PVT1 Assumes Signifying Capacity in Cervical Cancer Diagnosis.

Background: Cervical cancer (CC) is the second most prevalent malignancy among women, which severely threatens patients’ health. The study was conducted to determine the diagnostic role of plasmacytoma variant translocation 1 (PVT1) in CC to improve patients outcomes. Methods: The qRT-PCR was used to determine the expression level of PVT1 mRNA in CC samples and healthy controls. Chi-square test was used to determine the clinical effects of patients’ features on PVT1 expression. The receiver operating characteristics (ROC) curve with the area under the curve (AUC) was used as a tool for assessing the diagnostic role of PVT1 expression in CC. Results: The PVT1 mRNA level was signicantly higher in CC samples than healthy controls (P<0.0001). Large tumor size (P=0.006), positive uterus inltration (P=0.031) and advanced FIGO stages (P=0.011) were contributed to the elevated expression of PVT1 level. However, there was no close relationship between PVT1 expression and other clinical parameters, including age (P=0.205), family history (P=0.073), positive HPV infection (P=0.155 and histological type (P=0.159). The ROC curve showed the optimal cutoff point for PVT1 was 2.325, providing the sensitivity and specicity of 85.84% and 72.15%, respectively. Moreover, the AUC was 0.856, suggesting PVT1 level could be regarded as a diagnostic biomarker in CC (P<0.0001, 95%CI= 0.803-0.909). Conclusion: In summary, the level of PVT1 mRNA was signicantly increased in CC samples and the up-regulation of PVT1 could distinguish CC patients from healthy controls.


Background
Cervical cancer (CC) is considered to be the second most frequent malignancy in women worldwide, especially in developing countries and low-income districts [1][2][3]. As a malignant type of tumor, CC is found to be accompanied with high mortality and morbidity, and approximately 500,000 newly con rmed CC cases and 270,000 dead CC cases are reported every year according to the World Health Organization (WHO) all around the world [4,5]. At present, the commonly used methods for CC treatments, including surgical resection, chemotherapy and radiotherapy, have been improved in recent years [6,7]. However, the prognosis of CC patients are still dismal, especially those with advances stages, among whom the overall survival rate might be less than 40% [8,9]. Moreover, it is reported that CC is the only malignant tumor that could be preventable and curable if diagnosed early [10]. As a result, early diagnosis of CC is of great importance in the prevention and treatment of this disease. Therefore, biomarkers that could e ciently and timely diagnose CC are in urgent need.
Long non-coding RNAs (lncRNAs), longer than 200 nucleotides in length, are a series of RNAs that are lack of the protein-coding capacity [11,12]. A growing number of studies have demonstrated that lncRNAs are involved in various biological processes, such as gene expression and transcription, cell proliferation and metastasis, apoptosis, and disease development [13][14][15]. PVT1, a member of lncRNA family, maps to human chromosome 8q24, and could encode about 12 exons and several transcripts [16,17]. Besides, as an oncogene, PVT1 has been demonstrated to stimulate cell proliferation, mobility and cell cycle and so on [18,19]. Otherwise, emerging evidence has proved that PVT1 is dysregulated in a variety of malignancies, including gastric cancer, breast cancer and colorectal cancer [20][21][22].
In the retrospective study, we wanted to investigate the expression of PVT1 mRNA in CC samples and healthy controls, and then to explore the role of PVT1 in the diagnosis of CC.

Patients and specimens
Sampling of this study consisted of 192 women, including 113 CC patients who were pathologically con rmed in Harrison International Peace Hospital, and 79 blood donors in the same hospital. All the patients were graded according to the protocols of the International Federation of Gynecology and Obstetrics (FIGO). They received no radio-or chemo-therapy before investigation. And those patients with cancers other than CC were excluded from our study. Blood samples were obtained from both CC patients and healthy controls and stored at -80℃ until use. This study was supported by the Ethics Committee of Harrison International Peace Hospital. And all participants had provided the informed consents in advance.

Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from CC samples and healthy controls with the RNeasy Mini Kit (Qiagen, Germany) following the instructions. Then the reverse transcription was conducted to synthesize cDNA with 5µl of total RNA using SuperScript II RNses H-Rverse transcriptase (Invitrogen). Then real-time PCR was performed with cDNA using the Universal PCR Master Mix (Applied Biosystems) under optimal conditions. GAPDH was taken as an internal reference to normalize the PVT1 mRNA expression. The 2 -∆∆Ct method was applied to calculate the expression of PVT1.

Statistical analysis
All data were analyzed by Sigmaplot 12.5 and GraphPad Prism 5.0 softwares. Student's t-test was adopted to compare the difference of PVT1 expression between groups. Chi-square test was carried out to explore the association between PVT1 expression and clinical characteristics of patients. The diagnostic performance of PVT1 in CC was estimated by the receiver operating characteristic (ROC) curve with the area under the curve (AUC). The difference was statistical signi cant with the P value of less than 0.05.

Up-regulation of PVT1 mRNA in CC samples
The expression of PVT1 mRNA in CC samples and normal controls was examined via qRT-PCR. The level of PVT1 mRNA in CC patients was 3.04±0.71 (mean±SD), which was obviously higher than that in healthy controls (2.00±0.65), and the difference was statistical signi cant (P<0.0001). (Figure 1)

Association of PVT1 expression and clinical parameters
To determine the effects of clinical parameters on PVT1 expression, Chi-square test was conducted. Patients were rst classi ed by the median expression of PVT1 (3.02) into high PVT1 group (n=57) and low PVT1 group (n=56). According to the results in Table 1, PVT1 expression was signi cantly affected by large tumor size (P=0.006), uterus in ltration (P=0.031) and high FIGO stages (P=0.011). However, there was no link between PVT1 expression and other clinical characteristics, such as age (P=0.205), family history (P=0.073, positive HPV infection (P=0.155) and histological type (P=0.159).

Diagnostic performance of PVT1 in CC
To calculate the diagnostic signi cance of lncRNA PVT1 in CC, the ROC curve was performed. As shown in Figure 2, the optimal cutoff value of PVT1 in CC was 2.325, giving the sensitivity of 85.84% and the speci city of 72.15%. Furthermore, the AUC was 0.856, indicating PVT1 could discriminate CC patients from healthy individuals (P<0.0001, 95%CI=0.803-0.909).

Discussion
CC is one of the most frequent gynecologic malignancies in female patients globally, and the incidence rate exists obvious differences between regions. In recent years, with the prevalence of sexually transmitted diseases and their induction factors, such as human papillomavirus (HPV), CC becomes a heavy burden for female human beings. Besides, the onset ages of CC patients become younger and younger. What's more, CC is reported to be the only malignancy that could be preventable and curative if it was found at early stages. Therefore, early diagnosis and timely treatment are of great importance to improve the survival rate of CC patients. Recently, tumor biomarkers, such as chain antigen 19-9 (CA19-9), carcino-embryonic antigen (CEA) and cancer antigen 125 (CA125) are more and more used in the diagnosis and prognosis of various diseases, including CC [23,24]. However, the biomarkers are lack speci city for CC. So, we attempted to nd some speci c and effective candidates for CC.
LncRNAs, which are becoming more and more prevalent in the life science research in recent years, have been demonstrated to be potential targets in detection and treatment of cancers [25]. It has been con rmed that there are thousands of lncRNAs in mammalian genomes [26]. And plenty of lncRNAs have been investigated in various malignant diseases. For example, Ji et al. have reported that lncRNA TUG1 was involved in the cell transference and invasion in gastric cancer through regulating miR-144/c-Met axis [27]. Yang et al. claimed that lncRNA GAPLINC enhanced invasion of colorectal cancer via binding to PSF and NONO [28]. These studies indicated that lncRNAs might be related with the development and progression of malignancies. PVT1, a lncRNA, as mentioned previously, was observed to be elevated in various cancers. And Iden et al. also found that PVT1 was increased in CC tissues compared with healthy controls [25]. Thereby, we were engaged in exploring the role of PVT1 in CC diagnosis in this study.
We rst measured the expression of PVT1 mRNA in CC samples and healthy controls using the qRT-PCR method. And we observed that the PTV1 mRNA expression was signi cantly high in CC samples compared with healthy controls, which agreed with and con rmed the previous results. The following Chisquare test showed that elevated expression of PVT1 was signi cantly contributed to large tumor size, positive uterus in ltration and advanced FIGO stages. All the above results suggested that PVT1 expression might be related with the occurrence and development of CC. So we further detected the link between PVT1 expression and CC diagnosis. The nal ROC analysis illustrated that high expression of PVT1 could be regarded as a potential diagnostic marker for CC with high sensitivity and speci city.
Compared with miRNAs, the structure of lncRNAs is more complex, which leads to the diverse action modes of lncRNAs on tumors and genes, and more subtle regulation of the life processes. So far, the precise mechanism of PVT1 on CC tumorigenesis is still not well understood. In fact, multiple studies have provided the mechanistic data of PVT1 and suggested that it exerts its effect in a cell-type or disease-speci c manner. For example, PVT1 is reported to locate at about 55 kb downstream of the well known oncogene MYC, and is found to share close relationships with MYC in various cancers [29,30]. Thus , we sought to be better de ne the role of PVT1 in CC. This nding could provide a novel research area for our further studies.

Conclusions
In conclusion, PVT1 mRNA was highly expressed in CC samples compared with healthy controls. And the we could distinguish the CC patients from healthy individuals with the up-regulated expression of PVT1. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.

Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Competing interests
The authors declare that they have no competing interests.   Figure 1 The expression levels of PVT1 mRNA in CC samples and healthy controls were examined using qRT-PCR. The result showed that PVT1 mRNA was obviously elevated in CC patients (P<0.0001).