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ORIGINAL ARTICLE
Year : 2017  |  Volume : 13  |  Issue : 5  |  Page : 801-806

Long noncoding RNA activated by transforming growth factor-β promotes cancer development and is a prognostic marker in cervical cancer


The Third Department of Gynecological Cancer, Hunan Cancer Hospital, Changsha 410013, Hunan, China

Date of Web Publication13-Dec-2017

Correspondence Address:
Tianfang Peng
The Third Department of Gynecological Cancer, Hunan Cancer Hospital, No. 283 Tongzipo Road, Changsha 410013, Hunan
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_256_17

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 > Abstract 


Background: Long noncoding RNAs (lncRNAs) have been highlighted as crucial elements in cancer biology. LncRNA activated by transforming growth factor-β (ATB) was identified to promote the development of multiple cancers and may serve as a potential therapeutic target in human cancers. However, to the best of our knowledge, the functional role of ATB in cervical cancer has not been verified yet.
Materials and Methods: The expression of lncRNA ATB was evaluated by quantitative reverse transcriptase-polymerase chain reaction assay in cervical cancer cell lines and clinical specimens. The clinical significance of ATB was statistically evaluated by analyzing the relationship between ATB overexpression and the clinicopathological characteristics of cervical cancer patients. Moreover, Kaplan–Meier analysis and log-rank test were conducted to investigate the role of ATB in the overall survival (OS) and progression-free survival (PFS) of cervical cancer patients or subgroup patients. Furthermore, univariate and multivariate analyses were adopted to identify the risk factors of OS and PFS of cervical cancer patients.
Results: LncRNA ATB was upregulated in cervical cancer cell lines and tissues (P < 0.05). Statistical analysis revealed that ATB overexpression was correlated with higher squamous cell carcinoma antigen level, larger tumor size, lymph node metastasis, and more advanced International Federation of Gynecology and Obstetrics (FIGO) stage of cervical cancer patients (P < 0.05). In addition, survival analysis showed that ATB upregulation was associated with poorer OS in cervical cancer patients and in subgroup patients without/with lymph node metastasis and with FIGO Stage I/II (P < 0.05). Furthermore, high ATB expression was defined as an independent risk factor of poorer OS and early recurrence of cervical cancer patients (P < 0.05).
Conclusion: LncRNA ATB correlates with the malignant phenotypes and poor prognosis of cervical cancer. ATB may serve as a promising prognostic marker and therapeutic target of cervical cancer patients.

Keywords: Cervical cancer, development, long noncoding RNA activated by transforming growth factor-β, prognosis


How to cite this article:
Cao W, Peng T, Zhou Y. Long noncoding RNA activated by transforming growth factor-β promotes cancer development and is a prognostic marker in cervical cancer. J Can Res Ther 2017;13:801-6

How to cite this URL:
Cao W, Peng T, Zhou Y. Long noncoding RNA activated by transforming growth factor-β promotes cancer development and is a prognostic marker in cervical cancer. J Can Res Ther [serial online] 2017 [cited 2019 Dec 12];13:801-6. Available from: http://www.cancerjournal.net/text.asp?2017/13/5/801/220465




 > Introduction Top


Cervical cancer is the second most commonly diagnosed cancer and the third leading cause of cancer-related death among females in less developed countries, with an estimated 527,600 new cases and 265,700 deaths worldwide in 2012.[1] Although the incidence and mortality rates of cervical cancer have declined over the past several decades worldwide, the age-standardized incidence rates had a significant upward trend in China.[2] Curative resection and radiotherapy are both effective for early-stage patients, and the overall 5-year survival rate approaches 90%. However, there are limited treatment options for patients with advanced diseases, with a 5-year survival rate of <40%.[3],[4],[5] Therefore, the molecular mechanisms of cervical cancer initiation and development should be further elucidated to found new diagnostic markers and therapeutic targets for improving the prognosis of cervical cancer patients.

Long noncoding RNAs (LncRNAs), defined as transcripts longer than 200 nucleotides that lack protein coding potential, have been investigated to participate in almost every aspect of cell biology, including regulation of protein-coding genes, maintenance of genomic integrity, genomic imprinting, messenger RNA (mRNA) processing, cell differentiation, and development.[6],[7] Moreover, lncRNAs are found to play essential roles in carcinogenesis based on their influence on chromatin state and methylation, stability of proteins, and complexes or acting as a sponge for microRNA inhibition.[8] Besides, accumulating evidence suggested that lncRNAs could affect cancer development by regulating cancer self-sustained growth signaling, insensitivity to growth inhibition, apoptosis avoidance, uncontrolled proliferation, angiogenesis, and metastasis.[9]

LncRNA activated by transforming growth factor-β (ATB), a novel transforming growth factor (TGF)-β-induced lncRNA, was reported to promote the invasion-metastasis cascade by mediating epithelial–mesenchymal transition (EMT) and associate with prognosis in hepatocellular carcinoma.[10] Further studies have revealed lncRNA ATB as a prognostic marker in gastric cancer, nonsmall cell lung cancer, and colon cancer.[11],[12],[13] Thus, lncRNA ATB is considered as a promising prognostic marker and therapeutic target of human cancers.[14] However, to the best of our knowledge, the functional role of lncRNA ATB in cervical cancer has not been elucidated at present.

In this study, the relative expression of ATB was investigated in cervical cancer cell lines and clinical specimens. The clinical significance of ATB in cervical cancer was also illuminated. Moreover, ATB was found to be correlated with poor prognosis of cervical cancer patients. Besides, univariate and multivariate COX regression analyses discovered ATB as an independent risk factor of the prognosis of cervical cancer patients.


 > Materials and Methods Top


Cell lines and clinical tissues

Cervical cancer cell lines (SiHa, HeLa, CaSki, MS750, and C-33A) and normal human cervical epithelial cell line (H8) were all obtained from American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (Sigma, USA) or RPMI 1640 (Sigma, USA) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 U/ml) according to the manufacturer's instruction. All the cell lines were incubated in humidified air at 37°C with 5% CO2.

A total of 187 human cervical cancer tissues and paired tumor adjacent tissues were collected at Hunan Cancer Hospital from 2008 to 2012. Clinical samples were immediately frozen in liquid nitrogen and kept at −80°C until analysis. None of the patients had received antitumor therapy before diagnosis. The diagnosis was made based on pathological results. Written informed consents were obtained from all patients. This study was approved by the Ethics Committee of Hunan Cancer Hospital.

RNA extraction and real-time polymerase chain reaction

TRIzol reagent was utilized to isolate total RNA from cervical cancer cell lines and tissues according to the manufacturer's instruction. Then, the extracted total RNA (2 μg) was subjected to a reverse transcription (RT) system for first strand complementary DNA (cDNA) synthesis using the Prime-Script one-step RT-polymerase chain reaction (RT-PCR) kit (Takara, Dalian, China). Subsequently, synthesized cDNA (2 μl) was used for the real-time PCR on the Applied Biosystems 7900HT Real-Time PCR System (Applied Biosystems, USA) with the SYBR PrimeScript RT-PCR kit (Takara, Dalian, China). All the reactions were performed in triplicate with glyceraldehyde 3-phosphate dehydrogenase serving as an internal control. Relative expression level of the target gene was calculated using the 2−ΔΔCT method. The following primer sequences were used: ATB forward, 5'-TCCCTGACTCCTCTATGGCATCTGTGG-3'; ATB reverse, 5'-CCTTTGCTTCCTCTTTTCTCATCTACTC-3'; GAPDH forward, 5'-GCCGCATCTTCTTTTGCGTCGC-3'; GAPDH reverse, 5'-TCCCGTTCTCAGCCTTGACGGT-3'.

Statistical analysis

The continuous data were presented as mean ± standard deviations and compared by Student's t-test. Chi-square test was applied to analyze the correlation between ATB expression and the clinicopathological features of cervical cancer patients. Kaplan–Meier method was conducted to analyze the overall survival (OS) and progression-free survival (PFS), and the survival difference between groups was evaluated by the log-rank test. Univariate and multivariate COX regression analyses were used to assess the significance of the survival data. P < 0.05 was considered as statistically significant. All the statistical analyses were conducted with the SPSS 19.0 software.


 > Results Top


LncRNA activated by transforming growth factor-β is overexpressed in cervical cancer

To define the functional role of ATB in cervical cancer, the expression of ATB was assessed in cervical cancer cell lines. As showed in [Figure 1]a, cervical cancer cell lines, including SiHa, HeLa, C-33A, MS750, and CaSki, exhibited much higher ATB expression levels than normal human cervical epithelial cell line, H8 (P < 0.05). Furthermore, ATB expression was detected by quantitative RT-PCR in 187 paired cervical cancer tissues, which found an obviously upregulated ATB expression level in cervical cancer tissues in comparison with tumor adjacent tissues (P < 0.05) [Figure 1]b. Collectively, these results indicated that lncRNA ATB functions an oncogenic role in cervical cancer.
Figure 1: Long noncoding RNA activated by transforming growth factor-β is upregulated in cervical cancer. (a) The expression of long noncoding RNA activated by transforming growth factor-β was examined in cervical cancer cell lines and normal human cervical epithelial cell line, H8, by qRT-PCR assay. (b) qRT-PCR assay was conducted to detect the expression of activated by transforming growth factor-β in tumor adjacent tissues and paired cervical cancer tissues. (c-f) The expression of activated by transforming growth factor-β was assessed by qRT-PCR assay in cervical cancer patients with different squamous cell carcinoma antigen levels (c), tumor size (d), lymph node metastasis (e), and International Federation of Gynecology and Obstetrics stages (f). P < 0.05 based on Student's t-test. qRT-PCR = Quantitative reverse transcriptase-polymerase chain reaction

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LncRNA activated by transforming growth factor-β overexpression correlates with cervical cancer progression

To extend our understanding of the clinical significance of ATB in cervical cancer, the correlation between ATB expression and clinicopathological features of cervical cancer patients was further investigated. As expected, patients with higher squamous cell carcinoma antigen (SCCA) level, larger tumor size, lymph node metastasis, and advanced International Federation of Gynecology and Obstetrics (FIGO) stage displayed significantly higher ATB expression levels [Figure 1]c,[Figure 1]d,[Figure 1]e,[Figure 1]f.

Moreover, the patients were classified into the high ATB expression group and the low ATB expression group based on the cutoff value, which was determined as the average relative ATB expression level (T/A value) in cervical cancer tissue as compared with that in paired corresponding adjacent tissue. Statistical analysis revealed that high ATB expression associated with higher SCCA level, larger tumor size, lymph node metastasis, and more advanced FIGO stage [Table 1]. Together, ATB overexpression promotes the development and metastasis of cervical cancer.
Table 1: Correlation between long noncoding RNA ATB expression and cervical cancer clinicopathological characteristics

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LncRNA activated by transforming growth factor-β may be a potential prognostic marker of cervical cancer patients

Survival analysis indicated that patients with high ATB expression had a significantly shorter OS (P < 0.001) or PFS (P < 0.001) than those with low ATB expression [Figure 2]a and [Figure 2]b.
Figure 2: High long noncoding RNA activated by transforming growth factor-β expression correlates with poor OS and PFS of cervical cancer patients and subgroup patients. (a and b) Log-rank test was adopted to investigate the OS (a) and PFS (b) differences between the activated by transforming growth factor-β low expression group and the activated by transforming growth factor-β high expression group of cervical cancer patients. (c-f) The OS differences between the activated by transforming growth factor-β low expression group and the activated by transforming growth factor-β high expression group in the cervical cancer patients without lymph node metastasis (c), with lymph node metastasis (d), with International Federation of Gynecology and Obstetrics Stage I (e), and with International Federation of Gynecology and Obstetrics Stage II (f) were analyzed. P < 0.05 based on log-rank test. OS = Overall survival, PFS = Progression-free survival

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Both lymph node metastasis and FIGO stage are critical factors influencing the prognosis of cervical cancer patients. Survival analysis of the subgroup patients without/with lymph node metastasis showed that patients with high ATB expression suffered a shorter OS (P < 0.05) [Figure 2]c and [Figure 2]d. Meanwhile, similar result was observed in the subgroup patients with FIGO Stage I or II (P < 0.001) [Figure 2]e and [Figure 2]f.

In addition, univariate analysis discovered high ATB expression as a risk factor of cervical cancer OS (hazard ratio [HR] =1.168, 95% confidence interval CI = 1.059–1.289, P = 0.002) and PFS (HR = 1.156, 95% CI = 1.052–1.271, P = 0.003). Further analyzing with multivariate analysis found that high ATB expression was an independent risk factor of cervical cancer OS (HR = 1.116, 95% CI = 1.005–1.241, P = 0.041) and PFS (HR = 1.114, 95% CI = 1.007–1.232, P = 0.036). All these results showed in [Table 2].
Table 2: Univariate and multivariate analysis of clinicopathologic features for overall survival and progression-free survival of cervical cancer patients

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Taken together, ATB upregulation is associated with poorer OS in cervical cancer patients and in subgroup patients without/with lymph node metastasis and with FIGO Stage I/II. High ATB expression is an independent risk factor of poorer OS and early recurrence of cervical cancer patients.


 > Discussion Top


The essential role of lncRNA ATB in cancer development has been revealed in multiple cancers. Yuan et al.[10] first discovered that ATB was induced by TGF-β and upregulated in hepatocellular carcinoma metastasis and associated with poor prognosis. Further investigation by silencing ATB expression with ultrasound-targeted microbubble destruction-mediated siRNA transfection verified ATB may be a potential therapeutic target of hepatocellular carcinoma metastasis.[15] Subsequently, ATB was reported by Saito et al.[12] to be an independent prognostic risk factor (HR = 3.50, 95% CI 1.73–7.44, P = 0.0004) in gastric cancer. In addition, high ATB expression was indicated to be significantly associated with clinical development and poor outcomes in colorectal cancer, nonsmall cell lung cancer, renal cancer, glioma and prostate cancer.[13],[16],[17],[18],[19] A meta-analysis conducted by Fan et al.[14] confirmed that high ATB expression was associated with OS (HR = 3.11, 95% CI: 2.24–4.32, P < 0.00001), disease-free survival (HR = 2.75, 95% CI: 1.73–4.38, P < 0.0001), recurrence-free survival (HR = 3.96, 95% CI: 2.30–6.81, P < 0.00001), lymph node metastasis (OR = 4.07, 95% CI 1.74–9.53, P = 0.001), distant metastasis (OR = 3.21, 95% CI 1.06–9.72, P = 0.04), and high tumor stage (OR = 2.82, 95% 1.98–4.00, P < 0.0001) in cancer patients that excluded pancreatic cancer patients. Thus, lncRNA ATB is highlighted as a promising prognostic marker and therapeutic target in human cancers.

The present study defined the role of ATB in cervical cancer. Our results found that ATB was upregulated in cervical cancer cell lines and clinical specimens. Statistical analysis between ATB expression and clinicopathological features revealed that ATB overexpression was correlated with clinical progression of cervical cancer patients. Moreover, increased ATB expression was investigated to be associated with poor survival and early recurrence. Furthermore, univariate and multivariate COX regression analyses showed that ATB upregulation was an independent risk factor of cervical cancer prognosis. These findings shed new light on utilizing ATB as a potential prognostic marker and therapeutic target of cervical cancer patients.

Mechanistically, ATB was discovered to induce EMT and invasion by upregulating ZEB1 and ZEB2 expression through competitively binding the miR-200 family in hepatocellular carcinoma.[10] In addition, ATB could promote organ colonization of disseminated tumor cells by binding interleukin (IL)-11 mRNA, increasing IL-11 mRNA stability, causing autocrine induction of IL-11, and then activating STAT3 signaling.[10] In breast cancer patients, ATB was revealed to promote trastuzumab resistance and invasion-metastasis cascade by competitively binding miR-200c, upregulating ZEB1 and ZNF-217, and then inducing EMT.[20] Xu et al.[19] reported that ATB could promote the growth of prostate cancer cells via mediating cell cycle regulatory protein expression levels. In addition, ATB stimulated EMT associated with ZEB1 and ZNF-217 expression levels via ERK and PI3K/AKT signaling pathways. Besides, ATB could increase proliferation through directly binding with miR-141-3p in gastric cancer, and it was concluded that the lnc-ATB/miR-141-3p/TGF-β2 feedback loop was closely related to gastric cancer.[21] Based on the above evidence, it can be summarized that the functional role of ATB varies among different cancers by affecting cancer proliferation or metastasis through various signaling pathways. The detailed mechanisms of ATB promoting cervical cancer progression deserve further investigation.


 > Conclusion Top


ATB is upregulated in cervical cancer and correlates with cancer development. Moreover, ATB overexpression predicts poor OS and early recurrence. Besides, ATB was identified as an independent risk factor of poor prognosis and early recurrence of cervical cancer patients. LncRNA ATB may serve as a novel prognostic marker and therapeutic target of cervical cancer patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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