Identification of genetic variation in the lncRNA HOTAIR associated with HPV16-related cervical cancer pathogenesis.
OBJECTIVE
Previously, over-expression of the long noncoding RNA (lncRNA) HOTAIR has been found to be associated with the invasive and metastatic capacities of several epithelial cancers, including cervical cancer (CaCx). Here, we aimed at identifying functionally relevant genetic variants that may be employed to differentiate between clinically distinct CaCx subtypes, i.e., those exhibiting high HOTAIR levels and molecular signatures of metastasis and those lacking such signatures in the presence of low HOTAIR expression levels.


METHODS
Genomic DNA isolated from various cervical tissue samples (characterized by histopathology and HPV status) was used for HOTAIR amplicon sequencing, followed by validation of the findings by Sanger sequencing. The impact of the genetic variants found on the secondary structure of HOTAIR and the concomitant alterations in miRNA binding sites were determined through in silico analysis, followed by miRNA expression analysis by quantitative real-time PCR and confirmation of miRNA binding using a luciferase reporter assay.


RESULTS
We found that rs2366152C was over-represented [ORage-adjusted= 2.58 (1.23-5.57); p = 0.014] in low HOTAIR expressing HPV positive CaCx cases compared to HPV negative controls. This genetic variant showed the propensity of a secondary structure alteration and gain of a miR-22 binding site in HOTAIR, which was found to be concordant with miR-22 over-expression in low HOTAIR CaCx cases compared to controls. We found that miR-22 expression negatively correlated with HOTAIR and E7 expression in HPV16 positive cases and in an E7 transfected HPV negative CaCx-derived cell line (C33A), but was not altered 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.


CONCLUSIONS
Our data indicate that rs2366152C not only has the potential to serve as a marker for singling out CaCx cases lacking metastatic molecular signatures, but also to explain the functional inactivation of HOTAIR in these cases, including the mechanism of its down-regulation.
National Institute of Biomedical Genomics, Netaji Subhas Sanatorium, 2nd Floor, P.O.: N.S.S, Kalyani, 741251, West Bengal, India.
Sweta
S
Sharma Saha
Department of Gynecology, Saroj Gupta Cancer Centre and Research Institute, Kolkata, India.
Rahul
R
Roy Chowdhury
Department of Gynecology, Saroj Gupta Cancer Centre and Research Institute, Kolkata, India.
Nidhu Ranjan
NR
Mondal
Department of Gynecology, Saroj Gupta Cancer Centre and Research Institute, Kolkata, India.
Biman
B
Chakravarty
Department of Gynecology, Saroj Gupta Cancer Centre and Research Institute, Kolkata, India.
Tanmay
T
Chatterjee
Sri Aurobindo Seva Kendra, 1H, Gariahat Road (S) Jodhpur Park, Kolkata, 700068, West Bengal, India.
Sudipta
S
Roy
National Institute of Biomedical Genomics, Netaji Subhas Sanatorium, 2nd Floor, P.O.: N.S.S, Kalyani, 741251, West Bengal, India. ssg1@nibmg.ac.in.
Sharmila
S
Sengupta
D000328:Adult; D000368:Aged; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology* / Q000821:virology; D015536:Down-Regulation; D005260:Female; D052162:Human papillomavirus 16; D006801:Humans; D008875:Middle Aged; D030361:Papillomavirus Infections / Q000150:complications; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D062085:RNA, Long Noncoding / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000473:pathology* / Q000821:virology
C561566:HOTAIR long untranslated RNA, human; D062085:RNA, Long Noncoding
Cervical cancer; HOTAIR; HPV16 E7; SNP; lncRNA
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