Introduction
Cervical cancer is the fourth most frequent cancer in women worldwide with 570,000 new cases in 2018 representing 6.6% of all female cancers. Approximately 90% of deaths from cervical cancer occurred in low- and middle-income countries. In India, about 60,078 cervical cancer deaths occur annually (estimates for 2018), responsible for 7.5% of all female mortality from cancer. Cervical cancer ranks as the second leading cause of female cancer deaths in India in the 15 to 44 years age group.1
Cervical carcinogenesis is a multi-step process associated with refractory infection by high-risk human papillomavirus (HPV) types.2,3 This includes the transformation of normal cervical epithelium to cervical intraepithelial neoplasia (CIN), which progresses to invasive cervical carcinoma of the 200 HPV types known till date, fifteen (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) considered as high-risk types are associated with cervical cancer and precancerous lesions.4,5
Almost every sexually active individual will acquire at least one high-risk HPV infection during their lifetime. Fortunately, the majority of HPV infections are eradicated by the host immune system within 1-2 years of acquisition, and only <1% of infected people develop HPV mediated cancers.
Persistent infection with an oncogenic HPV type (especially HPV-16 and HPV-18) is the most important risk factor for progression to high-grade dysplasia and invasive cancer.6 Studies using quantitative type-specific PCR for high-risk HPV and low-risk HPV have shown that HPV-16 can attain very high viral loads compared to the other types.7 The integration of the high-risk HPV (hrHPV) DNA results in the constitutive expression of its oncogenes E6 and E7. HPV E6 oncoprotein binds to the tumor suppressor protein p53 and directs its ubiquitin-mediated proteolytic degradation whereas E7 binds to and inactivates another cellular tumor suppressor protein Rb, thereby interfering with the cell cycle control, consequently oncogenic growth.8
Protective immunity results from the interplay of nonspecific innate immunity and antigen-specific adaptive immunity. The innate immune system senses “ danger ” via signals from molecules that would normally not be found in the human body, such as damaged tissue, repetitive surface structures of bacterial cell walls, or DNA sequences containing typical viral sequence motives. These structures are recognized by pattern recognition receptors, such as e.g. Toll-like receptors (TLRs). Sentinel cells, such as dendritic cells (DCs) or Langerhans cells (LCs) in the skin and mucosa, continuously screen the environment and – if triggered - coordinate innate immune effectors and the initiation of an adaptive immune response.
HPV has evolved to evade human immune detection in multiple ways to establish an infection and maintain a persistent life cycle that leads to viral reproduction. This persistence is the greatest risk factor for the development of HPV-mediated invasive malignancies. The most important HPV immune evasion mechanism is to become invisible to the host immune system by not triggering any danger signals, such as cytolysis, cytopathic cell death, or inflammation. Suppression of the interferon response, resistance to immune-mediated apoptosis, and down-regulation of adhesion molecules for APCs, and active MHC class I down-regulation and impaired antigen presentation also play a critical role in HPV immune evasion.
Toll-like receptors (TLRs), a family of evolutionarily conserved pathogen recognition receptors, are emerging as key players in the pathophysiology of a host of human diseases, including cancer. TLR9 recognizes unmethylated CpG motifs present in bacteria and viruses. Human B cells can be activated by stimulation of TLR9 and results in innate immune responses in preclinical tumor models and in patients.9,10
Single nucleotide polymorphisms are the most common form of genetic variants in human genome, some of which can influence the susceptibility to human diseases including cancer. TLR 9 gene polymorphisms appear to have considerable role in disease susceptibility, including cancers.. The present study aims to identify the role of TLR 9 C2848T (rs352140) gene polymorphism in cervical cancer susceptibility in East Indian women.
Aims
To understand the role of innate immune response on the development of HPV related cervical cancers.
Objectives
To estimate the prevalence of HPV16/18 infections in various categories of cervical samples (grouped according to histopathology, presence/absence of HPV infection).
To evaluate whether TLR9 expression is deregulated among the Cancer Cervix cases compared to non malignant controls.
To determine whether the polymorphism such as rs352140 (C/T), within the TLR9 promoter, is associated with the risk of cervical cancer and to identify whether the significantly overrepresented genotype among cervical cancer compared to controls, correlate with TLR9 deregulation as well.
Materials and Methods
Recruitment of subjects
This is a case - control study, conducted in the period from October 2016 to April 2017. The study was conducted, on East Indian women of West Bengal. Study sample included 57 women; case group comprised 33 wom en, control group comprised 24 women, of which 15 were found to have HPV infection and 9 were HPV – ve. Women with history of chronic or recurrent pruritus vulvae and leucorrhoea, persistent abnormal vaginal bleeding (like post coital, post menopausal bleeding or menorrhagia) and persistent cervical lesions (cervical hypertrophy, erosion, ulceration, cervical growth noted after speculum examination of cervix) were included in the study. Women with history of recent childbirth, miscarriage/abortions (within previous 4 months), menstruation at the time of visit, prior treatment for cervical malignancy, pregnant & unmarried women were excluded from the study. A questionnaire was used to collect information from patients on clinical history, demographic data, life style and reproductive factors. Intervention was per speculum cervical examination, with cervical smear or cervical punch biopsy. Clinical examination findings and histopathological report of cervical biopsy samples, (noted from hospital records) were recorded in the questionnaire form.
Ethical consent
All samples were collected from the study participants with informed consent approved by the Institutional Ethical Committee.
Sample collection
Ecto-cervical and endo-cervical tissue samples were collected from subjects for cytopathological examination.
Detection of HPV positivity
DNA was isolated from all cervical tissue samples using the QIAamp DNA mini kit according to the manufacturer’s protocol. All samples were screened for the presence of HPV infection by PCR, using L1 consensus primers: MYO11 and MYO9. L1 negative samples were reamplified with nested GP 5/6 primers for further HPV screening. The amplified PCR products were subject to electrophoresis on a 2% agarose gel and amplified bands (150bp) were visualized under UV light after staining with ethidium bromide. The primer sequences are shown in Table 1. The samples that were negative for both the primers were considered to be HPV -ve. The samples that were positive for either primer were considered to be HPV +ve.
Table 1
List of primers used with amplicon length
Detection of HPV type 16 and 18
HPV +ve samples were typed by specific primers homologous to the E6 region of HPV-16 and 18. HPV 18 +ve were few, so the study was concentrated on HPV 16 +ve and HPV 16 –ve samples. Samples which were histopathologically confirmed squamous cell carcinoma were classified as the case group, non-malignant HPV-ve samples were classified as the control group and non-malignant HPV 16 +ve samples were classified as the intermediate group. Detection of genetic polymorphism of TLR9 gene rs352140 (C/T)
PCR amplification of DNA samples followed by RFLP (restriction fragment length polymorphisms).
PCR amplification of DNA samples
PCR amplification of DNA samples was done using 100ng of DNA. TLR9 gene (promoter region) was amplified. (Figure 1)
Performing RFLP (restriction fragment length polymorphisms)
The PCR fragments were digested by endonuclease BST UI (CG/CG) New England BioLabs, (Ipswich, USA). TLR9 C allele cleaved into 227 & 133bp fragments. TLR9 T allele remained uncut. DNA fragments were separated by gel electrophoresis on 2% agarose gel, visualized by ETBr staining. If one band was visualized, no allele was cut, the homozypous TT genotype was inferred; if two bands were seen, then homozygous CC genotype was inferred; if three bands were seen, then heterozygote CT genotype was inferred. (Figure 2)
Homogentisate preparation
The sample tissues was minced using sterile mortar and pestle. RLT buffer was added to the 1.5ml tubes and minced tissue was stored in RLT (lysis buffer) mix for overnight storage at -80ºc.
RNA isolation from tissue samples
RNA isolation was done using the RNeasy Mini kit, following the instructions of the manufacturer.
CDNA preparation
cDNA was prepared using random hexamer primers, as well as oligo –dT primers and reverse transcriptase enzyme (Fermentas). All cDNA samples were stored at -80°C for long term usage in small aliquots.
Estimation of TLR9 gene expression – by RT PCR (real time PCR)
2 samples of 0.5μl cDNA were taken in 2 PCR tubes, of which 1 was diluted 5 times with milliQ water. Syber green mix was prepared according to manufacturer’s instruction, using the following primers FP: 5’ TGG GAA GGG ACC TCG AG
RP: 5 ’ CAG GGT AGG AAG GCA GGC A3’
A 96 well plate was used – each well was loaded with cDNA 0.5μL (in replicate - undiluted and 5X diluted) and SYBR Green (Thermo Fisher Scientific) mix (4.5μl), which comprised syber green (2X) - 2.5 μl, 100ng/ml of primers, each of volume 0.05μl. Each well was carefully marked by sample number. After loading, the plate was sealed with paraffin film. The plate was centrifuged at 2000 rpm (pulse centrifuge), then loaded in thermocycler, setting the programme of TLR9 real time PCR in 3 stages- Stage I (denaturation) at 95°C for 5 min., Stage II of 40 cycles with denaturation at 95°C (30sec), annealing at 60°C (30sec) and elongation at 72°C (60sec) and a Stage III of dissociation - 95°C (15sec.) followed by 60°C (15sec) followed by 95° C (15sec).
(Figure 3,Figure 5) The (melting temperature) Tm of TLR9 gene = 86°C, Tm of GADPH gene = 79.7°C.
Statistical analysis
Categorical variables were expressed as Number of patients and percentage of patients and compared across the groups using Pearson’s Chi Square test for Independence of Attributes. The software used was SPSS version 20. Kolmogorov Smirnov Test (K-S TEST), was performed to determine whether the continuous variables in each sample categories followed normal distribution. The continuous variables did not follow normal distribution. Thus, Mann-Whitney U Test (SPSS version 20) was used to compare the median values between two different groups. An alpha level of 5% has been taken, i.e. if any p value is less than 0.05 it has been considered as significant.
Results
Samples belonging to case group (33), control group (9) and intermediates (15) were analysed. The TLR9 mRNA expression level was quantified by syber green assay using real time PCR. The real time PCR data, corresponding to TLR9 expression and GAPDH expression was represented as CT values, where CT defines the threshold cycle of PCR at which the amplified product is first detected. The delta CT value of TLR9 mRNA expression was obtained from the difference in CT value of TLR9 mRNA expression and CT value of GAPDH (housekeeping gene).
All the cDNA samples showed the presence of GAPDH housekeeping gene (quality control). The mean CT value of GAPDH did not differ significantly between case, (mean= 18.33+/- 1.78) intermediate (mean = 19.99 +/- 1.78) and control samples (mean = 19.57 +/- 0.03), suggesting a similar amount of input DNA for all 3 categories.
The delta CT values of TLR9 mRNA expression were compared between cases (n= 33) and controls (n= 15) but not found to be significantly different (p value 0.134). Similar analysis was carried out between case and intermediate samples, again no significant difference in their delta CT value of TLR9 mRNA expression was noted ((p value 0.857). Analysis between intermediate and control values of delta CT TLR9 mRNA showed no significant difference (p value 0.3734). The median values of delta CT values of TLR9 mRNA expression are shown in Figure 7.
As no significant difference in ΔCT values were found in TLR9 expression across categories, fold changes were used. To express the fold change in terms of 2-ΔΔCT, the median delta CT values of TLR9 mRNA expression of case, intermediate and control were used. Considering control value as calliberator, the TLR 9 expression of cases were 4.0062 folds that in the control group. Similarly TLR 9 expression of intermediates was 3.1156 folds that in the control group and TLR 9 expression of case was 1.2907 folds that in the intermediate group. (Table 2)
Table 2
Fold-changes and p-value testing for significance of the median values of TLR9 across different categories of samples
TLR9 mRNA expression was studied using median ∆CT values, comparing case and control, with respective genotypes, taking control group as calibrator. There was increased expression of TLR9 mRNA in the TT genotype among case group (1.496 fold) compared to controls. There was increased expression of TLR9 mRNA in the CT genotype among case group (3.578 fold) compared to controls. Table 3.
Table 3
Fold-changes and p-value testing for significance of the median values of TLR9 using different genotypes across different categories of samples
The case samples (n=33) were further studied according to their genotype. The relative TLR9 mRNA expression of the TT, CC and CT groups were 11.5257, 11.2337 and 11.8115 respectively. The fold change was calculated between TT and CC, with CC as calliberator. TLR9 expression of TT was decreased by 1.224 folds compared to the CC group. The fold change was calculated between CC and CT, with CC as calliberator-TLR9 expression of CT was decreased by 1.2191 folds compared to the CC group. Table 4
Discussion
TLR9 mRNA expression was analyzed in our study, using fold change of expression values. Greater the median ∆ Ct value of TLR9 mRNA, lesser will be the expression of the TLR9 gene. Our study showed increased expression of TLR9 in malignant group, compared to the control group. This indicates that TLR9 expression is upregulated in cervical cancer. TLR9 expression was also increased in the HPV +ve nonmalignant group, compared to HPV –ve controls, suggesting that TLR9 is upregulated in persistent HPV +ve infection. Our findings have been corroborated by other studies in different ethnic populations. Studies by Hasimu et al on Uighur women in China showed that expression of TLR9 can be upregulated by HPV 16 infection in CIN and in cervical squamous carcinoma cells.11 Lee et al studied Korean women in 2007and showed increased expression of TLR9 in cervical cancer patients.12 Hasan et al also showed increased expression of TLR9 in persistent and recurrent HPV infection;13 these findings are similar to our findings. Chen et al investigated the TLR9 - 1486T/C (rs187084), a potentially functional variant located in the promoter region, which is close to the region that interacts with HPV16 E6 and E7 oncoproteins.14 They found a significant increase in cervical cancer risk among the Chinese women carrying this variant in the TLR9 gene.
Hasimu et al found that the expression of TLR9 can be upregulated by HPV16 infection in CIN and in cervical squamous carcinoma cells.11 They also suggested that TLR9 may play important roles in the development and progression of CIN and cervical carcinoma. In contrast, Pandey et al. showed that the TT genotype of TLR9 (rs352140) displayed borderline significance in increased risk for advanced cervical cancer in a North India population.15
Correlating TT, CT, CC genotype with TLR9 expression analysis across malignant group and control group, there was increased expression of TLR9 among TT genotype and CT genotype compared to CC genotype (Statistically insignificant, p value >0.05, perhaps can be attributed to the low sample size). Our study showed that TLR9 C2248T polymorphism causes upregulation of TLR9 expression among cervical cancer patients. Our studies suggest that the TLR9 C2848T (rs352140) polymorphism may be a risk factor of cervical cancer in East Indian women.
Summary
Genetic variations such as single nucleotide polymorphisms (SNPs) greatly influence innate immune responses towards pathogenic challenges and disease outcome; therefore, a range of susceptibility to infections appears among people, with some of them being predisposed to certain infections while others are being protected. Several single-nucleotide polymorphisms (SNPs) within the TLR genes have been associated with altered susceptibility to infectious, inflammatory, and allergic diseases, and have been found to play a role in tumorigenesis.
Our study showed that TLR9 gene expression was increased in the malignant group, compared to controls and intermediate (HPV +ve nonmalignant) group. There was increase of TLR9 expression in TT genotype and CT genotype individuals among malignant group compared to similar genotypes in the controls. This upregulation was not statistically significant, possibly due to small sample size. Therefore this study should be replicated in a large cohort, and among varied ethnic population.
Acknowledgements
We acknowledge the contributions of Saroj Gupta Cancer Centre and Research Institute (SGCC & RI, Thakurpukur, South 24 Parganas, West Bengal, India), Calcutta Medical College Hospital(Kolkata, West Bengal, India) and Jawaharlal Nehru Medical College Hospital (Kalyani, Nadia, West Bengal, India) for providing us the clinical samples for the study; Dr. Sharmila Sengupta, National Institute of Biomedical Genomics for designing the study, Dr. Samsidhhi Bhattacharjee and Dr. Saroj Mohapatra of National Institute of Biomedical Genomics for helping with the statistical analysis.
