PAX9 functions as a tumor suppressor gene for cervical cancer via modulating cell proliferation and apoptosis

1 INTRODUCTION

Cervical cancer (CC), also known as carcinoma of the uterine cervix, refers to the malignant tumor occurred in the uterus or vagina of females, including the cervical canals, and has become a major threat to the female health.1 According to the World Health Organization (WHO)/International Agency for Research on Cancer (IARC), the estimated prevalence and mortality rate in 2020 (worldwide, both sexes, all ages) have been rising to 15.6% and 8.8%, respectively, while in China, the estimated age-standardized number of incident cases were 4,568,754, and the death toll was 3,002,899 (https://gco.iarc.fr/today/). CC develops as a consequence of multiple complicated molecular factors, including the activation of oncogene and the inhibition of tumor suppression gene.2, 3 A broad spectrum of genes or proteins have been identified involved in the development of CC with the constant progress of scientific research, which facilitates the diversity of CC treatment, such as gene-targeted therapy and hormonotherapy.4-6 As such, searching for markers for the diagnosis and prognosis of CC and clarifying the effective molecular targets have become the hot issue for medical scientists worldwide.

Paired box (PAX) family encodes a group of growth- and development-regulation-related transcription factors participated in the intracellular signal pathways to regulate the embryogenic development and the formation and differentiation of tissues and organs.7, 8 This family consists of nine members and further classified into four subclasses: Group 1 (including PAX1 and PAX9), which have only paired domain (PD) and octapeptide (OP); Group 2 (including PAX2, PAX5, and PAX8), which have PD, OP, and incomplete homeodomain (HD); Group 3 (including PAX3, PAX7), which have PD, OP, and complete HD; and Group 4 (including PAX4 and PAX6), which have PD and complete HD.9, 10 Of note, PAX9 (NM_001372076), located in q12-13 of human chromosome 14, possesses a DNA-binding paired domain consisting of 128 amino acids that can specifically bind to the DNA sequence.11, 12 Published literatures have reported that PAX9 was mainly expressed in the dental mesenchyme during the morphological development of tooth, while the mutation of PAX9 resulted in the dental hypoplasia, mainly the molar anodontia.13, 14 Recent findings also pointed out the abnormal expression of PAX9 in the development, progression, and treatment of several types of cancers, such as adenocarcinoma of esophagus,15 oral esophageal squamous cell cancer (OESCC),16 and lung cancer.17 Nevertheless, it is still unknown whether PAX9 is also expressed abnormally in CC development. Thus, this study was conducted to clarify the possible effects of PAX9 on the progression of CC both in vitro and in vivo.

2 MATERIALS AND METHODS 2.1 Ethical statement

This study conformed to the Guide for the Care and Use of Laboratory Animals published by the National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals,18 and all animal-related operations were conducted under the supervision of Ethical Board for Laboratory Animals of our hospital. This study gained the approval from the Ethical Board of our hospital, and all patients signed the written informed consents.

2.2 Subjects

In this study, CC tissues and tumor-adjacent normal tissues were collected from a total of 142 CC patients (49.2 ± 7.5 years old), and these specimens were preserved after rapid freezing in liquid nitrogen. Among 142 patients, there were 33 patients with adenocarcinoma, 104 with squamous cell carcinoma, and 5 with adenosquamous carcinoma, including 71 human papillomavirus 16 (HPV16)-positive patients, 36 HPV18-positive patients, 26 other HPV-positive patients, and 9 HPV-negative patients. According to the International Federation of Gynecology and Obstetrics (FIGO) stage,19 there were 102 cases ≤ IB1 stage and 40 cases > IB1 stage, and meanwhile, 33 cases had tumor size of over 40 mm and 109 cases were not over 40 mm. Besides, 109 cases had tumor infiltrating depth of ≤15 mm, and 33 cases were >15 mm. Of these patients, 85 cases had parametrium invasion, 47 cases had lympho-vascular space invasion, and 19 cases had tumor-positive lymph nodes.

2.3 Cell culture and detection

Human CC cell lines (C-33A, CaSKi, HeLa, and SiHa) and human cervical epithelial cells (HCerEpiC) were all purchased from American Type Culture Collection (ATCC, Manassas, VA). The SiHa (squamous cell carcinoma), HeLa (adenocarcinoma), and C-33A (squamous cell carcinoma) cell lines were propagated in an Eagle's minimal essential medium (EMEM) with 2 mM L-glutamine and Earl's balanced salt solution (BSS) containing sodium bicarbonate (1.5 g/L), nonessential amino acids (0.1 mM), sodium pyruvate (1.0 mM), and 10% fetal bovine serum (FBS). The CaSKi cell line (epidermoid carcinoma) was propagated in RPMI 1640 media with 2 mM l-glutamine containing sodium bicarbonate (1.5 g/L), glucose (4.5 g/L), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (10 mM), sodium pyruvate (1.0 mM), and FBS (10%). HCerEpiC was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 100 μg/ml streptomycin, and 100 IU/ml penicillin. All cell lines were grown in 75-cm2 culture flasks in 5% CO2 in air at 37°C to 90% confluence.

2.4 Cell grouping

Human CC cell lines (C-33A, CaSKi, HeLa, and SiHa) were grouped into the Vector group (cells were transfected by the control lentiviral vector, GeneChem, Shanghai, China) and PAX9 group (cells were transfected by lentiviral vector–mediated overexpression of PAX9, GeneChem, Shanghai, China). Vector information of PAX9: No.: GV492; element sequency: Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin; fluorescent label: gcGFP; clone site: BamHI/AgeI. Human embryogenic renal cells (293 T) were co-transfected by Lipofectamine 2000, and 8 h later, cells were cultured in the complete medium for 48 h, where the supernatant was concentrated to obtain the high-titer lentivirus (5 × 106 TU/ml). Human CC cell lines (C-33A, CaSKi, HeLa, and SiHa) in logarithmic phase, after digestion in trypsin, were maintained in complete medium, and cell density was adjusted to 104/ml. After mixture, 100 μl cell suspension was collected from each well and inoculated into a 96-well plate for culture. Twenty-four hours later, supernatant was discarded, and cells in the wells were rinsed in PBS twice and then mixed with the medium supplemented with polybrene and virus (80 μl of medium, 10 μl polybrene [50 μg/ml], and 10 μl of virus) for infection. Ten hours later, the medium was refreshed by the complete medium, and following experiments were conducted at 72 h.

2.5 Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted from CC cell lines by using the TRIzol® Reagent (catalog no. 15596–026, Thermo Fisher Scientific, China), and the concentration of RNA was determined by using the ultraviolet spectrometer. With RNA in appropriate volume, cDNA was prepared by using the SuperScript® III Reverse Transcriptase (Life Technologies, Carlsbad, USA) and then used to perform the polymerase chain reaction (PCR) with the primers designed by Primer 5.0 software (Table 1) and synthesized by Nanjing GeneScript Biotechnology Co., Ltd. Then, quantitative real-time (qRT)-PCR was performed as per the steps of the instructions of SuperScript® III Platinum® SYBR® Green One-Step qRT-PCR Kit (Catalog No. 11746–100, Thermo Fisher Scientific, China). Expression of targeted genes was calculated by using the formula of 2−△△Ct.20

TABLE 1. Primers used for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Gene GenBank assession Sequence (5′-3′) PAX9 NM_006194 Forward primer: GGAGGAGTGTTCGTGAACGG Reverse primer: CGGCTGATGTCACACGGTC Caspase-3 NM_004346 Forward primer: CATGGAAGCGAATCAATGGACT Reverse primer: CTGTACCAGACCGAGATGTCA PARP NM_001618 Forward primer: CGGAGTCTTCGGATAAGCTCT Reverse primer: TTTCCATCAAACATGGGCGAC Bax NM_138763 Forward primer: CCCGAGAGGTCTTTTTCCGAG Reverse primer: CCAGCCCATGATGGTTCTGAT Bcl-2 NM_000657 Forward primer: GGTGGGGTCATGTGTGTGG Reverse primer: CGGTTCAGGTACTCAGTCATCC GAPDH NM_001256799 Forward primer: GGAGCGAGATCCCTCCAAAAT Reverse primer: GGCTGTTGTCATACTTCTCATGG 2.6 Western blotting

Total proteins were extracted from tissue specimens and CC cell lines, and according to the instruction of bicinchoninic acid (BCA) Protein Assay Kit (23,224, Thermo Fisher Scientific, China), the concentration of protein was determined. Proteins were then separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (89888, Thermo Fisher Scientific, China) and then transferred onto the polyvinylidene fluoride membrane (88518, Thermo Fisher Scientific, China) by using the semi-dry transfer (Bio-Rad, USA). The membrane was blocked in 5% nonfat milk at 4°C overnight. Proteins on the membrane were then hybridized with the diluted PAX9 antibody (Product # PA5-28044, Thermo Fisher Scientific, China) at a dilution of 1:500 and β-actin (ab115777, Abcam, USA) at 1/200 dilution at room temperature for 1 h, followed by three washes in PBS, 5 min/wash. Thereafter, the immunoblots were detected by incubation with secondary antibodies at 37°C for 1 h, followed by three washes in PBS, 5 min/wash. Targeted proteins were then developed by horseradish peroxidase substrate (Bio-Rad), with β-actin as loading control. The relative expression of targeted protein was expressed by the ratio of the intensity of targeted band to that of β-actin.

2.7 EdU assay

Cell lines were collected and seeded to 96-well plates by 4000 cells/well. In each well of the plate, 100 μl EdU medium (HY-118411, MedChemExpress, China) was added to culture cells for 1 h, followed by washing with PBS for 1–2 times per 5 min. Then, paraformaldehyde-containing PBS (4%) was added to incubate cells for 30 min at room temperature, prior to the following procedures, including adding 2 mg/ml glycine to each well for 5 min, discarding glycine solution, adding PBS by 100 μl/well for 5 min, discarding PBS, adding penetrant (PBS containing 0.5%Triton X-100) by 100 μl/well for 10 min, washing once with PBS for 5 min, and adding 4′-6-diamidino-2-phenylindole (DAPI, 1 mg/ml, HY-D0814, MedChemExpress, China) by 100 μl/well for 10 min in dark environment at room temperature. At last, the staining solution was removed and 100 μl PBS was used for 1–3 times of cell washing. Staining results were observed with a fluorescence microscope (Olympus, Tokyo, Japan), and the experiment was conducted three times independently.

2.8 Annexin-V-FITC/PI detection

According to the instruction of Cell Apoptosis Kit with Annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) (V13242, Thermo Fisher Scientific, China), cell apoptosis was assessed. In brief, cells were rinsed in cold PBS for three times and then centrifuged to obtain the sediment, where the cells were resuspended in 1 × Annexin binding buffer (100 μl). In cell suspension, 5 μl FITC Annexin V and 1 μl PI working solution (100 μg/ml) were added, followed by incubation at room temperature for 15 min. Then, 1 × Annexin-binding buffer (400 μl) was added into the mixture and then placed on the ice for flow cytometry.

2.9 Construction of xenograft models on nude mice

For construction of xenograft models, 12 BALB/c mice aged at 4 weeks were purchased from Shanghai Laboratory Animal Center Co., Ltd, and, in the specific-pathogen-free environment, mice aged at 6 weeks were divided randomly into two groups, with 6 mice in each group. Xenograft CC models were constructed by subcutaneously injected with CaSki cells2, 21-24 transfected with PAX9-overexpression lentiviral vectors (PAX9 group) and control vectors (vector group). Cells were digested by trypsin and harvested for subcutaneous inoculation of 2 × 106 cells (150 μl cell suspension), and after the formation of subcutaneous tumors, tumor size (length and width) was measured every 7 days, by using the formula of V = L × W2/2. Four weeks later, mice were decapitated to dissect the tumor tissues completely to measure the weight of tumor, and the obtained tissues were subjected to HE staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining (apoptosis was quantified by counting the number of TUNEL positive cells in at least six high-power fields), and immunohistochemistry (IHC) to determine the expression of Ki-67, cleaved-caspase-3, Bax, and Bcl-2 (the positive cells were counted in six fields per tumor sample).

2.10 Statistical analysis

All data were processed by using the SPSS 21.0 software (SPSS, Inc, Chicago, IL, USA). The measurement data were expressed by mean ± standard deviation (SD) and compared by t-test. p < 0.05 suggested that the difference had statistical significance.

3 RESULTS 3.1 Expression of PAX9 in CC tissues

According to the database of Human Protein Altas (https://www.proteinatlas.org/ENSG00000198807-PAX9/pathology/cervical+cancer#ihc), PAX9 antibody (CAB016239) for IHC showed PAX9 was mainly expressed in the nucleus (Figure 1A). The survival analysis revealed that CC patients with the high PAX9 expression had a better prognosis as compared to those with low PAX9 expression (5-year survival rate: 88% vs. 59%; p = 5.40E-04, Figure 1B). Besides, PAX9 in the CC tissues was significantly lower than that in the tumor-adjacent normal tissues, as analyzed by western blotting (Figure 1C). Furthermore, the lower expression of PAX9 was significantly correlated with FIGO stage, tumor size, infiltration depth, parametrium invasion, lympho-vascular space invasion, and tumor-positive lymph nodes of CC patients, instead of age, histology, and HPV type (Table 2).

image

Expression of PAX9 in cervical cancer (CC) tissues. (A, B) The database of Human Protein Altas showed that PAX9 was mainly expressed in the nucleus of CC tissues (A), and the survival analysis revealed that CC patients with higher expression of PAX9 have a better prognosis as compared to those with lower ones (B); and (C) western blotting showed that PAX9 was lower in the CC tissues than that in the tumor-adjacent normal tissues, as shown in example tissues. Data are expressed in mean ± SD, n = 142. ****p < 0.001 versus the tumor-adjacent normal tissues (paired t test)

TABLE 2. The correlation between PAX9 expression in tumors and clinicopathological characteristics of patients with cervical cancer Clinicopathological characteristics No. PAX9 expressions F/t p Histology Adenocarcinoma 33 0.123 ± 0.021 Squamous cell carcinoma 104 0.103 ± 0.010 Adenosquamous cell carcinoma 5 0.092 ± 0.041 1.609 0.204 HPV type Type 16 71 0.105 ± 0.012 Type 18 36 0.099 ± 0.016 Other type 26 0.121 ± 0.024 Negative 9 0.143 ± 0.048 0.134 0.939 FIGO stage ≤IB1 102 0.345 ± 0.101 >IB1 40 0.228 ± 0.078 6.576 8.90E-10 Tumor size ≤40 mm 109 0.336 ± 0.092 >40 mm 33 0.231 ± 0.121 5.354 3.44E-07 Infiltration depth ≤15 mm 109 0.337 ± 0.097 >15 mm 33 0.230 ± 0.105 5.473 1.99E-07 Parametrium invasion No 57 0.394 ± 0.077 Yes 85 0.257 ± 0.090 9.343 1.99E-16 Lympho-vascular space invasion No 95 0.355 ± 0.087 Yes 47 0.225 ± 0.095 8.096 2.48E-13 Tumor-positive lymph nodes No 123 0.325 ± 0.093 Yes 19 0.227 ± 0.156 3.874 1.64E-04 Abbreviation: FIGO, International Federation of Gynecology and Obstetrics. 3.2 PAX9 expression in CC cell lines

The expression of PAX9 in the human CC cell lines (C-33A, CaSKi, HeLa, and SiHa) and HCerEpiC was determined by qRT-PCR and western blotting. Consequently, in the CC cell lines, PAX9 mRNA and protein expression were decreased, as compared to HCerEpiC cells (all p < 0.05), but no significant difference was detected among CC cell lines (all p > 0.05, Figure 2A–C). To avoid a possibility of off-target effects, C-33A, CaSKi, HeLa, and SiHa cells were transfected with PAX9-overexpression lentivirus vectors, and the PAX9 expression was evaluated by qRT-PCR and western blotting. Then, we found that PAX9 overexpression could up-regulate the expression of PAX9 in C-33A, CaSKi, HeLa, and SiHa cells (all p < 0.05, Figure 2D–F).

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The expression of PAX9 in cervical cancer (CC) cell lines. (A–C) Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were employed to determine the gene (A) and protein expression (B, C) of PAX9 in human CC cell lines (C-33A, CaSKi, HeLa, and SiHa) and human cervical epithelial cells (HCerEpiC); ***p < 0.005 or ****p < 0.001 versus the HCerEpiC cells (t test); (D–F) qRT-PCR and western blotting were employed to determine the gene (D) and protein expression (E, F) of PAX9 in those human CC cell lines (C-33A, CaSKi, HeLa, and SiHa) transfected with PAX9-overexpression lentivirus vectors; ****p < .001 versus vector group (t test). Data are expressed in form of mean ± SD, n = 3

3.3 Effect of PAX9 overexpression on the proliferation and apoptosis of CC cell lines

Edu staining and Annexin V-FITC/PI staining indicated that PAX9 overexpression inhibited the proliferation and facilitated the apoptosis of human CC cell lines (C-33A, CaSKi, HeLa, and SiHa) (all p < 0.05, Figure 3). Besides, qRT-PCR was carried out to detect the mRNA expression of caspase-3, PARP, Bax, and Bcl-2, and we found that after transfection of PAX9 overexpression lentiviral vectors, human CC cell lines presented with up-regulations of caspase-3, PARP, and Bax, but the down-regulation of Bcl-2 (all p < 0.05, Figure 4).

image

Effect of PAX9 overexpression on the proliferation and apoptosis of cervical cancer (CC) cell lines. (A, B) EdU staining was employed to detect the proliferation of human CC cell lines after transfection of PAX9 overexpression lentiviral vectors; nuclei (blue) were counterstained with 4′-6-diamidino-2-phenylindole (DAPI); red staining indicated cells proliferation. The EdU-positive index was expressed as the percentage of positive cell number /total cell number. (C, D) Annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining was employed to detect the apoptosis of human CC cell lines after transfection of PAX9-overexpression lentiviral vectors; ****p < 0.001 versus the vector group (t test). Data are expressed in form of mean ± standard deviation (SD), n = 3

image

Effect of PAX9 overexpression on the apoptosis-related genes in cervical cancer (CC) cell lines determined by quantitative real-time polymerase chain reaction (qRT-PCR). ***p < 0.005 or ****p < 0.001 versus the vector group (t test). Data are expressed in form of mean ± standard deviation (SD), n = 3

3.4 Effect of PAX9 on the formation of subcutaneous tumors in xenograft models

It has been confirmed that PAX9 has a pivotal role in the proliferation and apoptosis of CC cell lines in vitro. To verify the in vivo effect of PAX9 on the biological function of CC cell lines, nude mice were subcutaneously injected with the CaSki cells transfected PAX9 overexpression lentiviral vectors and control vectors. Consequently, the growth and volume of tumor in nude mice of PAX9 group were slower or lower than those in the vector group, and the weight was also lower (Figure 5A–C). HE staining (Figure 5D) also demonstrated that xenograft tumors in mice of vector group presented with necrosis and multiple cancer nests, which was alleviate in the PAX9 group. In addition, we noted that in PAX9 group, the positive expression of Ki-67, cleaved-caspase-3, and Bax in the tumor tissues was much lower, while TUNEL and Bcl-2-positive expression was much higher than that in the vector group (Figure 5D–G).

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Antitumor effects of PAX9 in vivo in mice. (A) PAX9 suppressed the in vivo cervical cancer (CC) tumor formation; (B, C) The xenograft tumor (B) and weight (C) at 35th day after inoculation in nude mice of PAX9 group and vector group; (D) hematoxylin and eosin (HE) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and IHC staining to detect the expression of Ki-67, Cleaved-caspase-3, Bax and Bcl-2 in the xenograft tumors; (E, F), Comparison of the Ki-67 positive ratio (%, E) and TUNEL-positive ratio (%, F) between the PAX9 group and the vector group; (G) comparison of the positive rats of cleaved-caspase-3, Bax, and Bcl-2 between the PAX9 group and the vector group. Data are expressed in form of mean ± standard deviation (SD), n = 6. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 versus the vector group (t test)

4 DISCUSSION

Recent studies have shown that some members of PAX family are associated with the CC,25, 26 like PAX1. There was evidence suggesting that PAX1 methylation and HPV infection demonstrated the synergistic effects on the carcinogenesis of CC,27 which activated multiple phosphatases and inhibited kinase cascades in CC.28 PAX9, as a member from the same group with PAX1, was found to be dysregulated in a variety of malignant tumors, which showed a close relation with the development, progression, and prognosis of tumors according to the latest evidence. For example, PAX9 was found to be lost or down-regulated in human ESCC tissue samples.29 Also, PAX9 expression has shown a correlation with the better survival and stronger sensitivity to the radiotherapy.30 Zhaohui Xiong and his group, using GEO datasets, confirmed the down-regulation of PAX9 in human ESCC and OSCC.16 Furthermore, the Human Protein Atlas database, a comprehensive database providing the protein expression profiles for a large number of human proteins, presented as immunohistological images in 64 cell lines, 48 normal human tissues, and 20 tumor tissues, demonstrated that PAX9 was mainly expressed in the nucleus, and those CC patients with higher PAX9 expression had a better prognosis (https://www.proteinatlas.org/ENSG00000198807-PAX9/pathology/cervical+cancer#ihc). In this regard, the expression of PAX9 was detected in the CC tissues and the tumor-adjacent normal tissues in our study, and as a consequence, PAX9 was not surprisingly down-regulated in the CC, showing a significant association with the FIGO stage, tumor size, infiltration depth, parametrium invasion, lympho-vascular space invasion, and tumor-positive lymph nodes. Moreover, PAX9 expression was either lost or significantly reduced in the majority of invasive carcinomas and epithelial dysplasias, the latter representing precancerous lesions.29 Thus, PAX9 may act as a potential tumor suppression gene to affect the progression of CC.

In the current research, the in vitro experiments were then performed in different HPV-genotype CC cell lines and human cervical epithelial cells, consistent with the results from our clinical samples that the gene and protein expression of PAX9 was also down-regulated in the CC cell lines, without evident differences among these cell lines, which suggested that HPV infection may not be a factor affecting the expression of PAX9. To further clarify the influence of PAX9 in CC, multiple human CC cell lines (C-33A, CaSKi, HeLa, and SiHa) were selected to transfect with PAX9-overexpression lentiviral vectors to avoid a possibility of off-target effects, and we found that PAX9 overexpression reduced the proliferation and promoted the apoptosis of CC cell lines. Bcl-2, one of the mostly emphasized oncogenes in the current research concerning the cell apoptosis, is believed to be an oncogene suppressing the apoptosis,31 which could regulate the permeability of mitochondria to resist the release of cytochrome C into the cytoplasm, and then activate some zymogens for members in Caspase family, thereby inhibiting the apoptosis.32 Biological experiments confirmed that Bax and Bcl-2 could regulate cell apoptosis via forming the homo- or heterodimer, and Bax/Bcl-2 balance determines cell survival or apoptosis.33 PARP, as a cleaved product from caspase (a core link in cell apoptosis), plays a key role in the DNA-injury repair and cell apoptosis.34 Besides, in this study, PAX9 was found to be able to up-regulate the expression of caspase-3, PARP, and Bax and down-regulate the expression of Bcl-2 in CC cell lines, suggesting that inhibiting tumor cell apoptosis may also be one of the mechanisms affecting the progression of CC. In PAX9-deficient mice, Zhaohui Xiong et al. observed a significant increase in the number of forestomach tumors and the incidences of esophageal and forestomach lesions (papilloma, dysplasia, and SCC).16 Similarly, we also constructed the in vivo xenograft models and found that PAX9 overexpression could inhibit the growth of tumor, concomitant with decreases in the positive expression of Ki-67, cleaved-caspase-3, and Bax in the tumor tissues of PAX9 group, while the apoptosis increased, with up-regulation of Bcl-2 positive expression. These results indicated that PAX9, possibly via inhibiting the proliferation and accelerating the apoptosis of tumor cells, can inhibit the growth of tumor. However, this study still has some limitations: (1) Clinicopathological features included in this study should be further comprehensively summarized and (2) the possible mechanism, which suggested that the abnormal expression of PAX9 in CC may be correlated with the methylation,35-37 can only be further explored in the follow-up research due to the limited time and funds.

In conclusion, PAX9 was decreased in the CC, which was correlated with the major clinicopathological features of CC patients. Besides, PAX9 overexpression could inhibit the proliferation and accelerate the apoptosis of CC cell lines, with up-regulations of caspase-3, PARP, and Bax and the down-regulation of Bcl-2. Moreover, PAX9 could also inhibit the in vivo growth of xenograft tumors of CC. sPAX9 may be a potential therapeutic target for the treatment of CC.

CONFLICT OF INTEREST

All authors declare no conflict of interest.

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