Background
Cervical cancer (CC) is one of the most common malignant gynecologic cancers and the leading reason of cancer-associated mortality among female population [
1,
2]. Despite great advancement in therapeutic approaches including surgery, radiotherapy and chemotherapy, a considerable patients’ long-term survival rate remains unsatisfactory due to recurrence and metastasis [
3‐
5]. Hence, it’s urgent to explore the underlying biological mechanisms in the progress of CC and develop a better therapeutic intervention for CC.
Recently, non-coding RNAs, which contains microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), have been recognized as novel candidates of signs or potential targets of treatment in multiple cancers [
6‐
8]. Accumulating evidence reported that aberrant lncRNAs play critical roles in diverse biological courses including cellular differentiation, proliferation, apoptosis, migration, invasion and stem-cell biology [
9‐
13]. For instance, Wang et al. showed that lncRNA CASC2 participated in tumor progression and exerted its inhibitory effects on hepatocellular carcinoma by CASC2/miR-367/FBXW7 pathway [
7]. Upregulation of lncRNA SNHG12 increased cell growth and invasion in cervical cancer through acting as a sponge for miR-424-5p [
14]. Recent studies demonstrated that lncRNA TPT1-AS1 was dysregulated in glioma [
15]. However, the expression level and effects of TPT1-AS1 in CC remain largely unknown.
Emerging evidence indicates that microRNAs participate in many physiological and pathological processes [
16‐
18]. miR-324-5p, a novel cancer-related miRNA, was dysregulated in many tumors [
19‐
22]. miR-324-5p inhibits HCC invasion by counteracting ECM degradation through regulating ETS1 and SP1 [
23]. Up-regulation of miR-324-5p suppressed cell growth and invasion of colorectal cancer cells by targeting ELAVL1 [
24]. However, its role in CC remains unclear.
In the present research, we attempted to explore the expression, clinical importance, functions and potential mechanisms of TPT1-AS1 in CC. Gain- and loss-of-function analysis confirmed the biological function of TPT1-AS1 in vitro and in vivo on CC development. Finally, we demonstrated that TPT1-AS1 promoted cell proliferation, colony formation, migration and invasion via TPT1-AS1/miR-324-5p/SP1 axis in cervical cancer.
Methods
Clinical samples and cell culture
The matched CC tissues and corresponding non-cancer tissues were obtained in our hospital. Informed written consent was acquired from each patient and this program was approved by the Ethics Committee of our hospital in accordance with Helsinki Declaration. Human CC cells (C33A, SiHa, HeLa, CaSki, ME-180) and immortalized cervical epithelium NC104 (Chinese Academy of Sciences, Shanghai, China) were cultured in incubator (37 °C, 5% CO2), and cultured in RPMI1640 (Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Invitrogen, CA, USA).
qRT-PCR
TRIzol reagent (Invitrogen, Carlsbad, CA) was used to isolate total RNA according to the manufacturer’s instructions. SYBR Premix Ex Taq II (TaKaRa, Dalian, China) was used to perform quantitative PCRs. qPCR primers were purchased from Genecopoeia (Guangzhou, China).
Immunohistochemical (IHC) staining
We conducted the samples in a procedure by dewax, dehydrate and rehydrate. Primary antibody (1:100, Cell Signaling, Danvers, MA, USA) was used and incubating at 4 °C overnight. The biotinylated secondary antibodies (Goldenbridge, Zhongshan, China) was used to perform SP-IHC assays. The staining scores was evaluated by the positive intensity and average percentage for 6 independent fields.
Western immunoblotting
RIPA lysis buffer (Pierce, Rockford, IL) was used to extract protein and BCA reagent (Rockford, IL, USA) was performed to quantify protein concentration. SDS-PAGE gel to separate the protein and detailed was performed as before [
25].
Transwell migration and invasion assay
The migration and invasion assays were performed using Transwell chamber (Millipore, Billerica, USA). The detailed was performed as previous studies [
25].
Colony assays and cell proliferation assays
Briefly, we plant cells at concentration 500 cells/well in six-well plate. After incubation for two weeks, we stained the colonies with 1% crystal violet and counted the number to quantify. EdU assays were conducted according to the protocols by manufacturer.
In vivo experiments
We purchased 4–6-week BALB/c nude mice from the Centre of Laboratory Animals of The Fifth Affiliated Hospital of Guangzhou Medical University to conduct the subcutaneous model and the tail vein injection of lung metastatic model. We used hematoxylin and eosin (H&E) staining to check the lung metastatic foci after 3 weeks’ injection. We determined the subcutaneous tumor volume = length × width × width/2. We maintained the animals in standard conditions.
Statistical analysis
All date is quantified as the Mean ± SD. The SPSS Version 13 (SPSS, Chicago, IL, USA) was conducted for the Pearson chi-square tests. A two-tailed Student’s t test was used to measure significance by GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). P < 0.05 was considered to be statistically significant.
Discussion
Numerous lncRNAs are aberrantly expressed in CC and play critical roles in the onset and progression of CC by acting as either tumor suppressor or oncogene [
28,
29]. Therefore, it may provide valuable therapeutic targets in the strategy for patients’ treatment to investigate the biological function and underlying mechanisms of lncRNAs in CC. In current research, we demonstrated for the first time that lncRNA TPT1-AS1 expression was up-regulated in CC tissues and cell lines. Its high expression was significantly associated with adverse clinical features, including FIGO stage, tumor size and lymph node metastasis and worse prognosis. These findings indicated that TPT1-AS1 serve as an oncogene and play critical role in CC progression.
To elucidate the biological function of TPT1-AS1 in CC, we performed gain- and loss-of-function experiment and showed that TPT1-AS1 overexpression promoted cell colony formation, proliferation, migration, invasion and EMT progress in vitro and in vivo. Moreover, we furthermore confirmed TPT1-AS1 was remarkably correlated with EMT markers in CC tissues. These data suggested that TPT1-AS1 play an important role in the biological function of CC development. Recently, accumulating studies reported that lncRNAs act as a ceRNA or miRNA sponge via interacting with miRNAs and suppressing their effects [
30]. Therefore, we assumed that TPT1-AS1 serve as a miRNA sponge in CC. To confirm the hypothesis, we conducted bioinformatic analysis and miR-324-5p contains the binding site of TPT1-AS1. Gain- and loss- experiment showed that TPT1-AS1 negatively regulated the expression of miR-324-5p in CC cells. In CC tissues, TPT1-AS1 existed an inverse correlation with miR-324-5p. Moreover, to confirm TPT1-AS1 could directly bind with miR-324-5p, luciferase reporter gene and anti-Ago2 RIP revealed that TPT1-AS1 directly targeted miR-324-5p in CC cells. In addition, we also confirmed that miR-324-5p was down-regulated in CC tissues and cell lines. Functional experiment showed that miR-324-5p inhibited cell colony formation, proliferation, migration, invasion and EMT progress in CC cells. Recent studies demonstrated that miR-324-5p play a key role in tumor progression [
31]. What’s more, rescue experiments in vitro experiments manifested that miR-324-5p was a mediator for TPT1-AS1 in CC cells. These data strongly demonstrated that TPT1-AS1 may act as a sponge for miR-324-5p in CC cells.
Furthermore, we attempted to explore the downstream target of miR-324-5p in CC cells, which may mediate TPT1-AS1/miR-324-5p axis. Then, bioinformatics tools combined with previous studies were employed for comprehensive analysis. According to the bioinformatics database, we found that SP1, a critical oncogene that regulated multiple cellular process in CC [
32], may be one of the candidate target o miR-324-5p. Previous studies confirmed that SP1 is a target of miR-324-5p in HCC cells [
23]. Luciferase reporter assays indicated that miR-324-5p directly targeted SP1 3’UTR. In addition, qRT-PCR and Western blot showed that miR-324-5p negatively regulated the expression of SP1 mRNA and protein. In CC tissues, the mRNA and protein of SP1 in high miR-324-5p tissues was lower than that in low miR-324-5p tissues. Moreover, miR-324-5p showed a negative relationship with SP1 in CC tissues. Finally, to investigate the role of SP1 in TPT1-AS1/miR-324-5p axis, we performed rescue experiment that reversed SP1 expression both abolished the biological function of TPT1-AS1 and miR-324-5p in CC cells. Previous studies demonstrated that SP1 could bind to the Snail promoter, which is a transcript factor of EMT-related protein, contribute to the tumor metastasis [
33,
34]. SP1 also affects the proliferation of cervical cancer in different mechanisms [
26,
35]. These findings suggest that SP1 mediated the TPT1-AS1/miR-324-5p-induced biological function on CC cells.
In conclusion, our study demonstrated for the first time that TPT1-AS1 is up-regulated in CC tissues and cells. Its high expression is associated with malignant clinical features and poor prognosis in CC patients. Gain- and loss-of-function confirmed that TPT1-AS1 promoted cell colony formation, proliferation, migration, invasion and EMT progress via miR-324-5p/SP1 axis, which could be a valuable and promising therapeutic target for CC treatment.
Conclusions
To conclude, our data offer the promising evidence that TPT1-AS1 overexpression acts as a predictor for indicating adverse clinical features and poor prognosis of CC patients. TPT1-AS1 facilitates CC cell colony formation, proliferation, migration, invasion and EMT progress in vitro and in vivo. MiR-324-5p was identified as not only a target but also a functional mediator of TPT1-AS1 in CC cells. miR-324-5p suppressed colony formation, proliferation, migration, invasion and EMT process of CC cells by directly targeting SP1. Restoration SP1 reversed the biological function of TPT1-AS1 and miR-324-5p on CC cells. To conclude, TPT1-AS1/miR-324-5p/SP1 axis suppressed cellular process of CC cells. These findings will improve understanding of mechanism involved in cancer progression and provide novel targets for the molecular treatment of CC.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (
http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.