LncRNA WT1-AS up-regulates p53 to inhibit the proliferation of cervical squamous carcinoma cells

We included 76 CSCC patients (all females, 20 to 63 years, 40.1 ± 6.1 year) from the 233 CSCC patients who were admitted by the Affiliated Tumour Hospital of Xinjiang Medical University between August 2010 and January 2014. Inclusion criteria: 1) the patients should be newly diagnosed CSCC patient by histopathological test, not recurrent CSCC; 2) the patients had not received any therapies for any clinical disorders within 3 months before this study. Exclusion criteria: 1) patients complicated with any other clinical disorders were excluded; 2) patients with a family history of malignancies were excluded; 3) patients with previous history of malignancies were excluded. HPV infections were detected by performing sensitive PCR. The results showed that 28 cases were HPV16 positive, 30 cases were HPV18 positive and 18 cases were negative for HPV. This study had been approved by Affiliated Tumour Hospital of Xinjiang Medical University Ethics Committee. All patients were informed with the whole operation protocol and signed informed consent.

All 76 CSCC patients were monitored for 5 years through telephone (or outpatient visit in some cases). The ones who were lost before the end of follow-up or died of other diseases, or accidence, such as traffic accidence were excluded from this study.

The cervical biopsy was applied to all the 76 CSCC patients before the initiation of any therapies. During biopsy, CSCC (cancer) and adjacent (within the region 5 cm around tumors) non-cancer tissues were obtained from all patients. All tissues were subjected to histopathological examinations, which were carried out by 3 experienced pathologists.

To study the effects of HPV, our study includes both SiHa, an HPV positive human CSCC cell line, and C-33A, an HPV negative human CSCC cell line. Cells were obtained from ATCC (USA). We received these two cell lines in January 2019 from ATCC. These two cell lines have been authenticated by STR analysis and morphology check. According to International Cell Line Authentication Committee C-33A was mislabeled as ovarian cancer cell line, but actually it is CSCC cell line. 10/% FBS was added into Eagle’s Minimum Essential Medium to cultivate SiHa and C-33A cells under the conditions of 5% CO₂ and 37 °C. Vectors (pcDNA3.1) expressing WT1-AS or p53 were provided by Sangon (Shanghai, China). Lipofectamine 2000 reagent (Invitrogen, USA) was used to transfect 2 μg WT1-AS or p53 expression vectors or pcDNA3.1 empty vectors (negative control, NC) into 10⁶ SiHa or C-33A cells. In this experiment, cells without any transfections were used as control (C).

RNAzol RT (Sigma-Aldrich, USA) was mixed with tissues (ground in liquid nitrogen before use) and cultivated SiHa and C-33A cells to extract total RNAs. After DNase I digestion, QuantiTect Reverse Transcription Kit (QIAGEN China, Shanghai, China) was used to carry out reverse transcriptions to synthesize cDNA. All qPCR mixtures were prepared using QuantiTect SYBR Green PCR Kit QIAGEN China, Shanghai, China) to detect the expression of WT1-AS and p53. Endogenous controls for WT1-AS and p53 were 18S rRNA and GAPDH, respectively. All reactions were replicated 3 times and Ct values were processed using the 2^-ΔΔCT method.

SiHa and C-33A were collected at 24 h after transfections, and 3 × 10⁴ cells were mixed with 1 ml Eagle’s Minimum Essential Medium (% 10 FBS) to prepare single-cell suspensions. To test cell proliferation ability, all cells were transferred to a 96-well plate, 100 μl per well. Under the conditions of 5% CO₂ and 37 °C, cells were cultivated and addition of 10 μl CCK-8 solution (Sigma,-Aldrich, USA) was performed every 24 h for 4 times. To reflect cell proliferation, OD values (450 nm) were measured.

SiHa and C-33A were collected at 24 h after transfections, and 10⁶ cells were mixed with 1 ml RIPA solution (Thermo Fisher Scientific) to extract total proteins. Protein samples with high quality were subjected to 10% SDS-PAGE gel electrophoresis after denaturing. After that, proteins were transferred to PVDF membranes through the semi-dry method, and 5% milk (non-fat) was used to incubate the membranes for 2 h at 22 °C. After that GAPDH (ab37168, 1:1500, Abcam) and p53 (ab131442, 1:1500, Abcam) primary rabbit polyclonal antibodies were used to incubate with the membranes overnight at 4 °C. After that, goat anti-rabbit IgG-HRP secondary antibody (MBS435036, 1:1500, MyBioSource) was used to further incubate with membranes at 22 °C for 2 h. Signals were developed using ECL (Thermo Fisher Scientific) and Image J v1.46 software was used to develop signals.

Experiments were repeated 3 times to calculate mean values. Correlations were analyzed by linear regression. The 76 CSCC patients were grouped into low (n = 40) and high (n = 36) WT1-AS level (in CSCC tissues) groups according to Youden’s index (cutoff value = 2.09). Based on survival data of two groups, survival curves were plotted using the Kaplan-Meier method and compared using log-rank test. The paired t-test was used to analyze differences between non-cancer tissues and CSCC tissues. One-way ANOVA and Tukey test were used to analyze differences among different groups of patients and different cell transfection groups. p < 0.05 indicated statistical significance.

WT1-AS in CSCC and non-cancer tissues of CSCC patients (n = 76) was detected by performing RT-qPCR. Among the 76 patients, 28 cases were HPV16 positive, 30 cases were HPV 18 positive and 18 cases were negative for HPV. Expression levels of WT1-AS were compared among these 3 groups by performing one-way ANOVA and Tukey test. It was observed that expression levels of WT1-AS in CSCC tissues (Fig. 1a) and non-cancer tissues (Fig. 1b) were not significantly different among 3 groups of patients. Expression levels of WT1-AS were compared between CSCC and non-cancer tissues by performing the paired t-test. It was observed that levels of WT1-AS were significantly lower in CSCC tissues than in non-cancer tissues (Fig. 1c, p < 0.05).

The 76 CSCC patients were grouped into low (n = 40) and high (n = 36) WT1-AS level (in CSCC tissues) groups. Based on survival data of two groups, survival curves were plotted using the Kaplan-Meier method and compared using log-rank test. It was observed that the overall survival rate of low WT1-AS level group was significantly lower than that of high WT1-AS level group (Fig. 2).

RT-qPCR was also performed to investigate the expression pattern of p53. Paired t-test analysis showed that levels of p53 mRNA were significantly lower in CSCC tissues than in non-cancer tissues of 76 CSCC patients (Fig. 3a, p < 0.05). Correlations between p53 mRNA and WT1-AS were analyzed by performing linear regression. In CSCC tissues, p53 mRNA and WT1-AS were significantly and positively correlated with each other (Fig. 3b). In non-cancer tissues, the correlation between these two factors was not significant (Fig. 3c).

WT1-AS and p53 expression vectors were transfected into SiHa and C-33A cells. It was observed that expression levels of WT1-AS and p53 were significantly increased at 24 h after transfections comparing to NC and C groups (Fig. 4a, p < 0.05). In addition, significantly up-regulated p53 was observed after WT1-AS over-expression at both mRNA and protein levels (Fig. 4b, p < 0.05), while p53 over-expression failed to affect WT1-AS (Fig. 4c). Moreover, p53 and WT1-AS over-expression resulted in the inhibited proliferation of CSCC cells (Fig. 4d, p < 0.05).