Novel Variant Identified in the Enhancer Region of Host Transcription Factor, BRN3A, is a Significant Risk Factor for HPV-Induced Uterine Cervix Cancer

Abstract

Among the HPV-mediated cervical cancers, cellular factor BRN3A has gained considerable attention due to its role in promoting an anti-apoptotic cellular environment and in facilitating epitheliotropic transformations of the host. The majority of previous studies looked at BRN3A's molecular characteristics; however, the possibility of genetic variations in BRN3A's auto-regulatory region in relation to cervical cancer risk has been underestimated until now. In a retrospective study in the Eastern UP population, India, we detected genetic variations in the cis-regulatory proximal enhancer region located around 5.6 kb upstream of transcription start site of BRN3A. Our analysis of PCR and DNA sequencing confirmed this novel SNP (BRN3A g.60163379A>G) within the auto-regulatory region of BRN3A. As compared to control subjects, cancer cases exhibited a 1.32-fold higher allele frequency (χ2 = 6.315, p = 0.012). In homozygous (GG) but not in heterozygous conditions, odds ratio (OR) analysis suggests a significant association of cancer risk with the SNP (OR = 2.60, p ≤ 0.004). We further confirmed using the functional analysis that this SNP increased the luciferase gene activity in HPV-positive cervical cancer SiHa cells that were exposed to progesterone. As a result of the association of polymorphisms in a non-coding region of an oncogene with increased cancer risks, we are suggesting that this genetic variation in non-coding region can be used in prediction, diagnosis, or predicting the progression of the disease.

Keywords: BRN3A; BRN3A proximal enhancer region; Cervical cancer; HPV; India; g. 6016 3379A >G.

Fig.1

Proximal enhancer region of BRN3A shows novel A>G variation in cervical tissue samples. Schematic representation of BRN3A regulatory enhancer regions and the novel variation within 302 bp proximal enhancer region through DNA sequencing, (a) Black rectangular box represents a sequence of 302 bp proximal enhancer region exhibiting multiple autoregulatory BRN3A protein binding sites (underlined sequences) and novel g.60163379A>G variation (mutation site shown in black circle) located 5692 bp from the transcription start site (b) Representative electropherogram of the automated sequencing highlights a single nucleotide substitution, g.60163379A>G (represented through black arrow) at 10 bp from 1st BRN3A binding site (Prox1) in the proximal enhancer of BRN3A

Fig.2

Genotyping study through RFLP, ARMS-PCR and Sangar sequencing. Digestion pattern of HpyCH4III restriction endonuclease within 221 bp segment of BRN3A proximal enhancer region. (a) Schematic presentation of HpyCH4III restriction endonuclease map in the reverse direction along with fragment size in wild-type and mutant variant. Mutation created an extra HpyCH4III restriction endonuclease digestion site. (b) Agarose gel image showing the 221 bp PCR product digested with HpyCH4III. Wild-type segment with A allele yields two DNA fragments of 142 bp and 79 bp (shown with white arrow in wild-type and absent in mutant) whereas, a mutant sequence containing G allele produces three DNA fragments of 142 bp, 61 bp, and 18 bp. (c) A 227bp ARMS-PCR amplified DNA product resolved on 2% agarose gel in different lanes with wild-type and mutant samples. In odd lane PCR products showing band with wild type specific forward primer whereas in even lanes amplified PCR product displaying bands with mutant specific forward. Interpretation of three genotype AA, GG and AG is based on the bands appeared in odd and even lane. Heterozygous (AG) is also confirmed through sanger DNA sequencing with reverse primer, here sample 2 is used for validation. DNA sequencing was done with reverse primer and thus chromatogram displaying reverse complementary sequences where T is indicating for A nucleotide and C is indicating for G nucleotide

Fig. 3

Distribution of SNP g.60163379 A>G variant in different ethnic group. Graph showing the distribution of SNP g.60163379 A>G variant in different ethnic group in the world retrieved through GnomAD database and compared with the control sample of the present study

Fig. 4

Cloning strategy for the wild-type (A) and mutant (G) variants in pGL3 promoter vector for the functional characterization of SNP g.60163379 A>G. Wild-type (g.60163379A) and mutant (g.60163379G) variant clones were generated and confirmed through Sanger sequencing shown in the electropherogram. The wild-type and mutant segments were cloned upstream of SV40 promoter and luciferase gene. Due to A>G point mutation, creation of any functional binding site was measured by differential luciferase activity in comparison to wild-type

Fig. 5

Functional characterization of SNP g.60163379 A>G variant through luciferase assay in cervical cancer cells. Dual-luciferase assay was performed after the transfection of wild-type and mutant clones in SiHa cells. Progesterone was given externally 6 h before harvesting the cells in different concentrations (1 µM, 2.5 µM, and 5 µM). The bar diagram represents the relative luciferase activity of the wild-type and mutant variant clones transfected in SiHa cells under different concentrations of progesterone. Each bar is showing the mean (±) SE of merged data of at least three independent experiments with three replicates. Groups compared are connected by a line displaying significance value above the bar. The table below the graph showing the relative mean luciferase activity in wild-type and mutant under given progesterone concentrations