Prognostic significance of miR 499 expression and Helicobacter pylori infection in malignant lesions of gallbladder cancer: a clinicopathological study

Background Gallbladder cancer (GBC) is an infrequent type of malignant neoplasm worldwide. There are a number of risk factors that increase a person’s likelihood of developing GBC. Gallbladder inflammatory (GID) diseases including cholelithiasis increase the risk of GBC, and this is further complicated by the fact that Helicobacter pylori ( H. pylori ) infection is extremely common in gastrointestinal tract in India. Since both miR 499 and H. pylori infection are found to be linked with tumor progression and metastasis, therefore there is a possibility that H. pylori might be involved in inflammation via dysregulation of miR 499. The study was designed to investigate the association of miR 499 expressions with H. pylori infection and their correlation with clinicopathological parameters of GBC. Material and methods The hundred three tissue samples used in this study are categorized into GID ( n = 55) and GBC ( n = 48). The expression of miR-499 was examined by using the Livak method for relative gene expression analysis. The presence/absence of H. pylori infection was examined by RT-PCR (Liferiver Helicobacter pylori RT-PCR Kit). Results Helicobacter pylori infection and GBC/GID cases were not significantly correlated. Decreased expression of miR 499 was observed in GBC (1.6 fold) as compared to GID patients ( P < 0.0001). Low miR 499 expression was found to significantly correlate with tumor differentiation ( P = 0.017), advanced staging ( P = 0.004) and liver metastasis ( P = 0.036). Multivariate regression analysis showed significant association of overall survival with low miR 499 expressions.


Introduction
Gallbladder cancer (GBC) is relatively rare, ranked 24th among the most common highly aggressive type of malignant neoplasm of gastrointestinal tract cancer (Globocan) [1].It has been reported that the incidence of GBC is almost 4-6 times greater in North Indian population than South India.(Delhi: 4.1/100,000 men, 9.5/100,000 women and Bengaluru 1.4/100,000 men, 1.7/100,000 women) [2].Most of the GBC cases are diagnosed at the advanced stages due to the lack of clinical appearance, which are nonspecific.The majority of cases are found co-incidentally at the time of histopathological examination of cholecystectomy [3].GBC is reported with high rate of recurrence and metastasis due to poor response to chemotherapy and radiotherapy.There are a number of risk factors responsible for the development of GBC.Cholecystitis is also often diagnosed in younger age group persons.Cholelithiasis (gall stones) has also been implicated in the development of GBC [4].Gallstones appear to be more common in ethnic groups with higher rates of GBC, where they appear to be more likely to be present and increase the risk of GBC by more than threefold.[5].This is made even more difficult by the fact that Helicobacter pylori (H.pylori) infection in the gastrointestinal tract is extremely widespread in India and is recognized as a certain cause of cancer by the International Agency for Research on Cancer.[6].It is now recognized as the main and specific infectious cause of stomach cancer in the world.It has been also reported that H. pylori is associated with pathogenesis of human cholelithiasis and cholecystitis and closely correlates with gastrointestinal cancers [7].H. pylori infections have also been seen in gallbladder, which results in chronic persistent inflammation.Furthermore, research performed emphasized that gallbladder metaplasia, adenomatous hyperplasia and dysplasia changes were more frequent in patients with positive H. pylori infection [8].
H. pylori infection has been reported to be correlated with dysregulation of some small non-coding miRNAs [9].The six biological capabilities acquired during tumorigenesis viz tumor proliferation, differentiation, evasion of apoptosis, defective cell cycle, an induction of metastasis and angiogenesis, have been described as hallmarks for human cancer [10].The role of miRs in various cancer types, including breast, squamous cell carcinoma of the head and neck, and bladder cancer, has been the subject of recent studies, but very few have focused on GBC [11][12][13][14][15]. miR 499 has been found to play role in modulation of expression of genes related to cancer resulting in tumorigenesis and resistance to chemotherapy, thus contributes to poor prognosis [16].Few studies have specifically highlighted the role of miR 499 expression role in various cancers.[17,18].
MiRs are involved in regulating the pro-inflammatory immune response induced during H. pylori infection.Cancer is caused by the accumulation of mutations, epigenetic modifications, and defective cellular function that may result from the long-term interaction of bacterial and inflammatory mediators with dysregulated miRNA expression.[19].Since both miR 499 and H. pylori infection, have been found to be involved in tumor progression and metastasis, there is a possibility that H. pylori might be involved in inflammation via dysregulation of miR 499.Thus the present study provides a hypothetical association of miR 499 expressions to H. pylori infection and their correlation with clinicopathological parameters of GBC.

Patients
The present study is a prospective study, including 103 subjects of gallbladder disease.All the recruited patients were grouped into GBC (n = 48); and GID (n = 55).The tissue samples were collected from August 2014 to September 2018.The formula n = Z2pq/L2 was used to determine the sample size, where (p = 57.83%,q = 100-p, Type I error α = 5%, Allowance error L = 1/4th of p, Power of study = 75%, Data loss = 10%) The final sample size is 48 for each group [20].
The tissue samples of both GBC and GID subjects were collected in two different containers.One portion of the tissue sample was collected in container containing formalin (10%) which was embedded in paraffin to prepare paraffin embedded blocks for histopathological examination.All histopathologically confirmed cases (GBC; n = 48 and GID; n = 55) were included in the study.The American Joint Committee on Cancer (AJCC) Tumor node metastasis (TNM) staging 7th edition recommended guidelines was used for pathological staging of cases [21].The other part of the tissue was collected in a container containing RNA later (Sigma-Aldrich, USA), stored at-80 °C for RNA isolation to study miR 499 expressions as well as DNA isolation H. Pylori detection.

Inclusion criteria
All patients who underwent biopsy of metastatic lesion or radial surgery of primary GBC and GID though laparoscopic or open cholecystectomy were included in the study.

Exclusion criteria
Patients diagnosed with any other types of cancer or who underwent chemotherapy or radiotherapy or were operated earlier and those with any immunodeficiency disorder were excluded from the study.

H. pylori detection
Helicobacter pylori real-time PCR kit (Liferiver Helicobacter pylori Real-Time PCR Kit, China) was used for DNA isolation as per the protocol provided by the manufacturer.The quality of the DNA was checked by agarose gel electrophoresis using a gel-doc system (UVP, DIGI DOC-IT System, USA).Nanodrop 2000 Spectrophotometer from Thermo Fisher, USA, was used to measure the concentration and purity of the obtained DNA.Quantitative real-time PCR was performed using (Applied Biosystems, StepOnePlus RT-PCR, version 2.3, Canada) to detect the H. pylori DNA in tissue samples of both GBC and GID.A 40 µl reaction mixture was prepared (36 µl master mix, 4 µl extracted DNA).The RT step was performed at 37 °C for 2 min(one cycle), followed by 2 min at 94 °C.The PCR step comprised 40 cycles of denaturation each for 15 s at 93 °C and annealing/extension for 1 min at 60 °C.Each run included negative and positive controls from the real-time kit to ensure that the sample processing, amplification, and detection steps were carried out correctly.

miR extraction and RT-PCR assay
The GBC/GID tissue samples stored in RNA later were used for the isolation of total ribonucleic acid (RNA) by using the mirVANA ™ miRNA isolation Kit, USA.The isolated RNA was quantified using nanodrop spectrophotometer (Thermo 2000C model, Thermo Fisher, USA) and the quality of RNA was checked by measuring A 260 /A 280 .The integrity of isolated RNA was also checked by agarose gel electrophoresis using 1.0% agarose gel stained with 0.5 μg/ml EtBr using gel doc (UVP, DIGI DOC -IT System, USA).The extracted total RNA was kept at − 20 °C.cDNA synthesis of miR 499 and RNU6B (internal reference gene) was synthesized from 2 µg of total RNA by using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, (USA) and TaqMan Universal PCR Master Mix, No AmpErase (Uracil-N glycoslyase).Individual RT-PCR assay were run in 20 μL reaction volume in a RT-PCR machine (Applied Biosystems StepOnePlus ™ system version 2.3, Canada) to detect miR 499 expressions in tissue.Each sample was examined in triplicate.The thermal cycling conditions consisted of optional AmpErase UNG activity and enzyme activation hold at (50 °C for 2 min, 95 °C for 10 min), respectively.Additionally, 40 PCR cycles of denaturation at were performed at 95 °C for 15 s followed by annealing and extension step at 60 °C for 60 s.The cycle threshold (Ct) value was recorded for both miR 499 and RNU6B, and the relative quantification of target miRNA expression was expressed as (2 −ΔΔCt ), where ΔCt = Ct miR 499-Ct RNU6B (Livak method for relative gene expression analysis) [22].The correlation was observed between Ct values of target miR with reference to RNU6B.The Livak method is used for relative quantification of miR 499 expression analysis.

Statistical analysis
Software used for the statistical analysis was SPSS (version 20; IBM, Chicago, IL, USA) Student t-test was carried out to compare the expression level of miR 499 between GBC and GID.Chi-square or Fisher exact test was performed for association between miR 499 and H. pylori infection with cliniopathological parameters.
The survival analysis was done by Kaplan-Meier analysis and Cox's multivariate regression using log-rank test, along with hazard ratio (HR) and corresponding 95% confidence interval (CI).

Association between downregulation of miR 499 and H. pylori positivity in GBC
Out of 103 gallbladder tissue samples, H. pylori infection was found positive in 25% (12/48) GBC, and in 23.6% (13/55) GID patients.There was no significant association was observed between H. pylori infection and GBC/GID cases.Decreased expression of miR 499 was observed in GBC (1.6 fold) as compared to GID patients (P < 0.0001).In relation with H. pylori infection, there was no significant difference in miR 499 expression between H. pylori present and H. pylori absent samples (P = 0.6455).The present data show no association between miR 499 and H. pylori infection in GBC.However, miR 499 expression levels were found to be statistically significant with GBC when compared with GID cases (Fig. 1a, b).

Association of miR 499 expression and H. pylori infection with clinicopathological parameters
All the clinical data including tumor size, differentiation, staging, lymphatic invasion and liver metastasis were obtained from patients' information sheet containing clinical records available in the department.No significant association was found between H. pylori infection and clinicopathological parameters in GBC patients.
All 48 (GBC patients) were divided into two groups using the median expression level of miR-499 (0.193) as a cutoff point.GBC patients who expressed values below the median expression level were placed in the low expression group (mean miR 499 expression value: 0.092; n = 24), and patients whose expression levels were higher than the cutoff value were placed in the high expression group (mean miR 499 expression value: 0.486; n = 24).Significant correlation was found between low expression of miR 499 with tumor differentiation (P = 0.017), advanced staging (P = 0.0004) and liver metastasis (P = 0.036) (Fig. 1c), whereas no significant correlation of miR 499 was observed with gallstones, tumor size, lymphatic invasion, tobacco pan masala chewers, age and sex (Table 2).

miR 499 downregulation is associated with poor prognosis of GBC patients
Patients of GBC were followed up to 36 months through phone calls and routine hospital visit.Kaplan Meier method and log-rank test were used to found the correlation between the median overall survivals of GBC patients with low miR 499 expression and those with high miR 499 expressions.Positive significant correlation was observed between miR 499 expressions and overall survival.Poor survival rates were observed in patients with low miR 499 expression than patients with high miR 499 expressions: P < 0.0001, HR 0.2486, 95% CI 0.1223-0.5056; 10 months (low miR 499) versus 24 months (high miR 499).However no significant correlation was found between H. pylori infection and overall survival status: P = 0.431, HR 0.743, 95% CI 0.3553-1.556(Fig. 2).
Clinicopathological parameters related to three years overall survivals of patients were examined by using univariate and multivariate analyses.Univariate analysis showed significant correlation between overall survival and tumor size, tumor differentiation, staging, lymphatic invasion, liver metastasis and low miR 499 expressions which were predictors of poor survival.association of overall survival with low miR 499 expressions and was found to be an independent risk factor for poor prognosis of GBC (Table 3).

Discussion
In the current study, miR 499 expressions were analyzed in GBC and GID.Due to inflammatory role of miR 499 in cancer, it was hypothesized that miR 499 expressions might be influenced by the H. pylori infection and it may play vital role in GBC progression.miR 499 was found to be downregulted in GBC patients (1.6 fold) versus GID patients, whereas no significant correlation was found between H. pylori infection and miR 499 expression.miR 499 plays a vital role in etiology of cancer by targeting the regulation of Forkhead box protein O4, Programmed cell death protein, and sex determining region Y-box 6 gene expression [23].Additionally, different types of miRs have also been shown to affect the progression of GBC [24,25].Particularly, miRNA-499 was found to act as a biological indicator for the prognosis of hepatocellular carcinoma.Previous research revealed that histone deacetylases (HDAC1-3) stimulated the proliferation of HCC cells by downregulating miR-499, indicating that miR-499 may function as a tumor suppressor [26].However, the precise molecular pathways by which miR-499 regulates the development of GBC remain unclear.
miR 499 and H. pylori infection are thought to important because they mediate the inflammatory process.Long-term infection of H. pylori leads to inflammation and inflammation in combination with oncogenic activation, promotes tumorigenesis [27].It has been reported that miR-146a negatively regulates H. Pylori-triggered interleukin (IL)-8 and plays a vital role in a negative feedback loop to modulate inflammation [28].
Very few studies have been done in relation to miR 499 expression in GBC cancers.Moreover, studies are available in other cancers, reported that miR 499 promote metastasis by targeting gene FOXO4 in colon cancer [18].Another study on non-small cell lung cancer patients has reported that miR 499 functions as tumor suppressor in vitro and in vivo by targeting VAV3 [29].Another study by Li et al. [30] has reported that miR 499 rs376444 polymorphism has been significantly associated with lung cancer.According to the Zhang et al., miR 499 rs3746444 polymorphism has been linked to a high risk of oral cancer [31].MiR 499 polymorphism has been found to be correlated with increased risk of hepatocellular carcinoma [32].Additionally, miR 499 in other cancers has been found to be significantly correlated with clinicopathological parameters.Li et al. have revealed a significant correlation between serum miR 499 expressions and advanced stage and poor prognosis in lung cancer [33].miR 499 has been found to be associated with stage I-IIIA in lung cancer [17].
In the present study, miR 499 expression levels was determined in GBC and GID tissues using RT-PCR.A statistically significant correlation was found between low miR 499 expression levels and tumor differentiation, staging and liver metastasis, while no significant correlation was found between miR 499 and variables such as gender, age, tumor size and lymphatic invasion.Furthermore, no significant correlation was observed between H. pylori infection and clinicopathological manifestations.A similar study has reported with high expression of miR 146a in primary gastric cancer with no clear link to H. pylori infection [28].Similar to our previous study, no significant association was observed between GBC and demographical parameters (gallstone presence, Smoking, Tobacco chewing, and pain in abdomen) [34].
Kaplan-Meier analysis on patients overall survival revealed its association with reduced miR 499 expression level when compared with high miR 499 levels in GBC patients.MiR 499 was observed as an independent predictor of prognosis in GBC patients by a multivariable Cox propositional hazards model.However, no significant correlation was found between overall survival and H. pylori infection in GBC.Similar results have been reported for association between low miR 499 expression levels and overall survival in non-small cell lung patients [29].According to the available literature related to GBC and miR 499, this is the first study, reporting a negative significant correlation between miR 499expression in GBC progression which is independent of H. pylori infection.

Conclusion
The outcome of the present study showed statistically significant correlation between low expression of miR 499 and tumor differentiation, higher stage and was found to be associated with GBC progression.Thus miR 499 may be considered as a useful prognostic biomarker in GBC progression.Further studies are required with a larger sample size with other miRNAs to validate the results with more specificity.This would lead to development of a novel prognostic and therapeutic biomarker for GBC.

Table 1
General characteristics of the study subjectsData are represented as n (%), Fisher Exact test.S.E Standard Error

Table 2
Correlation of miR 499 and H. pylori infection with various cliniopathological parameters for gallbladder cancer patients