Background
Cervical cancer is one of the most common gynecological malignancies worldwide, the morbidity and mortality of which increase year after year. To date, surgery, radiation therapy combined with chemotherapy is still mainly used for the treatment of patients with cervical cancer. Unfortunately, the effectiveness of radiotherapy and chemotherapy is poor and the five-year survival rate is only 40–50% [
1]. Reports have demonstrated that hypoxia is an important factor impacting the effectiveness of radiotherapy and causing the tumor cells obtaining radioresistance [
2]. Therefore, it is urgently required to elucidate the mechanisms underlying radioresistance of cervical cancer cells and improve the hypoxic condition in cells.
Owing to the rapid growth of tumor cells, vascular supplies are incapable of meeting the need of this growth, finally leading to hypoxia in tumor cells. The expression of several genes in hypoxic-tumor cells were altered, such as hypoxia-inducible factor 1 (HIF-1), causing the increase in cellular radioresistance [
3]. Hypoxia inducible factor-1α (HIF-1α) is a nuclear factor existing widely in hypoxic tissues. HIF-1α is a key regulatory factor of cellular response to hypoxia, mediating the gene expression for cell survival and resistance to oxidative stress. It has been shown that HIF-1α regulated the expression of genes involving angiogenesis, tumor invasion, metastasis, proliferation and apoptosis [
4]. Cui et al. reported that HIF-1α was highly expressed in cervical cancer tissues and associated with the decreasing radiosensitivity of cervical cancer [
5]. However, the regulation of HIF-lα in cervical cancer is still poorly understood.
HOX transcript antisense intergenic RNA (
HOTAIR) is an antisense transcript long noncoding RNA (lncRNA) located at the antisense strand of the HOXC gene locus in chromosome 12 q13.13 [
6]. It has been shown that overexpression of
HOTAIR contributed to the invasion and metastasis of several cancer cells, such as hepatocellular cancer, pancreatic carcinoma, breast cancer and colorectal cancer [
7]. Higher level of
HOTAIR was also found in colorectal cancer tissues comparing to that in the adjacent tissues, closely related to the patients’ age, clinical stages, invasive depth and lymphatic metastasis [
8]. Studies have shown
HOTAIR enhanced radioresistance in many cancers, such as breast cancer [
9], colorectal cancer [
10], pancreatic ductal adenocarcinoma [
11], etc. However, the function of
HOTAIR on the radioresistance of cervical cancer and the regulation of HIF-lα has not been reported.
In this study, we investigated the expression of HOTAIR in cervical cancer cells exposed to radiotherapy and analyzed the impact of HOTAIR and HIF-1α on the radiotherapy effect in cervical cancer cells.
Methods
Establishment of the nude mice model bearing cervical cancer cells
This experiment was performed in accordance with institutional guidelines of the Kaifeng Central Hospital and Use Committee guidelines. Six weeks old female BALB/c nude mice were randomly divided into two groups: control and radiation (
n = 6 for each). At first, HeLa cells (1 × 10
6 cells) were injected subcutaneously in the right posterior flank of the mice. After 10 days, mice were radiated with 10.8 Gy/each time for once a week, with totally four weeks (0, 7, 14 and 21 days, a total dose of 43.2 G) [
13]. The large dose of radiation was aimed to determine the effect of radiation alone on tumor growth, which can obviously observe the effect of
HOTAIR overexpression on radiation-inhibited tumor growth. The irradiation time lasted 30 min/each time. The X-ray was radiated by a Faxitron Cabinet X-ray System (Faxitron, IL, USA) (the dose rate = 0.36 Gy/min). Mice in control group did not receive radiation treatment. On day 26, all the mice were sacrificed and tumors were collected for measuring their volume (length×width
2)/2 and detecting the expression of
HOTAIR and HIF-1α.
Cell culture and treatment
Human cervical cancer cell HeLa and C33A, normal cervical epithelial cells (NCECs) were obtained from the American Type Culture Collection (ATCC, VA, USA) and were cultured in RPMI1640 medium (Gibco, Gaithersburg, USA) with 10% fetal bovine serum (FBS; Gibco Gaithersburg, USA), and incubated at 37 °C in a humidifed incubator at 5% CO2. For the irradiation treatment, the exponentially growing cells were seeded in the culture flasks or culture dishes. When the cell confluence reached 60%, the cells were treated with 2 Gy for 0, 6, 12, and 24 h.
Cell viability assay
3-(4,5-Dimethylthiazol-2-yl) 22,5-diphenyltetrazolium bromide (MTT) assay (ThermoFisher Scientific, CA, USA) was performed to detect the cell viability of cervical cancer cells as described by the manufacturer. HeLa cells or C33A cells were seeded into 96-well plates at an initial density of 3000 cells/well. After 48 h, the wells were incubated with MTT (5 mg/mL) for 4 h and the reaction was stopped by DMSO. Absorbance at 490 nm of the solution was read by using a spectrophotometric plate reader.
Cell apoptosis analysis
The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect the cell apoptosis using the In situ Apoptosis Detection Kit (Takara, Dalian, China). Cells were fixed by 4% paraformaldehyde/PBS solution for 30 min and washed by PBS for twice. After treated by 0.2% Triton X-100 for 5 min and washed by PBS for twice, cells were incubated with TdT reaction buffer and dUTP labelled by fluorescein for 1.5 h at 37 °C in humidifed chamber. Then washing buffer was added to stop the reaction. For viewing with a light microscope, the cells were incubated with converter-POD at 37 °C for 30 min in humidifed chamber. The reaction mixture was colored with DAB at room temperature for 10–15 min and terminated by washing with distilled water. The reaction mixture was counterstained with DAB for 20 min. TUNEL-positive nuclei were counted and detected with a light microscope (CX23, Olympus, Japan).
Quantitative real-time PCR
The total RNA from cervical tumors or cervical cancer cells was collected by the Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The purity and concentration of the total RNA was measured by using NanoDrop. The complementary DNA (cDNA) was synthesized using the First Strand cDNA Synthesis kit (Thermo Scientifc, USA) according to the manufacturers’ instructions. The expression of HOTAIR was quantified by real-time PCR using the SYBR EX TAQ (Takara, Dalian, China). GAPDH was used as the internal control. The relative expression of HOTAIR was calculated using the 2−ΔΔCt method. Primers were used as follows: LncRNA HOTAIR: 5’-GTGGTGCTGACAAAGCTTGGAA-3′ (forward), 5’-TCACTGGGTGCCA TCGTAAGAA-3′(reverse);GAPDH: 5’-GGAGCGAGATCCCTCCAAAAT-3′ (forward), 5’-GGCTGTTGTCATACTTCTCATGG-3′ (reverse).
Western blot
Total protein in cervical tumors or cervical cancer cells was extracted by using the RIPA buffer. Thirty micrograms of total protein were separated by 12% SDS-PAGE and then transferred onto the PVDF membrane (GE Health, London, UK). Following blocked in 5% non-fat milk for 1 h, the membrane was incubated with the primary antibodies overnight at 4 °C. The next day, the membrane was incubated with the horseradish peroxidaseconjugated second antibody for 2 h (Cell Signaling Technology, Boston, USA). Protein bands were visualized by using the ECL chemiluminescence kit (Pierce, IL, USA). The primary antibody for HIF-1α and β-Actin were purchased from Cell Signaling Technology (MN, USA).
Cell transfection
The HOTAIR overexpressing (Ad-HOTAIR), HIF-1α overexpressing (Ad-HIF-1α), HIF-1α silencing (si-HIF-1α) or scrambled oligonucleotides ligated into the adenovirus vector was prepared by Riobio Co. Ltd. (Guangzhou, China). HeLa cells or C33A cells were seeded at 4 × 104 cells/well in a 6-well plate, then transfected with Ad-HOTAIR, Ad-HIF-1α, si-HIF-1α or negative control adenovirus vector. The lenti-HOTAIR or scrambled oligonucleotides ligated into the lentiviral vector was prepared by Riobio Co. Ltd. (Guangzhou, China).HeLa cells transfected with Len-HOTAIR or Len-GFP were used to inject subcutaneously in the right posterior flank of the mice.
Statistical analysis
All statistical analyses were performed using SPSS 17.0 (SPSS, Chicago, USA). All data were presented as mean ± standard deviations. Differences among groups were assessed by unpaired Student’s t test and one-way ANOVA. P < 0.05 was considered to be statistically significant.
Discussion
To date, radiotherapy has been one of the major therapies for cancer treatment. Radiotherapy technology for cervical cancer is also increasingly innovated with the application of three dimensional conformal radiation therapy and intensity modulated radiationtherapy [
14,
15]. However, the radiosensitivity developed in tumor cells greatly compromises the effectiveness of radiotherapy. Long noncoding RNAs (lncRNAs) have been known to play a crucial role in the regulation of cellular processes, as well as the cancer radiation resistance [
16]. Among the extensively studied lncRNAs,
HOTAIR was found to be associated with the mechanism of radiosensitivity in several cancers [
17]. For example,
HOTAIR affected the radiosensitivity of pancreatic ductal adenocarcinoma by regulating the expression of Wnt inhibitory factor 1 [
18]. Chen reported that radiotherapy induced Lewis lung cancer cell apoptosis via inactivating β-catenin mediated by upregulating
HOTAIR [
12].
In the present study, we found that
HOTAIR was greatly decreased in cervical tumor from mice and cervical cancer cells exposed to radiotherapy. In addition,
HOTAIR overexpression abrogated the effect of radiation on the cell viability and cell apoptosis of HeLa and C33A cells.
HOTAIR overexpression slightly promoted the tumor growth in mice injected with Len-
HOTAIR comparing to that in mice injected with Len-GFP. Furthermore, Len-
HOTAIR abrogated the inhibition effect of radiation on tumor growth in mice. Li et al. reported that
HOTAIR induced radio-resistance and enhanced aggressive biological behaviors via inhibiting p21 in cervical cancer, which indicated that
HOTAIR might be a potent therapeutic strategy for those cervical cancer patients especially who accepted radiotherapy [
17]. Evidences have showed the significant diagnostic and prognostic value of
HOTAIR for cervical cancer [
8,
19].
Hypoxia is an important factor contributing to the radioresistance of tumor cells. Meanwhile, accumulating studies have revealed that depletion of oxygen leads to the inefficient of radiation on DNA strand breaks [
20]. Hypoxic stimuli might increase the cellular radioresistant phenotype through changing the cell death/survival signaling pathway and DNA damage repair pathway [
21]. In hypoxia-related tumor radioresistance, HIF-1α plays significant roles and is an important prognostic factor after radiation therapy [
22].
To investigate the mechanism of
HOTAIR-induced radio-resistance involving hypoxia, we explored the expression of HIF-1α and its regulation. Radiation decreased the expression of HIF-1α in cervical tumor from mice and cervical cancer cells.
HOTAIR overexpression significantly increased the protein level of HIF-1α in HeLa and C33A cells exposed to radiation. As HIF-1α was reported to upregulate the expression of
HOTAIR at transcriptional level in hypoxic cancer cells, we examined the effect of HIF-1α overexpression on the level of
HOTAIR in HeLa and C33A cells. However, HIF-1α overexpression could not reverse the downregulation of
HOTAIR induced by radiation in HeLa and C33A cells. We also found that HIF-1α knockdown reversed increasing cell viability and reducing apoptosis of HeLa and C33A cell induced by
HOTAIR overexpression. Bachtiary et al. reported that overexpression of HIF-1α had predictive and prognostic significance in cervical cancer patients receiving curative radiation therapy [
23]. HIF-1α also protected cervical carcinoma cells from apoptosis induced by radiation via modulation of vascular endothelial growth factor and p53 under hypoxia [
24]. In the current study, we found that
HOTAIR overexpression induced the radioresistance through upregulating the expression of HIF-1α in cervical cancer cells. However, the mechanism of how
HOTAIR interacted with HIF-1α in cervical cancer cells exposed to radiotherapy needs to be further investigated.
Conclusions
In conclusion, the present study not only reveals that radiotherapy inhibits the cervical cancer cell growth through downregulating HOTAIR to inhibit the expression of HIF-1α, but also sheds new lights on the molecular mechanisms related to radioresistance of cervical cancer cell. Furthermore, it implicates that HOTAIR-HIF-1α axis might be a potential target for cervical cancer radiotherapy.