CTCGCTTCGGCAGCACATATACT-3′, reverse 5′-ACGCTTCACGAATT-GCGTGTC-3′) as internal reference, respectively.

Cell culture

Cervical cancer cells (Hela, Caski, C-33A, ME-180) and normal cervical epithelial cell (HcerEpic) were obtained from the Shanghai cell Bank (Shanghai, China). Cell recovery was carried out at 37°C with a completed 10% FBS-containing DMEM culture medium (Gibco, USA). Then, the cells were further incubated at 37°C with 5% CO₂ until the cell density reached 80%.

Cell transfection

Cells were seeded in 6-well plates and were allowed to grow to 70% cell density. Cells were grouped as: CK, si-NC, si-AATBC (5′-CGGUCAUAUUUGAGCAUGATT-3′), miR-NC, miR-1245b-5p mimic (5′-UAGGCCUUUAGAUCACUUAAA-3′), miR-1245b-5p inhibitor (5′-UUUAAGUGAUCUAAAGGCCUA-3′), si-AATBC + miR-NC, and si-AATBC + miR-1245b-5p inhibitor. Cells were transfected or co-transfected with corresponding reagents with the help of Lipofectamine 2000 (Invitrogen, USA) at room temperature. The efficiency of cell transfection was estimated by the expression of AATBC and miR-1245b-5p.

Dual-luciferase reporter assay

According to the predicting binding sites, the wild-type AATBC and the mutant-type AATBC reporter plasmid were established with the pGL3 vector. The established plasmids were co-transfected with miR-NC, miR-1245b-5p mimic, or miR-1245b-5p inhibitor into the log phase cervical cancer cells for 48 h. Then, the transfected cells were lysed and analyzed with the dual-luciferase reporter kit to detect the relative luciferase activity of AATBC with Renilla as the internal reference.

CCK8 assay

Cells (5 × 10⁴ cells/well) were seeded into 96-well plates and were incubated for 0, 24, 48, 72, and 96 h in an incubator. Following this, CCK8 reagent was added the cell and incubated for 1 h in the dark. After incubation, absorbance at 450 nm was detected employing a microplate reader used to calculate the relative cell viability. There were four duplicate wells of each well.

Transwell assay

Cells (5 × 10⁴ cells/well) were seeded into the upper chambers of 24-well Transwell plates and incubated at 37°C with 5% CO₂. The upper chambers were filled with an FBS-free DMEM culture medium, while the bottom chambers were full of a 10% FBS-containing DMEM culture medium. Matrigel-coated chambers were used in the invasion assessment. After incubation for 24 h, the culture medium and the nonmetastasis cells in the chambers were removed. The metastasis cells were washed with PBS and stained with 0.1% crystal violet for 20 min at room temperature. The chambers were washed and viewed with a microscope after drying. Five fields were randomly selected for each chamber.

Statistical analysis

Experimental data were analyzed with Graphpad Prism 7.0 and SPSS 26.0 software and the difference was evaluated with Student's t-test (between two groups) or one-way ANOVA followed by Turkey's post-hoc test (among multiple groups). All data were expressed as mean ± SD. The p < 0.05 indicates statistical significance.

RESULTS AATBC and miR-1425b-5p expression levels and their correlation in cervical cancer

In tumor tissues, AATBC was found to significantly upregulate in comparison with normal tissues (Figure 1A), whereas miR-1245b-5p was downregulated (Figure 1B), which showed a significant negative correlation with AATBC expression levels (r = −0.676, Figure 1C).

FIGURE 1. Expression of lncRNA AATBC (A) and miR-1245b-5p (B) in cervical cancer tissues evaluated by PCR with GAPDH (for AATBC) and U6 (for miR-1245b-5p) as internal reference, and a negative correlation was observed between the expression levels of AATBC and miR-1245b-5p (C). ***p [less than] 0.001 by student's t-test

Significance of AATBC and miR-1245b-5p in clinical features of cervical cancer patients

According to the average expression of AATBC and miR-1245b-5p in tumor tissues, patients were categorized into high-AATBC, low-AATBC, high-miR-1245b-5p, and low-miR-1245b-5p groups. Both AATBC and miR-1245b-5p were found to be closely related to the FIGO stage (p_AATBC = 0.017, p_miR-1245b-5p = 0.025) and lymph node metastasis status (p_AATBC = 0.015, p_miR-1245b-5p = 0.020) of cervical cancer (Table 1). Additionally, higher levels of AATBC were observed in cervical cancer with advanced FIGO stage (Figure 2A) and positive lymph node metastasis status (Figure 2B), additionally, a lower expression level of miR-1245b-5p was seen (Figure 2D,E). No significant difference was observed in AATBC (Figure 2C) and miR-1245b-5p (Figure 2F) levels between cervical cancer patients with different tumor sizes (p > 0.05).

FIGURE 2. AATBC in patients with different FIGO stages (A), lymph node metastasis status (B), and tumor size (C). miR-1245b-5p in patients with different FIGO stages (D), lymph node metastasis status (E), and tumor size (F). ***p [less than] 0.001, nsp >0.05 by one-way ANOVA (for FIGO stage) and student's t-test (for lymph node metastasis and tumor size)

Significance of AATBC in the prognosis of cervical cancer patients

Based on the follow-up data of the enrolled patients, the overall survival information was plotted as a Kaplan–Meier curve (Figure 3A). Patients in the high-AATBC group showed a worse overall survival rate compared with patients in the low-AATBC group, and the difference was significant (log rank p = 0.027). Meanwhile, both AATBC and miR-1245-5p were shown to serve as independent indicators of cervical cancer prognosis together with FIGO stage and lymph node metastasis (Figure 3B).

FIGURE 3. Prognosis value of AATBC and miR-1245b-5p in cervical cancer patients. Kaplan–Meier curve of cervical cancer based on the expression of AATBC in tumor tissues (A). Cox regression analysis evaluated the prognostic value of clinicopathological features and the levels of AATBC and miR-1245b-5p (B)

Functions of AATBC and miR-1245b-5p in the cellular processes of cervical cancer

Increased AATBC levels (Figure 4A) and decreased miR-1245b-5p levels (Figure 4B) were observed in cervical cancer cells. The overexpression of miR-1245b-5p was found to dramatically suppress the luciferase activity of the AATBC WT vector in Caski and Hela cells (Figure 4C). Additionally, AATBC was inhibited by the transfection of its siRNA (Figure 4D). The knockdown of AATBC was found to enhance the expression of miR-1245b-5p in both Caski and Hela cells, which were reversed by the transfection of miR-1245b-5p inhibitor (Figure 4E).

FIGURE 4. Regulation of miR-1245b-5p by AATBC. Upregulation of AATBC (A) and the downregulation of miR-1245b-5p (B) were observed in cervical cancer cells. The interaction between AATBC and miR-1245b-5p was predicted by luciferase reporter assay (C) and further validated in Caski and Hela cells (D,E). ***p [less than] 0.001, nsp >0.05 relative to CK, ##p [less than] 0.01 relative to si-AATBC by one-way ANOVA.

Silencing of AATBC significantly suppressed the growth of Caski and Hela cells (Figure 5A); additionally, inhibition of Caski and Hela cell migration (Figure 5B) and invasion (Figure 5C) by AATBC knockdown were also observed, which was attenuated by the suppression of miR-1245b-5p.

FIGURE 5. Function of AATBC and miR-1245b-5p in the cellular processes of cervical cancer. The knockdown of AATBC dramatically suppressed the proliferation (A), migration (B), and invasion (C) of cervical cancer cells, which was attenuated by miR-1245b-5p. The proliferation of cervical cancer cells was analyzed at 0, 24, 48, 72, and 96 h. **p [less than] 0.01, ***p [less than] 0.001 relative to CK, #p [less than] 0.05, ##p [less than] 0.01 relative to si-AATBC by one-way ANOVA.

DISCUSSION

In most human diseases, there are few changes in cellular DNA; however, the expression levels of genes usually show obvious changes, which is associated with the disorder of epigenetics and further mediates disease pathogen and development.^16–18 Regulation of ncRNAs is one of the most critical mechanisms of epigenetics, and it not depend on changes in DNA sequences. LncRNA is a critical type of ncRNA and serves as a cellular regulator that responds to intracellular or extracellular signaling molecules and regulates the selective expression of downstream genes.^19,20 Dysregulation of lncRNAs would induce uncontrolled gene expression and result in the malignant development of diseases. Although there was no clear evidence suggesting the effect of ncRNA regulation in cervical cancer progression, several ncRNAs with abnormal expression have been demonstrated to be involved in the development of cervical cancer. For example, lncRNA LINC00116 has been demonstrated to promote the occurrence of cervical cancer by regulating the miR-106a/c-Jun pathway.²¹ Further, Peng et al. revealed the promoted effect of circular RNA circEPSTI1 on cervical cancer progression through the miR-375/409-3p/515-5p-SLC7A11 axis.²²

AATBC was identified to localize in human chromosome 21q22.3 with a length of 4622 bp and was primarily reported in bladder cancer. Subsequently, it attracted considerable attention and gradually become a research hot point.^15,23 Previous studies reported that AATBC served as an oncogene or suppressor in various human cancers, where it mediates disease development and possesses remarkable clinical significance. For instance, upregulation of AATBC was observed in breast cancer, which accelerates the metastasis of breast cancer by negatively modulating YBX1 and activating the YAP1/Hippo signaling pathway.¹³ Similarly, a promoted effect of AATBC has been observed in the proliferation and migration of prostate cancer cells via the regulation of the miR-1245b-5p/CASK axis.¹⁴ Based on previous findings that AATBC is a dysregulated lncRNA in cervical cancer, its potential functional role in cervical cancer was evaluated in the present study. In cervical cancer tissues, AATBC showed significant upregulation, which was found to be positively correlated with patients' disease development. Specifically, patients with advanced FIGO stage or lymph node metastasis were found to possess higher levels of AATBC. Additionally, the upregulation of AATBC was identified as an independent indicator of the adverse prognosis of cervical cancer patients.

Serving as a sponge of functional miRNAs is the major regulatory mechanism of lncRNAs.^24–26 AATBC has been disclosed to serve as endogenous ceRNA of various miRNAs and displays vital regulatory effects. In nasopharyngeal carcinoma, AATBC was identified as a potential biomarker that promoted cell metastasis and enhanced the expression of the desmosome-associated protein, which regulates epithelial-mesenchymal transition in nasopharyngeal carcinoma cells.¹² Moreover, it was reported that miR-1245b-5p served as a ceRNA of lncRNA HCG11 in osteosarcoma, which promoted the phenotypes of osteosarcoma through regulating cellular processes.²⁷ miR-1245b-5p was revealed to mediate the promoted effect on prostate cancer, where it could reverse the promotion of cell proliferation and migration by AATBC overexpression.¹⁴ In the present study, AATBC showed a significant negative correlation with the expression of miR-1245b-5p in cervical cancer tissues, which is consistent with the previous report.¹⁴ In vitro, the expression of miR-1245b-5p was regulated by the dysregulation of AATBC, and the inhibitory effect of AATBC knockdown was found to be alleviated by the downregulation of miR-1245b-5p. Therefore, miR-1245b-5p was speculated to be the underlying mechanism of AATBC function in cervical cancer.

Some limitations of this study must be noted. This is a single-center study with a relatively small sample size, and heterogeneity in the expression of lncRNAs and miRNAs may exist between different samples. Hence, larger sample sizes and multiple-center data are needed in future investigations, to comprehensively understand the relationship of the ATTB/miR-1245b-5p axis with the development of cervical cancer. Additionally, further molecular mechanisms has not been disclosed in the present study. miR-1245b-5p was reported to regulate CASK and therefore mediate the function of AATBC in prostate cancer.¹⁴ CASK might also be involved in their function in cervical cancer, which needs further validation.

Taken together, lncRNA AATBC was upregulated in cervical cancer, which was negatively correlated with the downregulation of miR-1245b-5p in cervical cancer. AATBC and miR-1245b-5p were associated with disease development and served as prognostic indicators of cervical cancer. Moreover, miR-1245b-5p mediated the tumor promoter role of AATBC in the processes of cervical cancer, which provides novel insights into the development of therapeutic strategies for cervical cancer (Figure 6).

FIGURE 6. Graphic abstract summarizing the major findings of this study.

CONFLICT OF INTEREST

All authors declare no conflict of interest.