MicroRNA-27a通过靶向PHLPP2促进胃癌细胞增殖和转移的机制研究

发布时间：2018-04-04 01:31

本文选题：miR-27a　切入点：PHLPP2　出处：《吉林大学》2017年博士论文

【摘要】：背景:胃癌是消化系统常见的恶性肿瘤之一。近来虽然胃癌预防诊治方面的进步增加了早期胃癌的存活率,但晚期胃癌是不可治愈的,死亡率仍然很高。胃癌细胞的过度增殖、侵袭转移、术后复发是胃癌患者死亡的主要原因。胃癌的发生是一个多因素参与、多步骤完成的生物化学变化过程。其过程涉及多个分子作用机制。因此探索阐明胃癌发生发展的分子机制有着重要的意义。令人信服的证据已经证实mi RNAs能够调节大多数肿瘤相关的生物学行为,包括细胞增殖、凋亡、分化、转移等。一般来说,mi RNAs通过抑制靶基因来行使它的功能。Micro RNA-27a(mi R-27a)在不同肿瘤中的作用是不同的。在一些肿瘤中它被认为是促癌的,例如骨肉瘤、喉癌。然而,在食管鳞状细胞癌和结肠癌中它被认为是抑癌的。越来越多的证据表明,mi R-27a在肿瘤的发生和进展中起着重要的作用,然而mi R-27a在胃癌中的作用还不清楚。mi R-27a在胃癌中的功能和其分子机制需要深入地探索。PH Domain Leucine-rich Repeat Protein Phosphatase2(PHLPP2)是一种蛋白磷酸酶,发挥着抑癌作用。它被报道可以引起细胞周期阻滞和凋亡并且在多种癌症中抑制肿瘤的转移。PHLPP2可以对Akt进行去磷酸化作用,进而抑制PI3K/Akt信号通路。在这里,我们探索了PHLPP2是否是mi R-27a的靶基因。我们的研究表明了mi R-27a在胃癌中通过靶向PHLPP2调控Akt通路来行使其促进胃癌细胞增殖和转移的功能。方法:应用Real-time PCR的方法来检测胃癌组织和胃癌细胞中mi R-27a和它的靶基因的表达水平。Mi R-27a在胃癌中的功能通过体外和体内实验来检测(MTT实验、克隆形成实验、流式细胞术、划痕愈合实验、transwell迁移和侵袭实验、免疫组化实验、免疫荧光实验、Western blot实验以及裸鼠成瘤实验)。应用荧光素酶报告实验对mi R-27a的靶基因进行验证。结果:第一部分Mi R-27a促进胃癌细胞的增殖和转移我们发现mi R-27a在胃癌组织中的表达水平高于与之相对应的癌旁组织。高水平的mi R-27a与肿瘤的临床分期、浸润、淋巴结转移和远端转移有关。在胃癌细胞系中mi R-27a的表达水平也普遍升高。我们选择mi R-27a表达相对较高和相对较低的SGC-7901和AGS细胞系进行后续的实验。我们进一步进行了相关的功能实验,来研究mi R-27a对胃癌细胞恶性生物学行为的影响。在SGC-7901细胞中,下调mi R-27a可以抑制胃癌细胞的增殖、克隆形成、划痕愈合、transwell迁移侵袭以及抑制胃癌细胞的EMT,使细胞周期阻滞,促进凋亡,同时影响Akt通路的相关蛋白的表达。在AGS细胞中,上调mi R-27a可以得到相反的结果。而且,裸鼠成瘤实验也显示下调mi R-27a可以抑制移植瘤的生长和转移。第二部分Mi R-27a在胃癌中靶向调控PHLPP2为了探索mi R-27a在胃癌中发挥作用的潜在的分子机制,我们运用生物信息学工具找到了mi R-27a的靶基因和相应的靶点。在预测的靶基因中我们选中了PHLPP2进行研究,因为有报道它在抑制肿瘤发展和调节细胞增殖迁移方面有重要的作用。荧光素酶报告实验显示在AGS细胞中,转染了mi R-27a agomirs的细胞与对照组相比,荧光素酶活性显著下降。在AGS细胞中上调mi R-27a,我们发现PHLPP2的蛋白水平和m RNA水平均下降。在SGC-7901细胞中下调mi R-27a时,可以看到相反的结果。此外,在胃癌组织中,mi R-27a的表达与PHLPP2成显著负相关。PHLPP2的低表达与胃癌的临床分期、浸润、淋巴结和远端转移相关。第三部分Mi R-27a通过靶向PHLPP2调控Akt通路蛋白参与胃癌的进展接下来,我们研究了在胃癌的恶性发展中,mi R-27a靶向抑制PHLPP2发挥了何种作用。为了验证mi R-27a是否通过PHLPP2行使功能,SGC-7901细胞共转染了mi R-27a antagomir和PHLPP2 si RNA。结果显示PHLPP2的敲减可以减轻mi R-27a antagomir对胃癌细胞增殖、凋亡、迁移和侵袭的抑制作用。我们的结果发现了mi R-27a的下调可以引起p-Akt、Cyclin D1、Vimentin、snail下调,p21、p27和E-cadherin上调,但PHLPP2的敲减可以减弱这些改变。这说明了PHLPP2/Akt轴参与了mi R-27a对胃癌细胞的调控。结论:总的来说,我们目前的研究证实了mi R-27a在胃癌中通过新的靶点PHLPP2发挥促癌效应。下调mi R-27a能够抑制多种恶性生物学行为,包括肿瘤细胞的增殖、迁移和侵袭。沉默PHLPP2能削弱mi R-27a下调引起的恶性生物学行为的改变。也就是说,mi R-27a能通过抑制PHLPP2调控Akt/GSK3β通路促进胃癌细胞的增殖和迁移。综上所述,我们的结果表明了mi R-27a在胃癌中发挥促癌基因的作用,并且阐述了其发挥作用的可能机制。随着研究的深入,mi R-27a可能成为胃癌防治的一个新靶标。
[Abstract]:Background: gastric cancer is one of the most common malignant tumor of the digestive system. Although recent advances in diagnosis and treatment of gastric cancer has increased the survival rate of early gastric cancer, but advanced gastric cancer is incurable, mortality is still very high. The excessive proliferation of gastric cancer cell invasion and metastasis, recurrence is the major cause of death in patients with gastric cancer gastric cancer. There is a multi factor, biochemical changes many steps to complete the process. The process involves multiple molecular mechanisms. So the study has important significance to elucidate the molecular mechanism of the occurrence and development of gastric cancer. The compelling evidence has confirmed that MI RNAs can regulate the biological behavior of most tumor related, including cell proliferation, apoptosis, differentiation, metastasis and so on. In general, MI RNAs through the inhibition of target gene to exercise its function of.Micro RNA-27a (MI R-27a) in different tumor cells is different in some. It is believed that tumor promoting cancer, such as osteosarcoma, laryngeal cancer. However, in esophageal squamous cell carcinoma and colon cancer it is considered a tumor suppressor. Increasing evidence suggests that MI R-27a plays an important role in the occurrence and development of tumor, but the role of MI R-27a in gastric carcinoma it is not clear how.Mi R-27a in gastric carcinoma and its molecular mechanism of.PH Domain Leucine-rich Repeat investigated Protein Phosphatase2 (PHLPP2) is a protein phosphatase plays a tumor suppressor role. It is reported that the transfer of.PHLPP2 can induce cell cycle arrest and apoptosis and inhibit tumor in a variety of cancers can be carried on the Akt dephosphorylation, and inhibition of the PI3K/Akt signaling pathway. Here, we explored whether PHLPP2 is the target gene of MI R-27a. Our study indicates that MI and R-27a in gastric cancer by targeting PHLPP2 in the regulation of Akt pathway To exercise its promoting cell proliferation and metastasis of gastric carcinoma. Methods: Real-time PCR method was used to detect gastric cancer and gastric cancer cell lines mi R-27a and its target gene expression level of.Mi R-27a in gastric carcinoma function detected by in vitro and in vivo (MTT experiment, clone formation assay, flow cytometry. Wound healing assay, invasion and migration experiment, Transwell immunohistochemistry, immunofluorescence assay, Western blot assay and nude mice). To verify the application of target gene luciferase reporter experiments on MI R-27a. Results: in the first part, Mi R-27a promotes gastric cancer cell proliferation and metastasis we found that the expression level of MI R-27a in gastric cancer higher than that in the corresponding adjacent tissues. The clinical Mi high level of R-27a and tumor staging, infiltration, lymph node metastasis and distant metastasis. In gastric cancer cells mi R-2 The expression of 7a was also increased. We choose mi R-27a for subsequent experimental expression of relatively high and low SGC-7901 and AGS cell lines. We further investigated the function of experiment, to study the effect of MI R-27a on the biological behavior of gastric carcinoma cells. In SGC-7901 cells, down-regulation of MI R-27a can inhibit gastric cancer cells the proliferation, clone formation, wound healing, Transwell migration and invasion and inhibition of gastric cancer cell EMT, cell cycle arrest, apoptosis, and expression of related protein Akt pathway. In AGS cells, upregulation of MI R-27a can get the opposite result. Moreover, the nude mice also showed down-regulation of MI can inhibit R-27a tumor growth and metastasis. The second part of Mi R-27a in gastric cancer and targeted expression of PHLPP2 in order to explore the molecular mechanism underlying mi R-27a play a role in gastric carcinoma, we use Bioinformatics tools to find the target gene of MI R-27a and the corresponding target. In the prediction of target genes in our selected PHLPP2 research, because it is reported in the inhibition of tumor development and regulation of cell proliferation and migration plays an important role. Luciferase reporter experiments showed that in AGS cells transfected with R-27a agomirs, compared to mi the cells with the control group, the luciferase activity was significantly decreased in AGS cells. Up regulation of MI R-27a, we found that the PHLPP2 protein level and m level of RNA was decreased. The down-regulation of MI R-27a in SGC-7901 cells, we can see the results instead. In addition, in gastric cancer tissue. The expression of PHLPP2 and MI R-27a into stage. Clinical expression and gastric cancer was negatively related to.PHLPP2 invasion, lymph node and distant metastasis. The next part of Mi R-27a in third by targeting PHLPP2 protein involved in the regulation of Akt pathway in gastric cancer, we In the study of the malignant development of gastric carcinoma, MI R-27a target role to inhibit PHLPP2. In order to verify whether mi R-27a through the PHLPP2 function, SGC-7901 cells were co transfected with the MI R-27a antagomir and PHLPP2 Si RNA. showed that PHLPP2 knockdown can reduce mi R-27a antagomir on apoptosis of gastric cancer cell proliferation, and inhibition of migration and invasion. Our results suggest that the down-regulation of MI R-27a can cause p-Akt, Cyclin D1, Vimentin, p21, p27 and down-regulation of snail, upregulation of E-cadherin, but the PHLPP2 knockdown can reduce these changes. This shows that the PHLPP2/Akt axis participates in the regulation of R-27a MI on gastric cancer cells. Conclusion: in general, our current the study confirmed that MI R-27a in gastric cancer by targeting PHLPP2 new play cancer promoting effect. The down-regulation of MI R-27a can inhibit the malignant biological behavior of tumor cells, including proliferation, migration and invasion The attack. The silencing of PHLPP2 can weaken the MI R-27a down the biological behavior of malignant changes. That is to say, MI R-27a can inhibit PHLPP2 regulation of Akt/GSK3 beta signaling promote the proliferation and migration of gastric cancer cells. In summary, our results suggest that MI R-27a plays a role in promoting cancer gene in gastric cancer, and describes their play may the mechanism of action. With the deepening of the study, MI R-27a may become a new target for gastric cancer prevention.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2

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