MiR-155-5p Expression And Its Role In Esophageal Squamous Cell Carcinoma

IntroductionEsophageal cancer is one of the most common cancers occurring in human, ranking the 6th worldwide with a high mortality rate. According to the latest statistics of WHO, approximately 80% of the esophageal cancer occurs in the developing countries, among which China belongs to the most prevalent one. Meantime, China has become one of the countries with high mortality rate. In China, almost 150 thousand patients died annually owning to esophageal cancer. In histology, esophageal cancer can be classified into two subtypes, namely esophageal squamous cell carcinoma and esophageal adenocarcinoma with a regional difference. The majority of esophageal cancer in our country belongs to esophageal squamous cell carcinoma, whereas esophageal adenocarcinoma in occident. With the character of high invasion and metastasis, esophageal cancer turns out to be difficult to be diagnosed until to the late stage, resulting in a relatively high postoperative mortality and only 15%-25% of five-year survival rate. Vital approaches to solve the low survival rate of late stage esophageal cancer are early detection, early diagnosis and early treatment. Up to date, five diagnostic methods are available:fiberscope detection; esophageal endoscopic ultrasonography; Cytological examination of esophagus; X-ray barium meal and CT scan of the chest. All the above approaches cannot detect early stage esophageal cancer effectively. Early diagnosis is the key for patients survival, and the detection of esophageal cancer marker miRNA genes or signals has important significance for false positives and unnecessary examination and treatment. Therefore, further exploration of the molecular mechanism underlying the development of esophageal cancer is still in urgent need to improve the early diagnosis rate and cure rate.Micro-RNA regulation has been regarded as the frontier of life science research. In mammal animals, mi-RNAs play a significant role in metabolism, differentiation, signal transduction, apoptosis and immune response and so on. And studies have suggested an important regulatory role of miRNA in the occurrence, development, metastasis, invasion and angiogenesis of cancer. Thus, research aiming at explaining the relationship between esophageal cancer and relative miRNA would contribute to our understanding of the pathogenesis of esophageal cancer, as well as the improvement of diagnostic technique, which may accelerate the application of this technology in clinical diagnostics and biomedical applications.MicroRNA (miRNA) is a class of non-encoding single chain small molecule RNA containing 18-25 nucleotides. Through binding to the 3-UTR or other complementary parts of target mRNA molecules, miRNAs could inhibit or enhance specific gene expression to participate the regulation of cell growth, development and differentiation. miRNAs play an important role in cell differentiation and proliferation, affecting cell apoptosis and metabolism as well. miRNAs could be regarded as oncogenes or tumor suppressor genes. Until now, no less than ten miRNAs have been investigated to have a close relationship with the occurrence, development, metastasis and apoptosis of many kinds of tumor, esophageal cancer included. However, the specific mechanism of the regulation of miRNA on gene expression still remains to be elucidated.MiR-155 is a typical multifunction miRNA. An increasing number of evidences have demonstrated that miR-155 exerts significant effect on cell metabolism, differentiation, apoptosis and immune response and so on. MiR-155 is located in the third exon of BIC that is situated in the non-coding region of the 21st chromosome, which was regulated by the translational level of BIC and miRNA process. BIC is a gene without open reading frame, with the overexpression of which could promote the abnormal cell proliferation. Studies have confirmed that miR-155 promote myc transcription and cell proliferation via inhibiting MX11 and MAD1 expression. Eis et al. have found that the expression level of miR-155 and BIC increased significantly in Diffuse large B lymphoma and hodgkin lymphoma. Results of a vast majority of researches turn out that miR-155 was overexpressed in various kind of tumor, playing an vital role in tumor occurrence and development. In addition, miR-155 functions as a cancer gene. Reportedly, the level of miR-155-5p increased in breast cancer, cancer of colon, lung cancer, carcinoma of endometrium and pancreatic cancer, and has a close relationship with the occurrence, development and prognosis of the tumor. Recently, Liu Hui et al. found that miR-155-5p was overexpressed in esophageal cancer, but with an unknown mechanism. In the present study, we applied qPCR to detect the level of miR-155-5p in esophageal squamous cell carcinoma. In addition, we also use miR-155 analogues and inhibitors to up-regulate and down-regulate the expression level of miR-155-5p in Eca109 cell lines to investigate its role in the proliferation, apoptosis and migration of esophageal squamous cell.Materials and Methods1.Sample collectionWe collect the esophageal squamous cell carcinoma tissue and the normal tissue adjacent to the diseased esophagus more than 5cm from 20 patients who underwent surgery in fengxian District Central Hospital of Shanghai and Shanghai Renji Hospital from December 2014 to June 2015. The age of the patients range from 43 to 74, and the mean age is 58.5. All of the patients involved did not have chemotherapy or radiotherapy before surgery, nor did the lymph node metastasis or distant metastasis was found. After admission, all patients went surgery, during which the esophageal cancer tissue and the adjacent normal tissue more than 5cm to the diseased esophagus were collected. All the specimens were frozen in liquid nitrogen. All 20 patients were diagnosed esophageal squamous cell carcinoma (ESCC).2.Cell cultureEsophageal squamous cell carcinoma cell line Eca109 was purchased from cell bank of Chinese Academy of Sciences, and were routinely grown in RPMI1640 medium supplement with 10% heat-inactivated bovine serum and 1% penicillin-streptomycin, cultured at 37℃ under humidified 5% CO2 atmosphere. When reached 80%, cells were digested with 0.25% trypsin at 37℃ for 10 minutes, followed by cell subculture or experiments.3.Detection of miR-155-5p level by real time fluorescent quantitative PCRWe measure the quality and concentration of total RNA after extraction. Then the reverse transcription was applied according to the instruction of miRNA cDNA Synthesis kit, and the specific primer of U6 was used as internal reference.4. Cell transfectionmiR-155-5p analogues, inhibitor, mimic-NC and inhibitor-NC was designed by Guangzhou Rui Bo Biological Technology company. We will divide human esophageal squamous carcinoma cells Eca109 into five groups, as follows: miR-55-5p mimic analog group (Lipofectamine2000+miR-55-5p mimic); NC (mimic) negative control group (Lipofectamine2000+NC (mimic)); miR-55-5p inhibitor inhibitor group (Lipofectamine2000+miR-55-5p inhibitor); NC (inhibitor) negative control group (Lipofectamine2000+NC (inhibitor)); untransfected group blank control group (plus equal only Lipofectamine2000 amount of liposomes).Cells were seeded into 6 well plate and went transfection by lipofectamine TM2000 after reaching 70-80%.5.Detection of miR-155-5p level in Eca 109 cells after transfected with miR-155-5p by real time fluorescent quantitative PCRWe collected all groups of cells after transfection for 24h in procedure 4, and detected the expression level of miR-155-5p according qPCR kit instructions.6.Cell proliferation assay(CCK8)We will divide human esophageal squamous carcinoma cells Eca109 into five groups.Cells were transfected with miR-155-5p mimic, NC (mimic)、inhibitor and NC (inhibitor) respectively with 5 holes each group. We used cells without transfection for blank control. Then all group of cells were seeded into 96 well plate after transfection for 6h, and went on culturing for Id,2d,3d respectively. The proliferation rate of all cells were detected by CCK8 kit according to its instruction and the OD value was measured at 450 nm.7. Cell apoptosis (Annexin V-FITC/PI)We will divide human esophageal squamous carcinoma cells Eca109 into five groups.After collecting Eca 109 cells after transfection for 24h, we centrifugated all group of cells at 1000 r/min for 5minntes. After collecting (1-5) x 105, we detected cell apoptosis rate according to the instruction of Annexin V-FITC/PI kit by using flow cytometry within 1h.8.The scratch experimentWe will divide human esophageal squamous carcinoma cells Eca 109 into five groups. All group of cells were seeded into 6 well plate and scratching in each of the wells using spearhead after transfection for 24h. Wash the shedding cells by medium without serum and then culture the cells with RPMI-1640 medium without serum at 37℃ under humidified 5% CO2 atmosphere. Then take photograph after scratching for 0 and 24h to evaluate the scratch healing ability.^StatisticsWe calculate the miRNAs copies of all groups by using Ct value after the differentially expressed miRNA screened by qPCR. The formula was expressed as 2-ΔΔCt(ΔCt= Ctsample-CtU6, ΔΔCt=Ctcancer-Ctnormal) Pairing (normal distribution) data using paired t-Test, Pairing (non normal distribution) data using U Mann-Whitney rank sum test. Single factor analysis of variance was used to compare the mean of multiple groups. We use SPSS 17.0 software for statistical analysis, and p<0.05 means the re statistics was significant.Results1.The level of miR-155-5p is higher in squamous cell carcinoma of esophagus than that of tissues adjacent to tumor.We will divide human esophageal squamous carcinoma cells Eca109 into five groups.We applied qPCR method to detect the level of miR-155-5p in squamous cell carcinoma of esophagus and in tissues adjacent to tumor from 20 patients. Compared to the adjacent tissues, the level of miR-155-5p in squamous cell carcinoma of esophagus in much higher (529.42%) ((P<0.01)).2.The transfection efficiency of miR-155-5p in Eca-109 cell lines.We will divide human esophageal squamous carcinoma cells Eca109 into five groups.Eca-109 cells were transfected with 50 nmol/L miRNA-155-5p analogue and 150 nmol/L inhibitor for 24h. After transfection, the level of miRNA-155-5p was measured by qPCR3. The effect of miR-155-5p on cell viability of esophageal squamous cell carcinoma cell line Eca109We will divide human esophageal squamous carcinoma cells Eca109 into five groups.After the transfection of miR-155-5p analogues and its inhibitors on Ecl109 cells for 24h,48h and 72h respectively, we measured the cell viability by using CCK8 methods. Our results showed that there were no significant difference of cell viability between all the transfection groups and the blank control (100%). This results implied that miR-155-5p analogues and its inhibitors may exert no effect on cell proliferation.4. The effect of miR-155-5p on apoptosis of esophageal squamous cell carcinoma cell line Eca109We will divide human esophageal squamous carcinoma cells Eca109 into five groups.After treating under various conditions, all groups of cells were collected to stained with Annexin V-FITC/PI, detecting cell apoptosis by flow cytometry. The results showed that the apoptotic rate was 5.43%±3.09% after transfected with miR-155-5p analogues, comparing to that was 5.67%±1.99% of control group (P>0.05). Moreover, it was also no significant difference between the inhibitor group (5.26%±1.98%,) and the control group (P>0.05). These results suggested that miR-155-5p had no obvious effect on cell apoptosis after transfected into Eca 109 cell for 24h.5. The effect of miR-155-5p on migration of esophageal squamous cell carcinoma cell line Eca109The scratch experiment indicated that the migration ability of Eca 109 was significantly improved after cells transfected with miR-155-5p analogues for 24h (p<0.05), while there was no big difference between the inhibitor group or the control (P>0.05). All the results suggested that enhanced miR-155-5p expression would promote Eca 109 cell migration.Conclusions1.The expression level of miR-155-5p was overtly increased, which may be related to the occurrence and development of esophageal squamous cell carcinoma.2.Enhanced expression of miR-155-5p have no effect on proliferation and apoptosis of Eca 109, but significantly accelerated cell migration3.Down expression of miR-155-5p have no effect on proliferation,apoptosis and migration of Eca 109.4.Change of miR-155-5p expression probably related to metastasis of esophageal squamous cell carcinoma.