| Background and objectiveEsophageal cancer is the world's fourth largest malignant esophageal disease, endemic in many parts of the world, especially in developing countries. "Esophageal cancer belt" extends from northeast China to the Middle East. Different countries and different regions in the same country differences in the incidence of the poor, can be a difference of 100-200 times. In China, Henan, Hebei, Jiangsu, Shanxi, Shaanxi, Anhui, Hubei and Sichuan provinces, the incidence and mortality in a variety of tumors, topped the rankings. Our country how to treat cancer have been designated as the focus of the study, but there is not one satisfactory kind of diagnostic methods and treatments. The 5-year survival rates were not above 50% actively, so looking for new treatments has become an urgent problems.miRNA-21 are non-coding small molecule RNA, composed of about 22 nucleotides involved in regulating gene transcription. People speculated that they play an important regulatory role in cell differentiation, proliferation, migration, metabolism, and death. Volinia foundmiR-20a, miR-21, miR-17-5p and so on were positively correlated with incidence of cancer on the study of lung, breast, gastric cancer cells. In recent years, studies have reported that miR-21 is highly expressed in esophageal cancer, and with the progress in cancer targeted therapy, miR-21 has become a key target genes in multi-cancer gene therapy. Antisense nucleic acid can be the target complementary with DNA or RNA bases and can bind to a DNA or RNA. Antisense technology is the use of antisense nucleic acids specifically inhibit the expression of certain genes. Through a variety of antisense research, it was discovered that antisense oligonucleotide (Aantisense oligodeoxy-nucleotides, ASODNs) can better reach the target site through the cell membrane, thus successfully transfected cells. The activating mutations of ras proteins have been previously implicated in all aspects of the malignant tumor, especially cellular proliferation, transformation, invasion as well as metastasis. Some studies have demonstrated that K-ras expression was regulated by miR-21. In Lung carcinoma, overexpression of miR-21 enhances tumorigenesis and that genetic deletion of miR-21 partially protects against tumor formation, which was achieved by regulation of K-ras expression. However, to date, there was no report about the role of miR-21 in ESCC, therefore, the purpose of the current study was to investigate the K-ras and miR-21 mRNA expressions in ESCC tissues by in situ hybridization, subsequently, K-ras protein was analyzed by immunohistochemistry methods. Further, anti-miR-21 sequences were transfected to EC9706 cells, and K-ras protein expression was detected by immuocytochemistry, in situ hybridization, semi-quantitative RT-PCR and Western blot methods. These findings will provide a theoretical basis for seeking new methods of clinical therapy.Materials and methods1. Materials:40 patients with esophageal squamous cell carcinoma,40 cases of normal esophageal mucosa were from the surgical resection specimens of An Yang Tumor Hospital from January 2009 to July 2010. Esophageal cancer cell line EC9706 was affored by the CAS Shanghai Institute of Cell Biology.2. Designed and synthesized miR-21 antisense oligonucleotide (ASODN) and an unrelated sequence oligonucleotide (N-ODN); separately used 3 different concentrations (150μg/ml,200μg/ml,250μg/ml)of miR-21 ASODN 1 and N-ODN to in vitro transfected cultured esophageal EC9706 group cells for 24h,48h and 72h. Seted up independent series of separate group.3. Observed miR-21 and K-ras mRNA expression in EC9706 cell by using in situ hybridization method in esophageal squamous cell carcinoma tissues. Observed K-ras protein expression in EC9706 cell by using immunocytochemistry and in situ hybridization methods.4. Observed K-ras protein and mRNA in EC9706 cells by using immuno-cytochemistry and in situ hybridization method in three group cells.5. K-ras mRNA and protein expressions were detected using RT-PCR and Western blot methods.6. Used SPSS 13.0 statistical software for statistical analysis, usedχ2 test for comparison between positive rates; measurement data was figured by mean standard deviation (X±S), and the means of groups were compared using t test (t-test); more than two groups were compared by using analysis of variance several (ANVOA);α= 0.05 is for the significance test.Results1. K-ras protein expression was localized in the cell nuclear of esophageal cancer EC9706 cells, showing brownish yellow granules; and K-ras mRNA was localized in the cytoplasm of esophageal cancer EC9706 cells, showing blue-purple granules.2. Esophageal cancer tissue and normal esophageal mucosa miR-21 expression were 77.5% and 25.0%, respectively, and the difference was statistically significant of two sets of data (P<0.05).3. K-ras protein expression was 62.50% and 0.00% in the esophageal cancer tissue and normal esophageal mucosa, respectively.4. K-ras mRNA expression was 62.50% and 0.00% in the esophageal cancer tissue and normal esophageal mucosa, respectively, and the difference was statistically significant of two sets of data (P<0.05).5.3 different concentrations of miR-21 ASODN on esophageal cancer EC9706 cells,the expressions of K-ras mRNA and protein were significantly inhibited. To 250μg/ml concentration for 72h of inhibitory effect of antisense oligonucleotide was the strongest; but different concentrations different miR-21 ASODN comparison between two, the difference was not statistically significant (P>0.05). N-ODN transfected group and control group by 3 different antisense oligonucleotide transfection compared to eath other in each experimental group, the differences were statistically significant (P<0.05).6. The results of semi-quantitative RT-PCR and Western blot demonstrated that the expressions of K-ras mRNA and protein were significantly decreased after transfection with miR-21ASODN (P<0.05), however, there was no difference between untreated group and nonsense group (P>0.05).Conclusion1. miR-21 and K-ras possibly participate in the occurrence and development of invasive ductal carcinoma.2. Down-regulation of miR-21 expression obviously inhibits the expression of K-ras on EC9706 cells. |
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