In total, 30 pairs of cervical cancer samples and adjacent normal tissues were collected from Gansu Provincial Cancer Hospital between June 2018 and June 2019. The clinical and pathological data of these patients (n=30) were collected with their written informed consent. The patient exclusion criteria were as follows: i) Patients (women) who suffered from other diseases and were currently under treatment; ii) pregnant and lactating women; iii) patients allergic to probiotics or have used/are using antibiotics recently; and iv) alcoholics (people who drink ≥5 bottles of beer at a time, or the alcohol content in the blood reaches ≥0.08). The patient inclusion criteria were as follow: Women (≥18 years old) who have been diagnosed with cervical cancer and have undergone surgery. Each tissue sample was stored at -80˚C until RNA extraction. In addition, the serum samples were collected from the patients. The present study was approved by the Ethics Committee of Gansu Provincial Cancer Hospital. The distribution of age and sex (30 females; mean age, 52 years; age range, 32-68 years) among the patients with cervical cancer was presented in Table I.

Meanwhile, serum was also collected from healthy donors (n=50; age, 21-58 years; sex, 28 males and 22 females). The informed consent was also obtained from healthy individuals for blood donation in the present study. The inclusion criteria of individuals without cervical cancer as control blood donors were as follows: i) Aged from 20-60 years old; and ii) no history of cancer.

The HeLa cell line was obtained from the Shanghai Cell Bank of Chinese Academy of Sciences. Cells were cultured in DMEM (Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 1% penicillin (Thermo Fisher Scientific, Inc.) and streptomycin (Thermo Fisher Scientific, Inc.) at 37˚C in the presence of 5% CO₂.

HeLa cells were seeded at a density of 3x10⁵ cells/well in a 6-well plate and cultured until 70% confluence. Then, the cells were transfected with small interfering RNA (si)STAT3 (10 nM), siJAK2 (10 nM) or negative control (empty vector, siNC, 10 nM) using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.). For siRNA knockdown, the sequence of siRNA targeting JAK2 (siJAK2) or STAT3 (siSTAT3) was designed and synthesized from Shanghai GenePharma Co., Ltd. The efficiency of transfection was detected by reverse transcription-quantitative PCR (RT-qPCR). The sequences of siRNAs were as follows: siNC, 5'-ACGUGACACGUUCGGAGAAUU-3'; siJAK2, 5'-ATCATGUUUGAGACCUUAAA-3'; siSTAT3, 5'-CUUUGAGGUCAGCCGACUCU-3'. After 24 h of transfection, transfected cells were used in subsequent experiments.

Total RNAs were extracted from tissues or cell lines with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RT-qPCR was conducted with PrimeScript RT Reagent kit (Takara Bio, Inc.) and SYBR Premix Ex Taq II kit (Takara Bio, Inc.). The temperature and duration of RT were as follows: 37˚C for 60 min and 85˚C for 5 min. The thermocycling conditions were as follows: Initial denaturation for 10 min at 95˚C; 40 cycles of 95˚C for 15 sec and 60˚C for 30 sec; and final extension for 1 min at 60˚C. The primers were purchased from Nanjing Jinsirui Biotechnology Co., Ltd. β-actin was used as the internal control. The primers were as follows: STAT3, 5'-CATCCTGAAGCTGACCCAGG-3'; STAT3 reverse, 5'-TCCTCACATGGGGGAGGTAG-3'; JAK2 forward, 5'-GAGACAACTGTGACGGGCTT-3'; JAK2 reverse, 5'-GCTCAGCTCCCACTCACATC-3'; IL-17 forward, 5'-CCTTGGAATCTCCACCGCAA-3'; IL-17 reverse, 5'-GAGCTCTTAGGCCACATGGT-3'; IL-17A forward, 5'-CTACAACCGATCCACCTCACC-3'; IL-17A reverse, 5'-AGCCCACGGACACCAGTATC-3'; IL-17F forward, 5'-CTGTGCCAGGAGGTAGTATGA-3'; IL-17F reverse, 5'-TTGATGCAGCCCAAGTTCCTA-3'; β-actin forward, 5'-GTCCACCGCAAATGCTTCTA-3'; and β-actin reverse, 5'-TGCTGTCACCTTCACCGTTC-3'. The 2^-ΔΔCq method (19) was used to measure relative expression.

HeLa cells (5x10³ per well) were plated into a 24-well Cell Culture Cluster. Once cells reached 80-90% confluence, the layer of cells was scratched perpendicular with a small pipette head. After washing with PBS for 3 times, serum-free medium was used for further culture, and the scratch widths at 0 and 24 h were recorded under an optical light microscope (magnification, x200). The experiment was repeated 3 times.

The levels of IL-17 in tissues of patients were detected using an ELISA kit (Hangzhou Multisciences Biotech Co., Ltd., cat. no. 70-EK117/2-96), according to the manufacturer's instructions.

For cell invasion analysis, Transwell assay was performed. The upper chamber was pre-treated with 100 µl Matrigel. HeLa cells were seeded into the upper chamber in medium with 1% FBS, and the density was adjusted to ~1.0x10⁶ cells per chamber. RPMI-1640 medium with 10% FBS was added to the lower chamber. After 24 h of incubation at 37˚C, the Transwell chamber was rinsed twice with PBS (5 min each time), fixed with 5% glutaraldehyde at 4˚C, stained with 0.1% crystal violet at room temperature for 30 min, washed twice with PBS and observed under an optical light microscope (magnification, x200). The number of cells invading the Matrigel was regarded to represent the invasion ability.

HeLa cells were seeded in 96-well plates (5x10³ per well) overnight. Then, cells were treated with 0, 5, 10, 50 or 50 ng/ml IL-17 for 72 h. Next, 10 µl CCK-8 reagent was added to each well and further incubated for 2 h at 37˚C. Finally, the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

Total protein was isolated from tissue or cell lysates using RIPA buffer (Shanghai GenePharma Co., Ltd.), and quantified using a BCA protein assay kit (Beyotime Institute of Biotechnology). Proteins (30 µg/lane) were resolved on 10% SDS-PAGE gel, and then transferred to PVDF membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were incubated with primary antibodies at 4˚C overnight, and then incubated with an HRP-conjugated secondary anti-rabbit antibody (1:5,000; cat. no. ab7090; Abcam) at room temperature for 1 h. Membranes were scanned using an Odyssey Imaging System and analyzed with Odyssey v2.0 software (LI-COR Biosciences). The visualization was performed using an ECL chemiluminescent kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The primary antibodies used in the present study were as follows: Anti-JAK2 (1:1,000; cat. no. ab108596; Abcam), anti-IL-17A (1:1,000; cat. no. ab79056; Abcam), anti-IL-17F (1:1,000; cat. no. ab168194; Abcam), anti-STAT3 (1:1,000; cat. no. ab68153; Abcam), anti-vascular endothelial growth factor (VEGF; 1:1,000; cat. no. ab32152; Abcam), anti-Akt (1:1,000; cat. no. ab8805; Abcam), anti-p65 (1:1,000; cat. no. ab32536; Abcam), anti-p-STAT3 (1:1,000; cat. no. ab267373; Abcam), p-p65 (1:1,000; cat. no. ab76302; Abcam), anti-p-JAK2 (1:1,000; cat. no. ab32101; Abcam), anti-p-Akt (1:1,000; cat. no. ab76302; Abcam) and anti-GAPDH (1:1,000; cat. no. ab8245; Abcam). GAPDH was used as an internal control.

Cervical cancer cells (1x10⁴ per well) were seeded in 24-well plates overnight and treated as following: Control, IL-17, IL-17 plus siRNA-NC, IL-17 plus siRNA-STAT3 or IL-17 plus siRNA-JAK2 for 72 h. After that, the cells were prefixed in 4% paraformaldehyde at 4˚C for 10 min, and fixed in pre-cold methanol at 4˚C for another 10 min. Next, cells were incubated with primary antibodies overnight at 4˚C: Anti-Ki67 (Abcam; cat. no. ab15580; 1:1,000). The nuclei were stained with DAPI (Beyotime Institute of Biotechnology). Goat anti-rabbit IgG antibody (Abcam; cat. no. ab150077; 1:5,000) was used as the secondary antibody. The samples were visualized by fluorescence microscope (magnification, x200; Olympus CX23; Olympus Corporation) immediately.

In total, 3 independent experiments were performed, and all data are expressed as the mean ± standard deviation. GraphPad Prism 7 (GraphPad Software, Inc.) was used for all statistical analyses. The comparison between two groups was analyzed using paired Student's t-test (Figs. 1A and 2) or unpaired Student's t-test (Fig. 1B). One-way ANOVA followed by Tukey's test was used for comparisons between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

In order to investigate the role of IL-17 in the progression of cervical cancer, RT-qPCR was employed. As indicated in Fig. 1A, the expression level of IL-17 was significantly upregulated in tumor tissues compared with that in normal tissues; to verify this result, ELISA was performed. The results demonstrated that the level of IL-17 in serum of patients with cervical cancer was significantly increased (Fig. 1B). Taken together, the results showed that IL-17 was upregulated during the tumorigenesis of cervical cancer.

For the purpose of exploring the role of JAK2/STAT3, NF-κB, VEGF and PI3K signaling in development of cervical cancer, western blotting was used. As shown in Fig. 2A-C, the expression levels of JAK2 and STAT3 in tumor tissues was notably increased compared with that in normal tissues. These data suggested that JAK2/STAT3 was involved in the pathogenesis of cervical cancer. Similarly, NF-κB, VEGF and PI3K/Akt signaling were also upregulated in tumor tissues (Fig. 2A and D-F). These results indicated that JAK2/STAT3, NF-κB, VEGF and PI3K/Akt were activated in the tumorigenesis of cervical cancer.

To verify the function of IL-17 in the progression of cervical cancer, CCK-8 assay was performed. As shown in Fig. 3A, IL-17 notably increased the proliferation of HeLa cells. Moreover, a concentration of 50 ng/ml exhibited the most proliferative effect. Therefore, 50 ng/ml was used in the following experiments. Next, RT-qPCR and western blotting were used to detect the transfection efficiency. The data demonstrated that the expression of JAK2 in HeLa cells was significantly decreased by knockdown of JAK2 (Fig. 3B-D). Similarly, the expression of STAT3 in cervical cancer cells was notably inhibited after STAT3 silencing (Fig. 3B-D). These results suggested that JAK2 and STAT3 siRNA were stably transfected into HeLa cells. Then, the results of Ki-67 staining demonstrated that IL-17 significantly promoted the proliferation of cervical cancer cells. However, knockdown of JAK2 or STAT3 partially rescued the proliferative effect of IL-17 (Fig. 3E). Overall, these data suggested that IL-17 could promote the growth of cervical cancer cells via activation of JAK2/STAT3 signaling.

To further investigate the effect of IL-17 on the migration and invasion of cervical cancer cells, wound-healing and Transwell assays were performed. As shown in Fig. 4A and B, the migration and invasion of cervical cancer cells were notably inhibited in the presence of IL-17, which were partially reversed by downregulation of JAK2 or STAT3. These data confirmed that IL-17 could promote the migration and invasion of cervical cancer cells via the JAK2/STAT3 signaling pathway.

IL-17A and IL-17F are two major isoforms of IL-17 that can regulate the cancer tumorigenesis (20,21). Thus, these two isoforms were selected for investigations. As indicated in Figs. 5A and B and S1A-D, the levels of IL-17A and IL-17F in cervical cancer cells were significantly upregulated by IL-17, while JAK2 or STAT3 knockdown reversed this phenomenon. Taken together, IL-17 promotes the tumorigenesis of cervical cancer via the upregulation of IL-17A and IL-17F.

To further verify the mechanism by which IL-17 modulates the progression of cervical cancer, western blotting was used. The results indicated that the expression levels of VEGF, phosphorylated (p)-JAK2, p-STAT3, p-Akt and p-p65 were significantly upregulated by IL-17, which was notably rescued by silencing of JAK2 or STAT3 (Figs. 6A and B, 7A and B). Taken together, the results confirmed that IL-17 promoted the progression of cervical cancer through the upregulation of JAK2/STAT3 signaling.