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目的 :探讨微RNA-374b-5p(micro RNA-374b-5p,miR-374b-5p)及其靶基因反转录富含半胱氨酸蛋白(reversion-inducing-cysteine-rich protein with Kazal motifs,RECK)在胃癌发生和发展中的作用。方法 :采用人胃癌MGC-803细胞建立BABL/c裸鼠移植瘤模型,当移植瘤体积达到60 mm3左右时,将小鼠随机分为3组(每组5只),即空白对照组、阴性对照组[体内转染无意义微RNA序列(miR-374b-5p negative control inhibitor)]和miR-374b-5p抑制组(体内转染miR-374b-5p inhibitor),并观察各组移植瘤的生长情况。在接种MGC-803细胞后第35天时(最后一次治疗3 d后)处死各组小鼠,苏木精-伊红(hematoxylin-eosin,HE)染色观察移植瘤标本的病理学特征;实时荧光定量-PCR(real-time fluorogenic quantitative-PCR,RFQ-PCR)检测移植瘤组织中miR-374b-5p的表达水平;荧光素酶活性检测验证miR-374b-5p是否靶向作用于RECK基因;分别采用RFQPCR和免疫组织化学法检测移植瘤中RECK mRNA和蛋白的表达情况。结果 :miR-374b-5p抑制组移植瘤体积在转染miR-374b-5p inhibitor 9 d后(即接种细胞的第23天后),开始明显小于空白对照组和阴性对照组(P<0.05),而空白对照组和阴性对照组小鼠的移植瘤体积差异无统计学意义。病理学检测结果提示,miR-374b-5p抑制组移植瘤的恶性程度较空白对照组和阴性对照组明显下降。miR-374b-5p抑制组移植瘤中miR-374b-5p的表达水平均较空白对照组和阴性对照组明显下调(P<0.05)。荧光素酶报告载体系统证实,RECK是miR-374b-5p直接调控的靶基因;miR-374b-5p抑制组移植瘤中RECK m RNA及蛋白的表达水平均显著高于空白对照组和阴性对照组(P均<0.05)。结论 :miR-374b-5p可能通过RECK通路在胃癌的发生和发展中扮演着癌基因的角色。
Objective: To investigate the expression of microRNA-374b-5p (miR-374b-5p) and its target gene reverse transcription-rich cysteine protein (reversion-inducing-cysteine-rich protein with Kazal motifs, RECK) in the occurrence and development of gastric cancer. Methods: The BABL / c xenografts in nude mice were established by using human gastric cancer cell line MGC-803. When the volume reached about 60 mm3, the mice were randomly divided into 3 groups (5 in each group), namely blank control group, negative The control group (miR-374b-5p negative control inhibitor) and the miR-374b-5p inhibitor group (miR-374b-5p inhibitor transfected in vivo) were used to observe the growth of xenografts Happening. The mice in each group were sacrificed on the 35th day after the MGC-803 cells were inoculated (after the last treatment for 3 days), and the pathological features of the xenografts were observed by hematoxylin-eosin (HE) staining. Real-time fluorescence quantitative The expression level of miR-374b-5p was detected by real-time fluorogenic quantitative-PCR (RFQ-PCR). Luciferase activity was used to detect whether miR-374b-5p targeted the RECK gene. The expression of RECK mRNA and protein in the transplanted tumor was detected by RFQPCR and immunohistochemistry. Results: The tumor volume of miR-374b-5p group was significantly lower than that of the blank control group and the negative control group (P <0.05) after transfection of miR-374b-5p inhibitor for 9 days (ie after inoculation of cells on day 23) However, there was no significant difference in the volume of xenografts between the blank control group and the negative control group. The results of pathology suggested that the malignant degree of miR-374b-5p inhibited group was significantly lower than that of blank control group and negative control group. The miR-374b-5p expression level in miR-374b-5p group was significantly lower than that in blank control group and negative control group (P <0.05). Luciferase reporter vector system confirmed that RECK is the direct target of miR-374b-5p regulation; miR-374b-5p inhibited the expression of RECK m RNA and protein expression levels were significantly higher than the blank control group and negative control group (P <0.05). Conclusion: miR-374b-5p may play an oncogene role in the development and progression of gastric cancer through the RECK pathway.