Identification of genetic variation in the lncRNA HOTAIR associated with HPV16-related cervical cancer pathogenesis: Beyond the Abstract

Long non-coding RNAs (lncRNAs) are more than 200 nt long transcripts that do not code for any protein. Deregulation of lncRNA expression in various disease conditions, including cancers, has been well documented in the existing literature [1-3]. Therefore, expression deregulation of lncRNAs could serve as biomarkers for diseases, specifically when such alterations can be detected in blood. 

HOTAIR is one such lncRNA which shows deregulated expression in a variety of cancer types and its over-expression has been shown to associate with invasive and metastatic abilities of several epithelial cancers, including cervical cancer (CaCx) [4-7]. Alteration in HOTAIR expression has not only been detected in CaCx tissues but also in the blood of women with CaCx [8]. Thus, HOTAIR expression status could serve as an important biomarker for the identification of metastatic cervical tumors.

Moreover, several GWAS approaches and candidate studies have identified the association of genetic variations in the non-coding genome with disease development, specifically cancer [9-10]. A few candidate approaches have also successfully identified genetic variations in HOTAIR that associate with CaCx development and are important regulators of HOTAIR expression [11-13].

In this study, the aim was to identify the functionally relevant genetic variations within the HOTAIR gene that may be employed to differentiate between clinically distinct CaCx subtypes, i.e., those exhibiting high HOTAIR levels with molecular signatures of metastasis and those harbouring low HOTAIR expression levels and lacking such signatures. The existence of such CaCx subtypes was established in a previous study from our laboratory [14].We therefore sequenced the full HOTAIR gene using a deep sequencing approach employing Ion Torrent based amplicon sequencing.

We identified 27 single nucleotide variations (SNVs), of which four were novel. Among these, rs2366152C was significantly over-represented among low HOTAIR expressing HPV16 positive CaCx cases compared to HPV negative controls. rs2366152C also showed the propensity of secondary structure alteration and gain of a miR-22 binding site in the 3' LSD1-complex binding domain of HOTAIR. miR-22 was over-expressed in low HOTAIR CaCx cases compared to controls and its expression negatively correlated with HOTAIR and E7 expression in HPV16 positive cases and in an E7 expression vector transfected HPV negative CaCx-derived cell line (C33A), but remained unaltered in high HOTAIR cases compared to controls. Reduced luciferase activity of a HOTAIR rs2366152C expression plasmid in C33A cells through miR-22 co-transfection confirmed the ability of miR-22 to specifically target rs2366152C-harbouring HOTAIR lncRNA in CaCx cells, ultimately leading to its down-regulation. Our data thereby justifies the potential of rs2366152C as a marker for singling out CaCx cases lacking metastatic molecular signatures and also explains the functional inactivation of HOTAIR in these cases, including the mechanism of HOTAIR down-regulation.

This study, therefore, highlights the importance of variations within the non-coding human genome during disease development, justifying how such variations could serve as potential genetic markers not only of the expression deregulation of lncRNAs but also for identifying the clinical subtypes among the cancer samples. The study also epitomises the strength of genome-level analysis, which identify a large number of mutations within the non-coding genome that could possibly be important drivers/regulators of disease development and could be utilized for genetic screening towards identification of disease status and patient stratification.

 Written by: Sweta Sharma Saha# and Sharmila Sengupta#*
# National Institute of Biomedical Genomics, Netaji Subhas Sanatorium, 2nd Floor, P.O.: N.S.S, Kalyani-741251, West Bengal, India, Kolkata, India.

References:
[1] M.E. Dinger, P.P. Amaral, T.R. Mercer, K.C. Pang, S.J. Bruce, B.B. Gardiner, et al., Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation, Genome. Res. 18, 1433–1445 (2008)

[2] R.A. Gupta, N. Shah, K.C. Wang, J. Kim, H.M. Horlings, D.J. Wong, et al., Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis, Nature. 464, 1071–1076 (2010)

[3] M. Huarte, J.L. Rinn, Large non-coding RNAs: missing links in cancer?, Hum. Mol. Genet. 19, R152–R161 (2010)

[4] R. Kogo, T. Shimamura, K. Mimori, K. Kawahara, S. Imoto, T. Sudo, et al., Long Noncoding RNA HOTAIR Regulates Polycomb-Dependent Chromatin Modification and Is Associated with Poor Prognosis in Colorectal Cancers, Cancer. Res. 71, 6320–6326 (2011)

[5] X. Li, Z. Wu, Q. Mei, X. Li, M. Guo, X. Fu, et al., Long non-coding RNA HOTAIR, a driver of malignancy, predicts negative prognosis and exhibits oncogenic activity in oesophageal squamous cell carcinoma, Br. J. Cancer. 109, 2266–2278 (2013)

[6] M. Ishibashi, R. Kogo, K. Shibata, G. Sawada, Y. Takahashi, J. Kurashige, et al., Clinical significance of the expression of long non-coding RNA HOTAIR in primary hepatocellular carcinoma, Oncol. Rep. 29, 946–950 (2013)

[7] L. Huang, L.M. Liao, A.W. Liu, J.B. Wu, X.L. Cheng, J.X. Lin, et al., Overexpression of long noncoding RNA HOTAIR predicts a poor prognosis in patients with cervical cancer, Arch. Gynecol. Obstet. 290, 717-723 (2014)

[8] J. Li, Y. Wang, J. Yu, R. Dong, H. Qiu, A high level of circulating HOTAIR is associated with progression and poor prognosis of cervical cancer, Tumor Biol. 36, 1661-1665 (2015)

[9] V. Kumar, H.J. Westra, J. Karjalainen, D.V. Zhernakova, T. Esko, B. Hrdlickova, et al., Human disease-associated genetic variation impacts large intergenic non-coding RNA expression, PLoS. Genet. 9, e1003201 (2013)

[10] B. Hrdlickova, R. Coutinho de Almeida, Z. Borek, S. Withoff, et al., Genetic variation in the non-coding genome: Involvement of micro-RNAs and long non-coding RNAs in disease, Biochim. Biophys. Acta 1842, 1910-1922 (2014)

[11] W. Pan, L. Liu, J. Wei, Y. Ge, J. Zhang, H. Chen, et al., A functional lncRNA HOTAIR genetic variant contributes to gastric cancer susceptibility, Mol. Carcinogen. 55, 90-96 (2015) 

[12] Y. Xue, D. Gu, G. Ma, L. Zhu, Q. Hua, H. Chu, et al., Genetic variants in lncRNA HOTAIR are associated with risk of colorectal cancer, Mutagenesis.  30, 303–310 (2015).

[13] M. Du, W. Wang, H. Jin, Q. Wang, Y. Ge, J. Lu, et al., The association analysis of lncRNA HOTAIR genetic variants and gastric cancer risk in a Chinese population, Oncotarget 6:31 (2015)

[14] S. Sharma, P. Mandal, T. Sadhukhan, R. Roy Chowdhury, N. Ranjan Mondal, B. Chakravarty, et al., Bridging Links between Long Noncoding RNA HOTAIR and HPV Oncoprotein E7 in Cervical Cancer Pathogenesis, Scientific Reports 5, 11724 (2015