Background
Despite the widespread screening programs and promotion of vaccine, cervical cancer (CC) remains the third most common cancer in developing countries [
1,
2]. Each year, over 260,000 women died of this disease, most of them from low-and-middle-income countries [
3]. Also, the prognosis of advanced patients with CC is still dismal. The 5-year survival rate of patients in advanced CC stage is approximately as low as 15% [
4]. A large set of molecular imaging biomarkers have been recognized and confirmed to predict outcome at baseline or early during cervical cancer therapy, which immensely contributes to cervical cancer diagnosis and prognosis [
2]. Based on the fact that CC is still a lethal cancer for women, there is a great need and urgency to identify novel treatment targets and prognosis biomarkers.
Long non-coding RNAs (lncRNAs) are biological regulators in a wide variety of important cellular processes including proliferation, migration and epithelial-mesenchymal transition (EMT) [
5,
6]. For example, lncRNA maternally expressed gene 3 (MEG3) inhibits cell proliferation and metastasis in gastric cancer [
7]. LncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) drives cell proliferation and EMT in breast cancer [
8].
Fetal-lethal non-coding developmental regulatory RNA (FENDRR) is a new-founded lncRNA which has been reported as a tumor suppressor in different cancers including non-small lung cancer, prostate cancer, breast cancer, cholangiocarcinoma and hepatocellular carcinoma [
9‐
12]. However, biological effects and its mechanism with respect to CC remain to be explored. Mechanistically, FENDRR can exert tumor-inhibitory functions in colon cancer through repression of Sry‑Related HMG‑BOX‑4 (SOX4) protein [
13]. Moreover, FENDRR post-transcriptionally regulate Runt-related transcription factor 1 (RUNX1) by sponging miR-18a-5p [
14]. In this study, we explored the functions of FENDRR in CC cell growth and migration. More importantly, we explored the downstream molecular mechanism of FENDRR in CC.
Methods
Clinical specimens and cell lines
The written informed consents were signed by 53 participants, and approval was acquired from the Ethics Committee of Minhang Hospital. 53 CC and adjacent non-cancer tissue samples were snap-frozen at − 80 °C in liquid nitrogen. CC cell lines including HeLa, SiHa, CaSki and C33A, as well as normal cell line Ect1-E6E7, were procured from the American type culture collection (ATCC) (Manassas, VA), incubated in the Dulbecco’s modified eagle medium (DMEM) adding the 10% fetal bovine serum (FBS) at 37 °C under 5% CO2.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Isolation of the total RNA from HeLa and SiHa cells were used to synthesize complementary DNA (cDNA) for conducting quantitative PCR using SYBR Green kit (TaKaRa, Shiga, Japan) on the Step-One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA). RNA expression level was calculated through 2−ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 small nuclear RNA (U6) were used as the corresponding internal genes for lncRNA/mRNA or microRNAs (miRNAs).
Transfection
Confluent CC cell lines were prepared in 24-well plates for 48 h of transfection utilizing Lipofectamine 2000 (Invitrogen, Carlsbad, CA). For gene overexpression, OE/FENDRR and negative control (NC), miR-15a/b-5p mimics and miR-NC, OE/TUBA1A and NC, were produced by Genepharma (Shanghai, China). For gene silencing, miR-15a/b-5p inhibitor and miR-NC, sh-TUBA1A and sh-NC, were acquired from Genechem Company (Shanghai, China).
CCK-8 assay
HeLa and SiHa were treated with cell counting kit 8 (CCK-8) reagents (Dojindo Molecular Technologies, Kumamoto, Japan) in the 96-well plates after transfection and detected at indicated times. Cell viability was monitored by detection of absorption at 450 nm.
Transfected CC cell lines were placed into the 6-well plates for 2 weeks, then treated with the fixation and staining using 4% paraformaldehyde and 0.1% crystal violet.
Flow cytometry analysis for apoptosis
Cells were reaped after transfection and fixed on ice for 1 h, followed by incubation with Annexin V-APC and 7-AAD kit in the dark. CC cells were exposed to analysis of flow cytometer.
Transwell assays
Cell invasion or migration assay was conducted with the 24-well transwell chamber (Costar, MA) coating matrigel or not. CC cells with invasive or migratory ability were dyed with 0.1% crystal violet solution after fixing for 30 min.
Western blot
Cellular protein sample was extracted for separation through electrophoresis on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sample on polyvinylidene fluoride (PVDF) membranes were treated with 5% nonfat milk and incubated with the specific primary antibodies (Abcam, Cambridge, MA) to E-cadherin, N-cadherin, Vimentin and GAPDH, subsequent to incubation with secondary antibody. Density of protein band was detected through enhanced chemiluminescence solution (Millipore, Billerica, MA).
Xenograft tumor assay
6 weeks old male BALB/C nude mice were bought from the National Laboratory Animal Center (Beijing, China) and employed under the approval from the Administrative Panel on Laboratory Animal Care of the Minhang Hospital. Xenograft tumor assay was performed through subcutaneous injection of transfected CC cells to mice. Following 4 weeks of injection, mice were killed before tumors were dissected and weighed.
Subcellular fractionation
PARIS™ Kit (Ambion, Austin, TX, USA) was acquired for Subcellular fractionation assay in CC cells. The expression levels of FENDRR, U6, GAPDH, were measured by qRT-PCR.
RNA immunoprecipitation (RIP) assay
EZMagna RIP Kit (Millipore), as well as human antibody against Ago2 or IgG were obtained for RIP assay in CC cells. The precipitated collected by beads were analyzed via qRT-PCR.
Luciferase reporter assay
Luciferase reporter vectors FENDRR Wt/Mut and TUBA1A Wt/Mut werr generated using the wild-type and mutant miR-15a/b-5p binding sites and pmirGLO Vector (Promega, Madison, WI). After 48 h of co-transfection with indicated plasmids, Dual-Luciferase Reporter Assay System (Promega) was used for luciferase activity.
RNA pull down
Protein extracts of CC cells were mixed with the Bio-miR-15a-5p or Bio-NC as control in the presence of beads for I h of incubation. The final mixture was subjected to analysis of qRT-PCR.
Statistical analysis
SPSS 17.0 software (SPSS, Inc., Chicago, IL) was employed in this study for data analysis with Student’s t-test for two groups and one-way or two-way ANOVA for more than two groups, with p < 0.05 as significance. Result from three different repeats was shown as the mean ± standard deviation (SD).
Discussion
Recently, more and more lncRNAs have been identified dysregulated in CC and regulating CC progression including XLOC_006390, highly upregulated in liver cancer (HULC), Growth arrest-specific transcript 5 (GAS5), Small nucleolar RNA host gene 20 (SNHG20), etc. [
15‐
18]. We found lncRNA FENDRR hasn’t been investigated in CC to date. Also, FENDRR was reported inhibiting various cancers such as colon cancer, hepatocellular carcinoma, etc. [
13,
19]. Similarly, FENDRR also had tumor suppressive property in CC, which was reflected by its downregulation in CC tissues and cells as well as the results of gain-of-function experiments.
Increasing studies have revealed that lncRNAs function as a ceRNA to sequester microRNAs (miRNAs) and thus modulate the downstream mRNAs [
20,
21]. For instance, lncRNA Plasmacytoma variant translocation 1 (PVT1) facilitates glioma by targeting miR-128-3p/Gremlin-1 (GREM1) axis [
22]. LncRNA Interleukin enhancer-binding factor 3 antisense RNA 1 (ILF3-AS1) accelates melanoma progression via miR-200b/a/429 axis [
23]. In our study, by means of StarBase, we found relevant miRNAs having binding sites with FENDRR. By examining the relative expression in tumor tissues and their adjacent normal tissues, we discovered miR-15a-5p and miR-15b-5p was prominently upregulated in tumor tissues. MiR-15a-5p and miR-15b-5p have been previously reported as promoters in diseases such as abdominal aortic aneurysm, osteoarthritis chondrocytes and colorectal adenocarcinoma [
24‐
26]. For further evidence, miR-15a/b-5p loss-of-function experiments were carried out and the oncogenic property of miR-15a/b-5p was confirmed. Next, the RIP assay proved miR-15a/b-5p and FENDRR coexisted in RISC and from pull down assay, we can observe the PCR product of miR-15a/b-5p is FENDRR, indicating that miR-15a/b-5p bind to FENDRR. Furthermore, luciferase reporter assay verified the interaction between miR-15a/b-5p and FENDRR. To select the downstream target gene of miR-15a/b-5p, we resorted to StarBase again and two mRNAs were finally selected. After testing the relative expression in CC tissues and their respective reaction to FENDRR overexpression, we confirmed TUBA1A was qualified target gene of miR-15a/b-5p. Studies have investigated the involvement of TUBA1A in ceRNA network. So next step, we applied gain-of-function experiments and observed cell viability, proliferation, migration and invasion ability decreased after overexpression of TUBA1A. Mechanism experiments further elucidated the ceRNA network constituted by FENDRR, miR-15a/b-5p and TUBA1A. Finally, the rescue experiments further demonstrated that knockdown of TUBA1A saved the function depletion caused by FENDRR overexpression.
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