Prevalence And Serum MicroRNA ExPression Of Concurrent Esophageal And Gastric Cardia Cancers From The Same Patient At High-incidence Area For Esophageal Squamous Cell Carcinoma

1 BackgroundEsophageal squamous cell carcinoma (ESCC) ranks the sixth of the most common malignant tumors and its obvious epidemiological characteristics are significant differences in regional distribution and coexistence with the high incidence of Gastric cardia adenocarcinoma (GCA).In high incidence area of ESCC, ESCC and GCA coexisting in the same patient is not uncommon that is defined as concurrent esophageal and gastric cardia cancers (CC).This thesis aims to clarify the prevalence of CC, and to provide the basis for prevention and treatment of the disease.The misdiagnosis of CC is still a serious proble with much poor prognosis.In addition to the clinical menifestions of the CC, exploring the pathogenesis and looking for specific tumor biomarkers will contribute to early diagnosis and treatment of the disease and improve patients'prognosis.MiRNA research in recent years becomes a hot field of life science. MiRNA is a large class of gene expression regulation factors, which plays a role in regulating approximately 1/3 of the human genome as oncogenes or tumor suppressor genes, and is closely related with the occurrence of tumor. MiRNA expression profiles play an important role in the pathogenesis of the tumor, and can be used as a tumor diagnostic marker in clinical medicine.More and more report show that blood and tissue miRNA expression profiles can be used to distinguish cancer from normal control, for example the miRNA can serve as biomarkers for breast detection and prognostic evaluation. We hypotheses that cancer-specific serum miRNA expression profiles exist in CC patients, and play a role in the diagnosis and pathogenesis.Blood miRNA expression profiles provide with broad prospects in cancer diagnostic marker of non-invasive exploration, we study serum miRNA expression of CC patients by the current mature chip technology, and explore miRNA diagnosis and pathogenesis significance in CC patients.2 Materials and methods2.1 SubjectsQuestionnaires were performed and confirmed by analyzing the pathological data from hospitals.The CC patients were interviewed at home. All the patients were from northern region of Henan province and Cixian in Hebei province.431 cases of CC were diagnosed initially, Information comes mainly from several local hospitals and related Cancer Registry institutions, and all patients were confirmed by histopathology.112 cases of CC were discovered through paraffin embedded slides serially and microscopic-observation of surgical specimens from 4722 patients including single GCA and ESCC, with a total of 543 cases.All the collected clinical data were input Excel sheet and analysed by SPSS15.0 statistics package, the standard test wasα= 0.05.We collected archived serum samples of 5 cases of CC,14 cases of ESCC and 13 cases of GCA patients and 16 normal control with complete clinical data in northern region of Henan province and Cixian in Hebei province,5mL non-fasting no anticoagulant venous blood were obtained from all subjects,standing 30min,4℃1600 transfer centrifugal separation of serum 5 minutes, the serum tube were placed EP-20℃kept inactive. According to the MiRNA amount extracted, some samples were made technology duplication to further eliminate of random error. 2.2 Affymetrix The GeneChip(?) miRNA Array MiRNA hybridizationSerum total RNA were extracted by proteinaseK digestion, and miRNA were isolated by ethanol precipitation, then the miRNA were hybridized with Affymetrix The GeneChip(?) miRNA Array MiRNA, and we started to scan after the chips were washed, stained.The software, Affymetrix(?) GeneChip(?) Command ConsoleTM 1.1,was used to process the original data,the data analysis software used:Significance Analysis of Microarrays software; Supervised Hierarchical Cluster Analysis and Results Display software; miRNA Chip Normalization Analysis and Quality Control software.3 Results3.1 The clinicopathological prevelence characteristics of CCCC occurred predomently in male, with a ratio of 5.12:1 for male:female. The peak age for CC patients was 60-70 years old. Staging analysis showed a high proportion of stageⅡboth in ESCC and GCA (55.56% vs 36.17%);The moderate differentiated type in ESCC was 62.81%,however, the predominent type for GCA was poorly differentiated (48.41%);ESCC located mainly in the middle part of the esophagus (66.67%);Clinicopathological changes were characterized by higher rate of medullary type and early cancer (both for 33.07%), the majority type of GCA was fungating (29.92%).124 cases of CC were followed up,10 cases lost, effective follow-up 114 cases,99 cases were male, with a median survival time 36.70 months, 15 were female, the median survival time was 22.00 months (P=0.263>0.05);106 cases of patients were both survey results of family history and follow-up data, the median survival time was 29.75 months in history-positive patients, median survival time of patients with negative was 41.30 months (P=0.424).The rate of positive family history of CC were 40.13%,male than in female (43.18%vs20.00% P=0.049). 5-year survival was 17.54%. Male and female, groups with positive and negative family history had no statistical significant in survival rate.3.2.Serum miRNA expression profiles of CC patients3.2.1 Serum miRNA expression profiles of CC group compared with normal control groupWe used SAM software to screen different miRNA expression for 2 group chip data (including biological and technical repetition), the screening criteria:reliability standard (P-value) was controlled less than 5%,and fold change was more than 1.5 times that Fold Change≥1.5 was upregulation and Fold Change≤0.667 was downregulation. CC group could be screened 12 miRNAs upregulation expression compared with the normal control group, follows were upregulated expression of miRNA, miR-320b, miR-320c, miR-320a, miR-92a, miR-486-5p, miR-627, miR-365, miR-1826, miR-923,miR-185,miR-129-star and miR-106a-star, the fold change located in 1.5192-3.8319.3.2.2 Serum miRNA expression profiles of CC group compared with ESCC groupWe screened a total of 12 upregulated miRNAs in CC group compared with ESCC group, including miR-320b, miR-320c, miR-320a, miR-92a, miR-486-5p, miR-627, miR-365,miR-1826, miR-185,miR-129-star, miR-558,miR-629-star, the fold change located in 1.5162-3.7357;miR-1184 downregulated with 0.5927 times.3.2.3 Serum miRNA expression profiles of CC group compared with GCA groupWe screened a total of 11 upregulated miRNAs in CC group compared with GCA group,including miR-320b,miR-320c, miR-320a, miR-92,miR-486-5p, miR-638, miR-627,miR-365,miR-1826,miR-923,miR-149-star, the fold change located in 1.5505-6.1185.4 ConclusionsThe misdiagnosis rate for CC is very high. The incidence rate of CC is higher in the male than in the female, and the male has a higher rate of positive family history than female. The 5-year survival rate for CC patients is low with a poor prognosis.There are 8 upregulated miRNAs of CC including miR-320b,miR-320c,miR-320a,miR-92a,miR-486-5p,miR-627,miR-365 and miR-1826,these findings suggest that the miRNAs might play a significant role in exploring biomarkers and pathogenesis of CC.