The Research Of The Expressions Of MiR-21and PTEN, And Common MicroRNA Single Nucleotide Polymorphisms In Head And Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a type of tumor which derives from the epithelium mucosa cell of oral cavity, oropharynx, laryngopharynx and larynx. And HNSCC is the fifth common malignant tumor worldwide. Tobacco use and alcohol consumption are considered as the most important risk factors of HNSCC. In addition, human papillomavirus (HPV) infection might be another important etiologic factor. Although comprehensive therapies including surgery, radiotherapy and chemotherapy was applied to increase the disease-free survival rate in recent years, patients with locally advanced HNSCC have a5-year overall survival rate hovering approximately30%. Therefore, there is a need to acquire deeper understanding of HNSCC biology and to identify predictive molecular markers, which might improve early diagnosis and patient selection for appropriate treatment and guide the development and evaluation of new therapies. In the past decades, researchers have mainly focused on the protein-coding genes including oncogenes and tumor suppressor genes, however, in recent years microRNAs as non-coding RNA gene products, have drawn much more attentions from researchers.MicroRNAs ware evolutionarily highly conserved, singled-stranded, short non-coding RNAs of about22nucleotides that mediate gene expression through completely or partially base pairing with target mRNA at the3'-untranslated region, leading to mRNA cleavage or translational repression. MicroRNAs, as a new family of gene regulators, play central roles in a broad range of physiologic and pathologic processes including cell proliferation, differentiation, apoptosis, immune/inflammation response system, stress resistance and metabolism. Although their precise biological mechanisms largely remain unclear, a great many studies have demonstrated that microRNAs might be involved in human tumorigenesis as tumor suppressors or oncogenes by affecting cell growth and development and altered expressions of microRNAs are associated with the etiology, diagnosis, progress and prognosis of numerous cancers including HNSCC.MiR-21is one of the most studied microRNAs in cancers and is upregulated in many solid tumors, including breast cancer, lung cancer, stomach cancer, glioblastoma, cholangiocarcinoma and pancreatic endocrine tumor. In head and neck cancer, high expression of MiR-21is also observed in tumor tissues. As microRNA expression is tissue-specific, the expression signatures could potentially suggest biological pathways/mechanisms that differ between the HNSCC subtypes. Phosphatase and tensin homologue (PTEN) plays an important role in tumorigenesis and decreased PTEN expression is associated with lymph node metastasis, tumor grade, tumor-node-metastasis (TNM) stage, and microvessel density. And several studies indicated that PTEN participated in mediating radiosensitivity and chemosensitivity in head and neck cancers. Most importantly, PTEN might be considered as one of target genes of MiR-21in HCC cell lines and some tumors. However, the association between MiR-21expression and PTEN expression has not been reported in Laryngeal squamous cell carcinoma (LSCC) and Hypopharyngeal squamous cell carcinoma (HSCC). Hence, in the present study we first investigated the expression of both MiR-21and PTEN and evaluated the association of MiR-21expression with PTEN expression in LSCC and HSCC to help us understand both function of MiR-21in tumorigenesis and its clinical significance.It has been demonstrated that HPV infection is an important etiologic factor of HNSCC, especially oral squamous cell carcinoma (OSCC). Most importantly, in response to infection during both the innate and acquired immune response, microRNAs may be involved in the regulation of immune/inflammation response system and apoptosis pathways. It is worth noting that both immune/inflammation response system and apoptosis pathways play key roles in the HPV clearance and escape of immune surveillance. Therefore, certain genetic alterations of microRNAs might have influence on the HPV status and the susceptibility to OSCC.A single nucleotide polymorphism (SNP) in microRNAs or their binding sites might affect the transcript of pri-microRNA transcripants, the processing of microRNA precursors to mature microRNAs, or microRNA-target interactions. To our knowledge, several SNPs in microRNAs and within their targets may have an effect on microRNA functions and their target gene expressions and thus may contribute to cancer susceptibility. Recent studies have investigated the association between HNSCC risk and several mircoRNA SNPs including miR146rs2910164, miR149rs2292832, miR196rs11614913, and miR499rs3746444polymorphisms. However, these studies show conflicting results. Considering the important roles of HPV in the development of OSCC, we evaluated the joint effects of HPV16serology status and these four common microRNA SNPs on risk of OSCC.Part1Altered expressions of MiR-21and PTEN in human laryngeal and hypopharyngeal squamous cell carcinomasObjective:To examine the expressions of MiR-21and PTEN in laryngeal squamous cell carcinoma (LSCC) and hypopharyngeal squamous cell carcinoma (HSCC), and assess the associations of their expressions with clinical characteristics of patients as well as the association between the expressions of MiR-21and PTEN.Methods:1. We collected60cases of primary HNSCC tumor tissue including30LSCC and HSCC, and morphologically normal mucosa epithelium tissue adjacent to carcinomas (at least2cm from tumor margin) as control.2. Total RNA enriched with small RNA was isolated from fresh frozen tumor tissue and adjacent normal mucosal epithelium tissue. 3. Real-time RT-PCR for MiR-21expression was analyzed by using Taqman microRNA assays and ABI7900HT Sequence Detection.4. Immunohistochemistry Staining was carried out to study altered PTEN protein expression in60tumor tissues and60matched adjacent normal mucosa epithelium tissues.5. Statistical analysis was performed using SPSS software (version13.0, SPSS Inc., Chicago, IL, USA). The relative expression of MiR-21was presented as mean±standard deviation (SD). The differences of MiR-21expressions between tumor and matched adjacent normal tissue were assessed by the Paired-Student's t test and the differences of PTEN expressions were analyzed by Wilcoxon Singed Ranks Test. Differences between two groups were examined by the Independent Samples t Test or Mann-Whitney U test. Multiple group comparisons were analyzed using One-way ANOVA or Kruskall-Wallis test. The criterion for statistical significance was set at P<0.05.Results:1. MiR-21was up-regulated in LSCC and HSCC compared to adjacent non-tumor tissues (P<0.05), and the up-regulated expression of MiR-21was associated with clinical stage (P=0.001), T classification (P=0.007), pathologic differentiation (P=0.025), and lymph node positivity (P=0.002).2. PTEN IHC staining was notably weaker in tumor tissues than that in matched non-tumor tissues (P<0.05), and the down-regulated expression of PTEN was correlated with tumor staging (P=0.025), the extent of tumor (P=0.017), and lymph node positivity (P=0.040).3. The level of MiR-21was reversely correlated with PTEN expression (P=0.006).Conclusion:1. MiR-21might play a central role in the progression of LSCC and HSCC.2. PTEN was down-regulated in LSCC and HSCC.3. The expression of MiR-21was negatively associated with the expression of PTEN in LSCC and HSCC. Part2MicroRNA Variants Increase the Risk of Squamous Cell Carcinoma of Oropharynx Associated with HPV16Objectives:To test the hypothesis that microRNA polymorphisms might modify the association of HPV16seropositivity with OSCC risk.Methods:1.325non-Hispanic white patients with primary squamous cell carcinoma of oral and335cancer-free control individuals were selected by frequency matching. And all study participants signed an informed consent forms and completed an epidemiological questionnaire regarding demographic and risk factors including smoking and drinking status.2. HPV16serologic detection was tested in the plasma of study participants by using a standard ELISA.3. Genomic DNA was extracted from3ml of whole blood with the DNA Blood Mini Kit.4. A polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) assay was applied to amplify the fragments that contain polymorphisms of miR146rs2910164, miR149rs2292832, miR196rs11614913, and miR499rs3746444.5. The differences of the distributions of selected demographic variables, tobacco smoking, alcohol consumption, and microRNA genotypes frequencies between HPV16seropositivity and HPV16seronegative cases were evaluated using the chi-square test of association. To evaluate the association of serologic HPV16status and microRNA genotypes with OSCC risk, the odds ratios (ORs) and95%confidence interval (CIs) were calculated using both univariate and multivariate logistic regression analyses. We also assessed the joint effects of HPV16serology and microRNA genotypes on OSCC risk, and the joint effects were further stratified by smoking status and tumor site. Statistical Analysis System software (Version9.1; SAS Institute, Cary, NC) was used for all the statistical analyses. All tests were two-sided, and a p<0.05was considered the cutoff for statistical significance.Results:1. HPV16seropositivity was significantly more common in patients than that in controls.2. Overall, no significant association of these four microRNA polymorphisms with the risk of OSCC was observed3. Compared with individuals with miR499rs3746444TT genotype and HPV16seronegativity, an increased risk of OSCC was found among those with TT or CC genotypes and HPV16seronegativity (OR=1.4,95%CI=1.0-2.0), TT genotype and HPV16seropositivity (OR=3.4,95%CI=2.0-5.6) and CT or CC genotypes and HPV16seropositivity (OR=4.1,95%CI=2.1-8.0), respectively. Similar results were found for miR146rs2910164, miR149rs2292832and miR196rs11614913polymorphisms.4. With further analysis stratified by smoking status and tumor site, we observed that each microRNA polymorphism altered risk of HPV16-associated OSCC, and such effect modification was particularly prominent in the never smokers, and for oropharyngeal cancer as opposed to oral cavity cancer.Conclusion:The risk of oropharynx squamous cell carcinoma (OPSCC) associated with HPV16seropositivity could be modified by microRNA polymorphisms. Larger studies are needed to verify our findings.