Genetic Variants of MicroRNA-146a and MicroRNA-196a2 are Associated with Poor Outcome but not Risk of High-Grade B-Cell Non-Hodgkin Lymphoma

Background: Polymorphisms in microRNAs (miRNAs) encoding genes are involved in carcinogenesis. However, their relation to lymphomagenesis is still unclear. Aim: To investigate the influence of miRNA-146a rs2910164 G/C polymorphism and miRNA-196a2 rs11614913 C/T polymorphism on risk and clinical outcome of high-grade B cell non-Hodgkin lymphoma (HGB-NHL). Methods: Seventy-five patients with HGB-NHL and 100 matched controls were screened for miRNA-146a rs2910164 G/C and miRNA-196a2 rs11614913 C/T polymorphisms by Polymerase Chain Reaction-Restriction Fragment length Polymorphism (PCR-RFLP). Results: The two studied miRNA polymorphisms were not associated with the risk of NHL. The GG genotype of miRNA-146a rs2910164 was associated with a worse disease-free survival (DFS) compared to the GC and CC genotypes (HR =5.7; 95% CI=1.05-31.09; p=0.044). The miRNA-196a2 rs11614913 CC genotype was associated as well with worse DFS compared to the CT and TT genotypes (HR=10.37; 95% CI=1.80-59.62; p = 0.009). No significant association was found between the studied miRNA polymorphisms and patients’ overall survival. Conclusions: miRNA-146a rs2910164 G/C and miRNA-196a2 rs11614913 C/T polymorphisms may be associated with shorter DFS in HGB-NHL.


Introduction
MicroRNAs (miRNAs) are endogenous short (21-24 nucleotides) single stranded non-protein coding RNA molecules that control gene expression at the posttranscriptional level 1 . They bind either to the 3'untraslated end of their target messenger RNA (mRNA) with subsequent translational repression or to the open reading frame resulting in mRNA degradation 2 . Therefore, they are responsible of many physiological processes including cell proliferation, differentiation and apoptosis 3 .
MiRNAs have specific expression patterns in different cancers and therefore they may be considered as non-invasive tumour markers for cancer diagnosis and prognosis 4 . Single nucleotide polymorphisms (SNPs) in genes coding miRNAs may disrupt their expression and maturation and might be implicated in the process of carcinogenesis 5 . MiRNA-146a rs2910164 G/C polymorphism and miRNA-196a2 rs11614913 C/T polymorphism have been found to be associated with susceptibility to a variety of solid tumours like colorectal cancer 6,7 , stomach cancer 8,9 , hepatocellular carcinoma 10, 11 , breast cancer 12 and papillary thyroid carcinoma 13

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Little is known about the association between these miRNA gene polymorphisms and haematological malignancies like leukemia and lymphoma. B-cell non-Hodgkin lymphoma (B-NHL) is a genetically heterogeneous B-cell neoplasm in which molecular biomarkers have been widely used for diagnosis and prognosis 14 . Few studies have explored the association between miRNA-146a and miRNA-196a2 polymorphisms and NHL focusing mainly on disease susceptibility [15][16][17] . We performed this work to study the relation between miRNA-146a rs2910164 G/C polymorphism and miRNA-196a2 rs11614913 C/T polymorphism and risk of highgrade B-NHL and to identify their influence on disease outcome.

Methods
This single institution observational case-control study was conducted during the period from January 2017 through January 2019 at Kasr Al-Ainy School of Medicine, Cairo University, Egypt.

Participants
The study included 75 patients with high grade B-NHL and 100 age and sex matched healthy controls. Patients were enrolled in the study during their follow up visits at Kasr Al-Ainy Centre of Clinical Oncology and Nuclear Medicine (NEMROCK), Cairo University. Healthy controls were invited to participate in the study during their outpatient clinic visits at the New Kasr Al-Ainy Teaching Hospital for routine check-up provided that they have normal complete blood count.

Management of non-Hodgkin lymphoma
According to the world health organization (WHO) classification, the diagnosis and classification of NHL was based on histopathological examination and immunohistochemical (IHC) studies of lymph node biopsy 18 . Due to logistic reasons, BCL2 and cmyc were done in some patients by IHC to detect double expressor diffuse large B-cell lymphoma (DLBCL). All patients were evaluated by history and clinical examination. Laboratory work up included complete blood picture, renal and hepatic profile, serum uric acid, lactate dehydrogenase (LDH), bone marrow biopsy and screening for hepatitis B surface antigen. In addition to echocardiography, patients had computerized tomography (CT) with contrast of the chest, abdomen, and pelvis, positron emission tomography (PET/CT) was done if feasible. Clinical staging was assessed according to the Ann Arbor classification system 19 21 . Patients were assessed clinically before each treatment cycle, by CT scan or PET/CT after 3 cycles and at the end of treatment plan. Assessment of response was performed according to Lugano criteria 22 .
Non-bulky early disease (stages I and II) received either R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone) for 3 cycles and involved field radiotherapy or 6 cycles of R-CHOP, while patients with bulky disease receive 6 cycles of R-CHOP and radiotherapy. Patients with advanced disease (stages III and IV) received 6 cycles of R-CHOP. R-EPOCH (rituximab plus etoposide, prednisone, vincristine, cyclophosphamide and doxorubicin) regimen was used in patients with primary mediastinal large B-cell lymphoma and double expressor lymphoma.
Genetic study Three mL of venous blood was collected on ethylene diamine tetra-acetic acid (EDTA) by sterile venepuncture using a sterile vacutainer tube. By using Gene JET Whole blood Genomic DNA purification Mini kit (Thermo Fisher Scientific, USA), genomic DNA was extracted from whole blood. Genotyping of miRNA-146a rs2910164 and miRNA-196a2 rs11614913 was performed by Polymerase Chain Reaction-Restriction Fragment length Polymorphism (PCR-RFLP) assay as described before 12 . The PCRs were done in a total volume of 25 µL containing 100 ng genomic DNA, 25 pM of each primer and 12.5 µL PCR master mix.
The PCR conditions comprised of an initial denaturation step at 94°C for 5 minutes followed by 30 cycles of denaturation at 94°C for 60 seconds, annealing at 62°C for miRNA-146a and 63°C for miRNA-196a2; for 60 seconds and extension at 72°C for 30 seconds and final extension at 72°C for 10 minutes. The presence of the PCR amplicon (147 bp for miRNA-146a and 149 bp for miRNA-196a2) was confirmed on 2% agarose gel electrophoresis. The PCR products were digested overnight at 37°C with the appropriate restriction enzyme (SacI for miRNA-146a and MspI for miRNA-196a2). Visualization of digested PCR products were performed on ethidium bromide stained 3% agarose gel electrophoresis.

Statistical analyses
Numerical data were presented as mean and standard deviation and compared by student's t test, while qualitative data were described as number and percentage and compared by Chi-square /   were not associated with risk of high-grade B-NHL.
In agreement with our study, these polymorphisms were not associated with susceptibility to acquired immune deficiency syndrome (AIDS) associated NHL in a previous multicentre study 17 . In contrast to our findings, the CC genotype of miRNA-146a rs2910164 polymorphism was found to be associated      The G allele of miRNA-146a rs2910164 was found to be associated with increased miRNA-146a expression 13 . Upregulation of miRNA-146a was detected in DLBCL cases suggesting a role of this polymorphism in pathogenesis of DLBCL 15 . The CC/CT genotypes of miRNA-196a2 rs11614913 were found to be associated with indictors of poor NHL prognosis like advanced stage, bone marrow invasion, and B symptoms. This was attributed to higher expression of miRNA-196a2 that was detected among patients carrying the C allele of miRNA-196a2 rs11614913 16 .

Conclusions
The results of the current study provide initial evidence that miRNA-146a and miRNA-196a2 polymorphisms may have a negative impact on NHL prognosis. Further larger scale studies are warranted to validate our findings.