PVT1 Assumes Signifying Capacity in Cervical Cancer Diagnosis.

doi:10.21203/rs.3.rs-57365/v1

Download PDF

Research article

PVT1 Assumes Signifying Capacity in Cervical Cancer Diagnosis.

https://doi.org/10.21203/rs.3.rs-57365/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

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 significantly higher in CC samples than healthy controls (P<0.0001). Large tumor size (P=0.006), positive uterus infiltration (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 specificity 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 significantly increased in CC samples and the up-regulation of PVT1 could distinguish CC patients from healthy controls.

Cancer Biology

Oncology

PVT1

Cervical cancer

Diagnostic

ROC

Cervical cancer (CC) is considered to be the second most frequent malignancy in women worldwide, especially in developing countries and low-income districts [1-3]. As a malignant type of tumor, CC is found to be accompanied with high mortality and morbidity, and approximately 500,000 newly confirmed 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 efficiently 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 [13-15]. 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 [20-22].

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 confirmed 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 significant 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 significant (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 first classified 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 significantly affected by large tumor size (P=0.006), uterus infiltration (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 significance 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 specificity 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).

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 specificity for CC. So, we attempted to find some specific 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 confirmed 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 first 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 significantly high in CC samples compared with healthy controls, which agreed with and confirmed the previous results. The following Chi-square test showed that elevated expression of PVT1 was significantly contributed to large tumor size, positive uterus infiltration 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 final ROC analysis illustrated that high expression of PVT1 could be regarded as a potential diagnostic marker for CC with high sensitivity and specificity.

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-specific 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 define the role of PVT1 in CC. This finding could provide a novel research area for our further studies.

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.

Cervical cancer (CC)

plasmacytoma variant translocation 1 (PVT1)

receiver operating characteristics (ROC)

area under the curve (AUC)

World Health Organization (WHO)

Long non-coding RNAs (lncRNAs)

Federation of Gynecology and Obstetrics (FIGO).

Quantitative real-time PCR (qRT-PCR)

human papillomavirus (HPV)

chain antigen 19-9 (CA19-9)

carcino-embryonic antigen (CEA)

cancer antigen 125 (CA125)

Ethics approval and consent to participate

This study was supported by the Ethics Committee of Harrison International Peace Hospital and also has been carried out in accordance with the World Medical Association Declaration of Helsinki.

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.

Funding

Not applicable.

Authors’ contributions

S.C. design of the work; S.C. the acquisition, analysis, L.Z. interpretation of data; L.Z. the creation of new software used in the work; S.C. have drafted the work or substantively revised it. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Yu Q, Lou XM, He Y: Preferential recruitment of Th17 cells to cervical cancer via CCR6-CCL20 pathway. PloS one 2015, 10(3):e0120855.
Moshkovich O, Lebrun-Harris L, Makaroff L, Chidambaran P, Chung M, Sripipatana A, Lin SC: Challenges and Opportunities to Improve Cervical Cancer Screening Rates in US Health Centers through Patient-Centered Medical Home Transformation. Advances in preventive medicine 2015, 2015:182073.
Chitapanarux I, Tharavichitkul E, Nobnop W, Wanwilairat S, Vongtama R, Traisathit P: A comparative planning study of step-and-shoot IMRT versus helical tomotherapy for whole-pelvis irradiation in cervical cancer. Journal of radiation research 2015, 56(3):539-545.
Shen HW, Tan JF, Shang JH, Hou MZ, Liu J, He L, Yao SZ, He SY: CPE overexpression is correlated with pelvic lymph node metastasis and poor prognosis in patients with early-stage cervical cancer. Archives of gynecology and obstetrics 2015.
Liu L, Xia M, Wang J, Zhang W, Zhang Y, He M: CISD2 expression is a novel marker correlating with pelvic lymph node metastasis and prognosis in patients with early-stage cervical cancer. Med Oncol 2014, 31(9):183.
Marinescu S, Anghel R, Gruia MI, Beuran M: Involvement of reactive oxygen species in the mechanisms associated with cervical cancer specific treatment. Chirurgia (Bucur) 2014, 109(6):806-811.
Yousif NG, Sadiq AM, Yousif MG, Al-Mudhafar RH, Al-Baghdadi JJ, Hadi N: Notch1 ligand signaling pathway activated in cervical cancer: poor prognosis with high-level JAG1/Notch1. Archives of gynecology and obstetrics 2015, 292(4):899-904.
Fujiwara H, Shimoda A, Ishikawa Y, Taneichi A, Ohashi M, Takahashi Y, Koyanagi T, Morisawa H, Takahashi S, Sato N et al: Effect of providing risk information on undergoing cervical cancer screening: a randomized controlled trial. Archives of public health = Archives belges de sante publique 2015, 73(1):7.
Sun B, Ye X, Lin L, Shen M, Jiang T: Up-regulation of ROR2 is associated with unfavorable prognosis and tumor progression in cervical cancer. International journal of clinical and experimental pathology 2015, 8(1):856-861.
Behnamfar F, Azadehrah M: Factors associated with delayed diagnosis of cervical cancer in Iran--a survey in Isfahan City. Asian Pacific journal of cancer prevention : APJCP 2015, 16(2):635-639.
Wu Y, Wang YQ, Weng WW, Zhang QY, Yang XQ, Gan HL, Yang YS, Zhang PP, Sun MH, Xu MD et al: A serum-circulating long noncoding RNA signature can discriminate between patients with clear cell renal cell carcinoma and healthy controls. Oncogenesis 2016, 5:e192.
Cui D, Yu CH, Liu M, Xia QQ, Zhang YF, Jiang WL: Long non-coding RNA PVT1 as a novel biomarker for diagnosis and prognosis of non-small cell lung cancer. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2016, 37(3):4127-4134.
Xie Z, Chen X, Li J, Guo Y, Li H, Pan X, Jiang J, Liu H, Wu B: Salivary HOTAIR and PVT1 as novel biomarkers for early pancreatic cancer. Oncotarget 2016.
Gibb EA, Brown CJ, Lam WL: The functional role of long non-coding RNA in human carcinomas. Molecular cancer 2011, 10:38.
Perry MM, Tsitsiou E, Austin PJ, Lindsay MA, Gibeon DS, Adcock IM, Chung KF: Role of non-coding RNAs in maintaining primary airway smooth muscle cells. Respiratory research 2014, 15:58.
Ilboudo A, Chouhan J, McNeil BK, Osborne JR, Ogunwobi OO: PVT1 Exon 9: A Potential Biomarker of Aggressive Prostate Cancer?International journal of environmental research and public health 2016, 13(1):ijerph13010012.
Yu YH, Hu ZY, Li MH, Li B, Wang ZM, Chen SL: Cardiac hypertrophy is positively regulated by long non-coding RNA PVT1. International journal of clinical and experimental pathology 2015, 8(3):2582-2589.
Ding C, Yang Z, Lv Z, Du C, Xiao H, Peng C, Cheng S, Xie H, Zhou L, Wu J et al: Long non-coding RNA PVT1 is associated with tumor progression and predicts recurrence in hepatocellular carcinoma patients. Oncology letters 2015, 9(2):955-963.
Yang YR, Zang SZ, Zhong CL, Li YX, Zhao SS, Feng XJ: Increased expression of the lncRNA PVT1 promotes tumorigenesis in non-small cell lung cancer. International journal of clinical and experimental pathology 2014, 7(10):6929-6935.
Ding J, Li D, Gong M, Wang J, Huang X, Wu T, Wang C: Expression and clinical significance of the long non-coding RNA PVT1 in human gastric cancer. OncoTargets and therapy 2014, 7:1625-1630.
Zhang Z, Zhu Z, Zhang B, Li W, Li X, Wu X, Wang L, Fu L, Dong JT: Frequent mutation of rs13281615 and its association with PVT1 expression and cell proliferation in breast cancer. Journal of genetics and genomics = Yi chuan xue bao 2014, 41(4):187-195.
Takahashi Y, Sawada G, Kurashige J, Uchi R, Matsumura T, Ueo H, Takano Y, Eguchi H, Sudo T, Sugimachi K et al: Amplification of PVT-1 is involved in poor prognosis via apoptosis inhibition in colorectal cancers. British journal of cancer 2014, 110(1):164-171.
O'Brien DP, Sandanayake NS, Jenkinson C, Gentry-Maharaj A, Apostolidou S, Fourkala EO, Camuzeaux S, Blyuss O, Gunu R, Dawnay A et al: Serum CA19-9 is significantly upregulated up to 2 years before diagnosis with pancreatic cancer: implications for early disease detection. Clinical cancer research : an official journal of the American Association for Cancer Research 2015, 21(3):622-631.
Suzuki M: Targets in schizophrenia. Psychiatry and clinical neurosciences 2015, 69(5):241-242.
Iden M, Fye S, Li K, Chowdhury T, Ramchandran R, Rader JS: The lncRNA PVT1 Contributes to the Cervical Cancer Phenotype and Associates with Poor Patient Prognosis. PloS one 2016, 11(5):e0156274.
Liu E, Liu Z, Zhou Y: Carboplatin-docetaxel-induced activity against ovarian cancer is dependent on up-regulated lncRNA PVT1. International journal of clinical and experimental pathology 2015, 8(4):3803-3810.
Ji TT, Huang X, Jin J, Pan SH, Zhuge XJ: Inhibition of long non-coding RNA TUG1 on gastric cancer cell transference and invasion through regulating and controlling the expression of miR-144/c-Met axis. Asian Pacific journal of tropical medicine 2016, 9(5):508-512.
Yang P, Chen T, Xu Z, Zhu H, Wang J, He Z: Long noncoding RNA GAPLINC promotes invasion in colorectal cancer by targeting SNAI2 through binding with PSF and NONO. Oncotarget 2016.

Table 1. Relationship of PVT1 expression and clinical characteristics

Clinical features	Case	Expression		c²	P value
	NO.	High	Low
Age (years)				1.610	0.205
≦50	68	31	37
>50	45	26	19
Family history				3.207	0.073
No	49	20	29
Yes	64	37	27
HPV infection				2.025	0.155
Never	61	27	34
Ever	52	30	22
Histological type				1.983	0.159
SCC	53	23	30
AC	60	34	26
Tumor size (cm)				7.439	0.006
≦4	56	21	35
>4	57	36	21
Uterus infiltration				4.674	0.031
Absent	55	22	33
Present	58	35	23
FIGO stage				6.486	0.011
I,II	59	23	36
III,IV	54	34	20

SCC: squamous cell carcinoma; AC: adenocarcinoma.

HPV: human papillomavirus

Download PDF

Version 1

posted

You are reading this latest preprint version

PVT1 Assumes Signifying Capacity in Cervical Cancer Diagnosis.

Status:

Version 1

Abstract

Figures

Background

Materials And Methods

Results

Discussion

Conclusions

Abbreviations

Declarations

Reference

Tables

Status:

Version 1