差异表达的miR-223对胃癌细胞增殖、迁移功能的影响及其机制研究

发布时间：2019-05-24 15:02

【摘要】：背景胃癌是全球最常见的消化道恶性肿瘤之一。亚洲日本、韩国及我国是胃癌高发区,我国每年新发病例约40万例,占世界总发病例数的42%。早期胃癌患者术后5年生存率可达90%以上,但绝大多数胃癌在确诊时属进展期,或已经发生了肿瘤转移,5年生存率不足30%。要减少胃癌的死亡率,最有效的措施就是早发现和早治疗,分子生物学的发展为胃癌机制的研究提供了先进的技术手段和方法学,对胃癌治疗方案的制定具有重要的指导意义。miRNA是一类长约18~22个核苷酸的保守的非编码小分子RNA,miRNA在转录或转录后水平负调控蛋白质编码基因的表达:通过与其靶基因m RNA非完全互补或近似完全互补结合,导致m RNA降解或抑制其翻译。从miRNA被发现以来,许多研究报道了肿瘤组织中miRNA异常表达,并证实了miRNA与一些抑癌因子和促癌因子有着显著相关性。目的本研究通过检测胃癌患者癌组织与癌旁组织差异表达的miRNA,探讨其表达水平与肿瘤大小、TNM分期的相关性及与转移状态的相关性。采用生物信息学手段预测筛选出与胃癌侵袭和转移相关性最大的靶基因,并探讨其如何通过靶基因影响胃癌细胞的增殖和侵袭能力。方法(1)本研究选取3对胃癌和癌旁组织样本进行芯片检测,通过q RT-PCR方法对上一步筛选出的27个miRNA在胃癌组织及癌旁组织的表达水平进行验证。(2)为了探索miRNA与胃癌发生发展的关系,对发生转移和未发生转移的胃癌样本中的miR-223、miR-20a和miR-150这3种miRNA的表达情况以及患者的生存情况进行分析统计。实时荧光定量PCR检测84个已发生转移胃癌样品和48个未发生转移胃癌样品中miR-223、-20a和-150的的表达。(3)为了检测miR-223对胃癌细胞增殖能力的影响,选用GT3TKB和MKN45作为研究对象。首先,测定了原始的GT3TKB和MKN45中miR-223的表达水平。接下来在GT3TKB和MKN45细胞系中分别转染了化学合成的miR-223的拟似物(mimics)和抑制物RNA(inhibitor)。其次,对转染后的细胞进行Transwell小室侵袭实验及划痕愈合实验。(4)为了寻找miRNA-223的靶基因,用生物信息学手段预测并根据经验规则和已发表的研究成果筛选出与胃癌侵袭和转移相关性最大的靶基因PAX6。在GT3TKB和MKN45细胞系中分别转染了miR-223 mimics和miR-223 inhibitor,提取了转染后GT3TKB和MKN45以及对应的对照组细胞的总RNA,用RT-PCR方法检测PAX6的表达水平,并用Western blot方法检测PAX6的表达情况。进一步用q RT-PCR方法及Western blot方法检测132例临床胃癌组织样品中miR-223和PAX6表达水平,并进行两者相关性分析。结果(1)胃癌细胞中差异表达miRNA的筛选研究:结果显示,13个miRNA在胃癌组织中的表达显著上调,14个miRNA在胃癌组织中表达呈下调。进一步验证发现,有21个miRNA与芯片实验结果相符。其中10个在胃癌组织中高表达,11个在胃癌组织中低表达。6种miRNA的表达在胃癌与癌旁组织中的差异无统计学意义。其中,差异最显著的3个miRNA分别为miR-223、miR-20a和miR-150。(2)异常表达的miRNA在发生转移的胃癌组织中的表达情况及患者生存分析:结果显示,miR-223表达量与肿瘤大小、TNM分期显著相关(P0.05),其与转移状态的相关性极显著(P0.01);miR-20a与TNM分期显著相关(P0.05),与转移状态相关性极显著(P0.01);miR-150仅与TNM分期相关性显著(P0.05)。接着分析了76例具有严重的组织学性状的胃癌样品的miR-223表达水平,使用Kaplan-Meier生存分析对这些样品的miR-223与生存率相关性进行分析,结果显示miR-223低表达组的无病生存率显著高于miR-223高表达组。这些结果表明miR-223表达量与胃癌发展有关。(3)miR-223对胃癌细胞迁移和侵袭生物学行为作用的研究:通过实验发现高侵袭力的MKN45的miR-223表达水平比GT3TKB的显著上调(P0.01)。GT3TKB在转染miR-223的mimics后第3天增殖活性显著上调;与此相反,MKN45在转染了miR-223的inhibitor后第3天增殖活性降低。通过转染miR-223 mimics上调GT3TKB内源性miR-223的表达,相同倍数显微镜下,我们观察到穿过侵袭小室基质胶的转染mimics的GT3TKB细胞比对照组GT3TKB细胞密集,数量也显著增多(150:100;p0.01),提示侵袭能力提高。相反地,利用miR-223 inhibitor下调MKN45的miR-223表达后,其穿过基质胶的细胞数少于对照组(150:200;p0.05),表明侵袭力下降。转染了miR-223 mimics的GT3TKB培养1天后,划痕愈合情况明显优于对照组,说明miR-223提高GT3TKB的划痕愈合速度,即转移能力;相反,转染了miR-223 inhibitor的MKN45在培养1天后,划痕愈合情况明显比对照组差,表明了miR-223在MKN45的划痕愈合中起到重要作用。(4)miR-223促进胃癌细胞迁移和侵袭的分子机制研究:结果显示转染了miR-223mimics的GT3TKB细胞系的PAX6表达水平显著低于未转染的GT3TKB对照组;而转染了miR-223 inhibitor的MKN45细胞系的PAX6表达水平高于对照组MKN45。Western blot的实验也显示出与此一致的结果:PAX6在miR-223上调的胃癌细胞中表达量下降,两者呈负相关。结论(1)胃癌组织中差异表达的miRNA共21个,差异最显著的miRNA为miR-223、miR-20a和miR-150。(2)miR-223表达水平与肿瘤大小、TNM分期显著相关,其与转移状态的相关性极显著;miR-20a与TNM分期显著相关,与转移状态相关性极显著;miR-150仅与TNM分期相关性显著。进一步分析显示miR-223的高表达与组织学性状的严重程度和低生存率有关。(3)miR-223可促进胃癌细胞的增殖,并促进胃癌细胞侵袭和转移。(4)PAX6是miR-223在胃癌细胞中的靶基因。(5)miR-223可通过下调PAX6,增强胃癌细胞的增殖和侵袭能力。因此,miR-223和PAX6可能成为胃癌治疗的靶点。
[Abstract]:Background Gastric cancer is one of the most common malignant tumors in the world. In Asia, Japan, South Korea and our country are the high-risk areas of the stomach cancer, and about 400,000 new cases in our country each year, accounting for 42% of the total number of cases in the world. The 5-year survival rate of the patients with early gastric cancer can reach more than 90%, but most of the gastric cancer is in the progress of the diagnosis, or the tumor metastasis has occurred, and the 5-year survival rate is less than 30%. To reduce the mortality of gastric cancer, the most effective measure is the early detection and early treatment, the development of molecular biology provides the advanced technical means and methodology for the research of the gastric cancer mechanism, and has important guiding significance for the development of the gastric cancer treatment plan. The miRNA is a conserved non-coding small-molecule RNA of about 18-22 nucleotides, and the miRNA regulates the expression of the protein-encoding gene at the post-transcriptional or post-transcriptional level: the m-RNA is degraded or suppressed by a non-fully complementary or nearly completely complementary combination with the target gene m RNA. Since miRNAs have been found, many studies have reported the abnormal expression of miRNAs in tumor tissues, and confirmed that miRNAs have a significant correlation with some of the cancer-inhibiting factors and the cancer-promoting factors. Objective To study the correlation between the expression level and the tumor size, TNM stage and the correlation between the tumor size and TNM stage by detecting the expression of the differentially expressed miRNA in the cancer tissue and the adjacent tissue of the gastric cancer. Bioinformatics is used to predict the target gene which is the most relevant to the invasion and metastasis of gastric cancer, and how to influence the proliferation and invasion ability of gastric cancer cells through the target gene. Methods (1) In this study,3 samples of gastric cancer and non-cancerous tissue were tested, and the expression levels of 27 miRNAs screened by the previous step were verified by the q-RT-PCR method. (2) In order to explore the relationship between the development of miRNA and gastric cancer, the expression of miR-223, miR-20a and miR-150 in the sample of gastric cancer with metastasis and metastasis and the survival condition of the patient were analyzed. The expression of miR-223,-20a and-150 in 84 cases of metastatic gastric cancer and 48 non-metastatic gastric cancer samples was detected by real-time fluorescence quantitative PCR. (3) To detect the effect of miR-223 on the proliferation of gastric cancer cells, GT3TKB and MKN45 were used as the research object. First, the level of expression of miR-223 in the original GT3TKB and MKN45 was determined. The mimetics of the chemically synthesized miR-223 and the inhibitor RNA were subsequently transfected into the GT3TKB and the MKN45 cell lines, respectively. Secondly, the transwell-cell invasion experiment and the scratch-healing experiment were performed on the transfected cells. (4) In order to find the target gene of the miRNA-223, the target gene PAX6 with the largest correlation with the invasion and metastasis of the gastric cancer was selected by means of bioinformatics and according to the experience rules and the published research results. The expression level of PAX6 was detected by RT-PCR and the expression of PAX6 was detected by Western blot. The expression levels of miR-223 and PAX6 in 132 patients with gastric cancer were detected by the method of q-RT-PCR and Western blot. Results (1) The screening of differentially expressed miRNAs in gastric cancer cells showed that the expression of 13 miRNAs in gastric cancer was up-regulated and 14 miRNAs were down-regulated in gastric cancer tissues. Further validation found that 21 miRNAs were in line with the results of the chip test. Among them,10 of them were highly expressed in gastric cancer tissues and 11 were low-expression in gastric cancer tissues. The expression of 6 miRNAs was not statistically significant in gastric cancer and adjacent tissues. Among them, the most significant three miRNAs were miR-223, miR-20a and miR-150, respectively. (2) The expression of the abnormal expression of the miRNA in the tissue of the gastric cancer with metastasis and the survival analysis of the patient: The results showed that the expression of miR-223 was significantly correlated with the size of the tumor and the TNM stage (P0.05). The correlation between the expression of the miR-223 and the metastasis was very significant (P0.01), and the miR-20a was significantly related to the TNM stage (P0.05). The correlation between miR-150 and TNM was significant (P 0.01), and the correlation between miR-150 and TNM was significant (P0.05). The miR-223 expression level of 76 gastric cancer samples with severe histological characteristics was then analyzed, and the correlation between the miR-223 and the survival rate of these samples was analyzed using the Kaplan-Meier survival analysis, and the results showed that the disease-free survival rate of the miR-223 low-expression group was significantly higher than that of the miR-223 high-expression group. These results show that the expression of miR-223 is related to the development of gastric cancer. (3) The study of the effect of miR-223 on the migration and invasion of gastric cancer cells: The expression of miR-223 of MKN45 with high invasiveness was significantly up-regulated than that of GT3TKB (P0.01). The proliferation activity of MKN45 on day 3 after transfection of an inhitor of miR-223 was reduced. The expression of the endogenous miR-223 of the GT3TKB was up-regulated by transfection of miR-223 misitics, and under the same multiple microscope, we observed that the GT3TKB cells transfected with mimics, which passed through the matrix of the invasion cell, were more dense and the number of GT3TKB cells in the control group (150:100; p0.01), suggesting an increase in the invasion ability. On the contrary, after the miR-223 expression of MKN45 was down-regulated by miR-223 inhitor, the number of cells that passed through the matrix gel was less than that of the control group (150:200; p0.05), indicating a decrease in the invasion force. After 1 day after transfection of the GT3TKB of miR-223 misitics, the scratch-healing condition was better than that of the control group, which indicated that miR-223 increased the scratch-healing speed of the GT3TKB, that is, the transfer capacity; on the contrary, the MKN45 transfected with the miR-223 inhitor was significantly worse than the control group after 1 day of culture, It is shown that miR-223 plays an important role in the scratch healing of MKN45. (4) The molecular mechanism of miR-223 to promote the migration and invasion of gastric cancer cells showed that the expression of PAX6 of the GT3TKB cell line transfected with miR-223mmics was significantly lower than that of the untransfected GT3TKB control group. The expression of PAX6 in the MKN45 cell line transfected with miR-223 inhitor was higher than that of the control group (MKN45). Conclusion (1) There are 21 miRNAs differentially expressed in gastric cancer, and the most significant miRNAs are miR-223, miR-20a and miR-150. (2) The expression level of miR-223 was significantly correlated with tumor size and TNM stage, and the correlation between miR-20a and TNM was very significant, and the correlation between miR-20a and TNM was very significant; and miR-150 was associated with TNM stage only. Further analysis showed that the high expression of miR-223 was related to the severity of the histological character and the low survival rate. (3) miR-223 can promote the proliferation of gastric cancer cells and promote the invasion and metastasis of gastric cancer cells. (4) PAX6 is the target gene of miR-223 in gastric cancer cells. (5) The miR-223 can enhance the proliferation and the invasion ability of the gastric cancer cells by down-regulating the PAX6. Therefore, miR-223 and PAX6 may be the target for gastric cancer treatment.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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