Gene Microarray Analysis Of LncRNA And MRNA Expression Profiles And The Expression Patterns Of PHLPPs In Patients With Hypopharyngeal Squamous Cell Carcinoma

PART 1:Gene Microarray Analysis of LncRNA and mRNA Expression Profiles in Patients with Hypopharyngeal Squamous Cell CarcinomaBackground:Long non-coding RNAs (lncRNAs;> 200 nucleotides), which have emerged as one of the largest and most diverse classes of cellular transcripts, are defined as non-coding RNAs (ncRNAs) distinct from housekeeping RNAs and small RNAs. LncRNAs play important roles in almost every aspect of cell biology, including chromosome remodeling, transcription, and post-transcriptional processing. Studies have shown that lncRNAs are involved in the development and progression of many types of cancer. However, the mechanisms by which lncRNAs influence development and progression of hypopharyngeal squamous cell carcinoma (HSCC) are unclear.Method:We investigated differences in lncRNA and mRNA expression profiles between 3 pairs of HSCC tissues and adjacent nontumor tissues by microarray assays and bioinformatics analyses, such as Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, Gene Ontology (GO) analysis, Enhancer lncRNA profiling analysis, Intergenic lncRNA profiling analysis, and HOX cluster profiling analysis.Results:1. In HSCC tissues,1299 lncRNAs were significantly upregulated (n=669) or downregulated (n=630) compared to levels in adjacent nontumor tissues. Moreover, 1432 mRNAs were significantly upregulated (n=684) or downregulated (n=748) in HSCC tissues.2. We randomly selected 2 differentially expressed lncRNAs (AB209630, AB019562) and 2 differentially expressed mRNAs (SPP1, TJP2) for confirmation of microarray results using qRT-PCR. The qRT-PCR results matched well with the microarray data.3. The differentially expressed lncRNAs and mRNAs were distributed on each of the chromosomes, including the X and Y chromosomes. Pathway analysis indicated that the biological functions of differentially expressed mRNAs were related to 48 cellular pathways that may be associated with HSCC development. GO analysis revealed that 593 mRNAs involved in biological processes,50 mRNAs involved in cellular components, and 46 mRNAs involved in molecular functions were upregulated in the carcinomas; 280 mRNAs involved in biological processes,58 mRNAs involved in cellular components, and 71 mRNAs involved in molecular functions were downregulated in the carcinomas.4. In addition,8 enhancer-like lncRNAs and 21 intergenic lncRNAs with their adjacent mRNA pairs were identified as coregulated transcripts.Conclusion:These findings provide insight into the mechanisms underlying HSCC tumorigenesis and will facilitate identification of new therapeutic targets and diagnostic biomarkers for this disease.PART 2:Aberrant Expression of PHLPP1 and PHLPP2 Correlates with Poor Prognosis in Patients with Hypopharyngeal Squamous Cell CarcinomaBackground:The PHLPP(Pleckstrin Homology [PH] Domain Leucine rich Repeat Protein Phosphatase)family, which represents a family of novel Ser/Thr protein phosphatases, is composed of 2 members:PHLPP1 and PHLPP2. PHLPPs partake in diverse cellular activities to exhibittheir antitumor and metastasis suppressor functions. It is necessary to investigate the expression patterns of PHLPP1 and PHLPP2 in hypopharyngeal squamous cell carcinomas (HSCCs) and clarifytheir clinical significance.Methods:A total of 138 patients with primary HSCC who underwent curative surgical treatment as an initial treatment were enrolled in this study. A total of 138 HSCC specimens and 64 adjacent noncancerous mucosal epithelial tissues were collected. The expression levels of PHLPP1 and PHLPP2 were examined by quantitative reverse transcription polymerase chain reactionand immunohistochemistry assays. Correlations between clinicopathological parameters of the patients were further evaluated.Results:1. Both of PHLPP1 and PHLPP2 mRNA transcript levels were significantly lower in tumor samples than in paired adjacent nontumor mucosae (P<0.0001, both). Positive correlations were observed between the mRNA levels of PHLPP1 and PHLPP2 in HSCC tissues (correlation coefficient r=0.678, P<0.001) and in adjacent nontumor mucosae (r=0.460, P<0.001).2. The majority of the noncancerous tissues showed higher expression levels of PHLPP1 (87.5%,56/64) and PHLPP2 (85.9%,55/64). However, the expressions of PHLPP1 and PHLPP2 were significantly decreased in 83.3% (115/138) and 82.6%(114/138) of tumor tissues, respectively (P<0.0001, both).3. The expressions of both PHLPP isoforms were significantly related to the tumor clinical stage, differentiation, and cervical lymph node metastasis (P<0.05, all). It was PHLPP1 but not PHLPP2 that was significantly related to the tumor T stage.4. Lower PHLPP 1 and PHLPP2 expressions were associated with poor overall survival (OS) in HSCC patients (P=0.004, P=0.008, respectively). Multivariate analysis revealed that PHLPP1 was an independent prognostic factor for OS.Conclusion:This study indicates that, in HSCC, aberrant expressions of PHLPP1 and PHLPP2 are common events, and loss of PHLPPsmight identify patients with poor prognostic outcomes.