Predictive Value Of LINC01133 For An Unfavorable Prognosis Was Impacted By Alcohol In Esophageal Squamous Cell Carcinoma

BackgroundEsophageal carcinoma?EC?is one of the most common cancer and leading cause of cancer-related deaths according to the latest global and Chinese cancer statistics^[1,2].Approximated 50%of esophageal carcinoma occurs mainly in China,and 90%of cases are esophageal squamous cell carcinoma?ESCC?,the predominant histological type of EC^[3].Despite the mortality rates of ESCC have begun to decline due to recent advances in diagnostic and therapeutic techniques,the 5-year overall survival?OS?for patients with resectable ESCC is only 35%-45%which is still dismal^[4,5].One of the main reasons is probably that TNM staging system could not accurately predict the prognosis of patients with resectable ESCC^[6].Some of them with early-stage ESCC rapidly developed local and regional recurrences and distant metastasis after esophagectomy^[7].Therefore,it is imperative to identify novel prognostic biomarkers to assist oncologists in improving the predictive accuracy of TNM staging system for ESCC.Over the past decade,long noncoding RNAs?lncRNAs?are attracting considerable research attention^[8].Although lncRNAs are lack of protein-encoding capacity,it is well-recognized that lncRNAs drive many critical biological processes including tumor growth,invasion and metastasis^[9].For instance,the lncRNA HOTAIR?HOX antisense intergenic RNA?is one of the most important oncogenic lncRNAs in several human tumor types,including breast cancer,hepatocellular carcinoma and colorectal cancer^[9].HOTAIR regulated tumor proliferation,migration and chemotherapeutic sensitivity by modulating gene expression and chromatin dynamics^[17].In addition,the lncRNA XIST?X-inactive specific transcript?,conducted by genetic imprinting,appears to be dysregulated in different cancers including lung cancer,gastric cancer and ESCC^[10-12].Owing to their powerful functions and tissue-specific expression,lncRNAs were considered as most promising biomarkers in cancer diagnosis,therapy and prognosis^[13].mong these lncRNAs,LINC01133,a novel lncRNA,located in human chromosome1q23 that encodes a long intergenic noncoding RNA?Table S1?.High expression of LINC01133 was firstly reported in lung squamous cell cancer?LSCC?^[14].Knockdown LINC01133 significantly inhibited the migration and invasion capacity of lung cancer cells and osteosarcoma cells through targeting EZH2,LSD1 and sponging miR-422a,respectively^[15,16].Interestingly,two recent studies showed that LINC01133 expression was lost in colorectal cancer?CRC?,and silence of LINC01133 suppressed the metastasis and epithelial�mesenchymal transition?EMT?by interacting with SRSF6 protein^[18,19].These contradicting reports suggested that LINC01133 exerts both a positive and negative effect on migration and invasion in a tumor type-dependent manner.However,very little is known about the expression level and prognostic significance of LINC01133 in ESCC.ObjectiveTo study the expression and clinical predictive value of LINC01133 in esophageal squamous cell carcinoma?ESCC?.Methods1.ESCC tissue specimens.The ESCC tissues and paired normal esophageal epithelial tissues from 149 patients,were surgically collected form Sun Yat-Sen University Cancer Center between January 2007 and December 2009.The eligibility criteria of ESCC patients were as follows:?1?no distant metastases or not receiving any local and/or systemic treatment before the operation;?2?all the samples were histologically diagnosed by two pathologists;?3?no serious complications or other malignant disease.The clinical stage was determined according to the 7th edition of the TNM classification of the International Union against Cancer?UICC?.Overall survival?OS?was defined as the time from the date of radical surgery to the date of death or last follow-up,and progression-free survival?PFS?time was from the date of radical surgery to the date of relapse,metastasis or last follow-up.The study was reviewed and approved by the medical ethics committee of Sun Yat-Sen University Cancer Center.Informed consents were obtained from all patients before the surgery.2.ESCC cell lines and cell culture.Nine ESCC cell lines?KYSE-140,KYSE-150,KYSE-180,KYSE-30,KYSE-410,KYSE-510,KYSE-520,EC-18 and HKESC1?and an immortalized esophageal epithelial cell line NE-1 were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen?DSMZ,Braunschweig,Germany?.These cancer cells were cultured at 37�C and 5%CO2 in RPMI-1640 medium?Gibco,Carlsbad,CA?supplemented with 10%fetal bovine serum?FBS?.NE-1 cell line was maintained in a 1:1 mixture of defined keratinocyte serum free medium with growth supplements?Gibco?and EpiLife medium?Cascade Biologics?.All experiments were performed during the exponential phase of cell growth.3.Quantitative real-time PCR?qRT-PCR?.Total RNA from ESCC tissues and cells was extracted using TRIzol reagent?Invitrogen,Carlsbad,CA,USA?according to the manufacturer's protocol.Total RNA quality and concentration were examined by the260/280 nm absorbance with a NanoDrop-2000 spectrophotometer?NanoDrop products,DE,USA?.RNA was reverse transcribed to cDNA using PrimeScriptTM RT Master Mix?Takara,Dalian,China?.For quantitative real-time PCR?qRT-PCR?,the cDNA was quantified in triplicate using SYBR Green Mix?Promega,Madison,WI,USA?on a Light Cycler 480 instrument?Roche,Basel,Switzerland?.4.Statistical analysis All statistical analyses were carried out with SPSS version 23.0statistical software package?SPSS,Chicago,IL,USA?.Relative expression of LINC01133 in ESCC tissues and paired normal tissues was performed by paired-sample t test.The Chi-square test or Fisher's exact test was conducted to examine the correlation between LINC01133 expression and the various clinicopathological variables.Survival analysis was performed using the Kaplan�Meier method,and the log-rank test was used to assess the differences between patient groups or subgroups.Univariate and multivariate Cox proportional hazards regression analyses were performed to identify the independent prognostic factors that predict OS and PFS.All P-values were two sided and P-values of less than 0.05 were defined as statistically significant. Results In summary,our findings indicated that relative expression of LINC01133 was significantly underexpressed in ESCC.LINC01133 functions as a tumor suppressor and plays a critical role in early stage development of ESCC.More importantly,our data demonstrated for the first time that drinking status can influence the predictive value of LINC01133 in ESCC.The combination of LINC01133 expression,drinking status and TNM stage better identified ESCC patients at high risk after esophagectomy,and more aggressive postoperative therapy are recommended for these patients.However,elucidating how alcohol regulates the biological functions of LINC01133 in ESCC need future mechanistic studies.Conlusion1.LINC01133 is lowly expressed in ESCC tissue and cell lines.2.Reduced expression of LINC01133 is positively correlated with cancer progression and unhealthy lifestyles of ESCC patients.3.LINC01133 downregulation is associated with adverse prognosis in ESCC.4.Prognostic significance of LINC01133 is influenced by drinking status in ESCC patients.5.LINC01133 expression combined with drinking status and TNM stage better predicts patient survival in ESCC.