摘要
背景和目的
食管癌是全球第四大恶性肿瘤,在全球许多地区流行,特别是在发展中国家。食管癌高发区在我国从中国东北部延伸至中东部地区。不同的国家和在同一国家的不同地区发病情况差别悬殊,可相差100-200倍以上。在中国的河南、河北、江苏等地区,其发病率、死亡率在各种肿瘤中高居首位。我国对如何治疗食管癌已经列为了研究的重点,但目前任何一种治疗方法和手段都不能令人满意,其5年生存率均未达到50%,所以积极寻找新的治疗手段已成为亟待解决的问题。
miR-21是由约22个核苷酸组成的参与转录后基因调控的非编码小分子RNA。人们推测它们在细胞的分化、增殖、迁移、代谢和死亡中起重要调节作用。近几年有研究报道miR-21在食管癌组织中高表达,且随着恶性肿瘤靶向治疗的进展,miR-21也成为多种癌症基因治疗中的一个关键靶点。反义核酸是指与靶DNA或RNA碱基互补,并能与之结合的一段DNA或RNA。反义核苷酸技术是指利用反义核酸特异性地抑制某些基因的表达。经过对多种反义核酸的研究,人们发现反义寡核苷酸(Antisense oligodeoxy- nucleotides, ASODNs)能较好的穿过细胞膜到达靶位点,从而成功的转染细胞。已有研究表明,激活的Ras蛋白的突变与多种恶性肿瘤密切相关,尤其是细胞增殖、转化、侵袭和转移。而在一些肿瘤细胞中K-ras的表达受miR-21调控。在肺癌中,过表达的miR-21能在体内增强肿瘤的致瘤性,而缺失miR-21从某种程度上来说抑制肿瘤的生长,这些通过调控K-ras的表达来实现。然而,迄今为止,miR-21在食管癌中是通过什么机制起作用国内外尚未见报道。为了初步了解miR-21在食管鳞癌中的可能作用,本研究首先采用原位杂交技术检测食管鳞癌组织中K-ras和miR-21的表达,接着采用免疫组化分析食管癌组织中K-ras蛋白的表达。进一步从细胞水平利用反义核苷酸技术抑制miR-21的表达来分析食管癌组织中K-ras mRNA和蛋白的表达,该研究有望为寻求食管癌等恶性肿瘤的临床治疗新方法提供理论依据。
材料和方法
1.材料：40例食管鳞癌组织、40例正常食管黏膜组织为河南省安阳市肿瘤医院2009年1-2010年7月间新鲜手术切除标本,食管癌细胞株EC9706由中国医学科学院肿瘤医院分子肿瘤学国家重点实验室惠赠。
2.设计合成1条miR-21反义寡核苷酸(ASODN)及1条无关序列寡核苷酸(N-ODN);分别采用150μg/m1、200μg/m1、250μg/ml 3种不同浓度的miR-21ASODN和N-ODN体外分组转染培养的食管癌EC9706细胞株24h、48h和72h。另设无关系列组及空白对照组。
3.采用原位杂交法检测食管鳞状细胞癌组织中miR-21及K-ras的表达。采用免疫组化方法检测食管鳞状细胞癌组织中K-ras蛋白的表达。
4.采用免疫细胞化学方法及完整细胞原位杂交方法观察各组miR-21 ASODN转染后的EC9706细胞中K-ras蛋白及mRNA的表达。
5.采用RT-PCR和Western blotting技术检测食管鳞癌中转染miR-21 ASODN后,Kas mRNA和蛋白相对表达的变化。
6.运用SPSS 13.0统计软件进行统计学分析。阳性率之间的比较采用χ2检验；计量资料以均数标准差(X±S)表示,两组均数的比较运用t检验(t-test);两组以上均数比较采用方差分析(ANVOA);以a=0.05为显著性检验标准。
结果
1. K-ras蛋白阳性表达位于食管癌EC9706细胞的细胞质中,呈棕黄色颗粒；K-ras mRNA定位于食管癌EC9706细胞细胞质内,呈蓝紫色颗粒。
2.食管癌组织、正常食管粘膜组织中miR-21的表达率分别为77.5%和25.0%,两组间比较,差异具有统计学意义(P<0.05)。
3.食管癌组织、正常食管粘膜组织中K-ras的蛋白阳性表达率分别为67.5%和5.0%,两组间比较,差异具有统计学意义(P<0.05)。
4.食管癌组织、正常食管粘膜组织中K-ras的mRNA阳性表达率分别为62.5%和0.0%,两组间比较,差异具有统计学意义(P<0.05)。
5.3种不同浓度的niR-21ASODN对食管癌EC9706细胞中K-ras的蛋白及mRNA表达均有显著的抑制作用,且以浓度为250gg/ml的miR-21ASODN作用72h抑制效应最强；但不同浓度的niR-21 ASODN之间两两比较,差异具无统计学意义(P>0.05)。各实验组与N-ODN转染组或对照组两两相比,差异均具有统计学意义(P<0.05)。
6.半定量RT-PCR和Western blotting的结果表明：转染miR-21 ASODN后,EC9706细胞中K-ras mRNA和蛋白的相对表达均显著降低(P<0.05),而未处理组和无义组之间K-ras的表达无差异(P>0.05)。
结论
1.miR-21及K-ras可能参与了食管鳞癌发生、发展的过程。
2.miR-21表达的下调可抑制食管鳞癌EC9706细胞中K-ras的表达。
Background and objective
Esophageal 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 methods
1. 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.
Results
1. 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).
Conclusion
1. 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.
引文
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