本文選題：肝癌　切入點：微小核糖核酸　出處：《內(nèi)蒙古大學(xué)》2017年碩士論文

【摘要】：肝細(xì)胞癌(Hepatocellular carcinoma,HCC)是一種常見的惡性腫瘤,多發(fā)于成人,肝癌的發(fā)生、發(fā)展和轉(zhuǎn)移是一個復(fù)雜的過程,受多個因子和基因的調(diào)節(jié)[1]。微小核糖核酸RNA(MicroRNA,miRNA)是一類長度在19-24個核苷酸范圍內(nèi)的內(nèi)源性非編碼RNA,miRNA能夠通過與靶基因mRNA3' UTR互補結(jié)合來抑制翻譯或者促進(jìn)mRNA降解,從而調(diào)控相關(guān)基因的表達(dá)。大量研究報道稱miRNA在許多腫瘤細(xì)胞中都存在差異性表達(dá),并且在腫瘤的發(fā)生發(fā)展中都可能發(fā)揮著重要的作用。我們實驗室前期的研究發(fā)現(xiàn)一定濃度的臍帶間充質(zhì)干細(xì)胞(Umbilical cord-derived mesenchymal stem cells,UC-MSCs)條件培養(yǎng)基能夠抑制肝癌細(xì)胞HepG2的增殖,并且通過高通量測序技術(shù),分析了 UC-MSCs條件培養(yǎng)基共培養(yǎng)后HepG2 miRNAs的差異表達(dá)譜,找到了一些可能相關(guān)的miRNAs。其中miR-3065-5p和miR-1307-5p在UC-MSCs條件培養(yǎng)基處理HepG2后表達(dá)水平顯著上調(diào)。首先,我們通過qRT-PCR檢測了三種肝癌細(xì)胞系(HepG2、MHCC97-L、MHCC87-H)和正常肝細(xì)胞系L02中miR-3065-5p的表達(dá)是否存在差異;結(jié)果顯示,相較正常肝細(xì)胞L02,miR-3065-5p在肝癌細(xì)胞系中的表達(dá)明顯下調(diào),接下來我們通過體外合成miR-3065-5p質(zhì)粒轉(zhuǎn)染三種肝癌細(xì)胞,結(jié)果發(fā)現(xiàn)過表達(dá)miR-3065-5p能夠顯著抑制三種肝癌細(xì)胞的增殖、侵襲、克隆形成能力。此外,我們通過生物信息學(xué)預(yù)測到SATB1可能是miR-3065-5p的相關(guān)靶基因,通過雙熒光素酶報告基因?qū)嶒炦M(jìn)一步檢測了預(yù)測的可靠性,實驗結(jié)果顯示SATB1與miR-3065-5p存在互補結(jié)合位點。qRT-PCR與Western blot結(jié)果顯示過表達(dá)的miR-3065-5p能夠顯著抑制SATB1表達(dá)水平。最后通過體外合成siRNA干擾SATB1在肝癌細(xì)胞中的表達(dá),結(jié)果顯示,干擾SATB1后也能顯著抑制肝癌細(xì)胞的增殖,其效果與miR-3065-5p類似。在miR-1307-5p的研究中,我們發(fā)現(xiàn)過表達(dá)的miR-1307-5p也能顯著抑制肝癌細(xì)胞的增殖、侵襲等生物學(xué)特性,而在靶基因的篩選驗證中,我們通過生物信息學(xué)軟件預(yù)測了 PIM3可能是miR-1307-5p的靶基因,但是雙熒光素酶報告基因?qū)嶒炐Ч⒉幻黠@,說明PIM3并不是miR-1307-5p的相關(guān)靶基因,其靶基因有待進(jìn)一步篩選驗證。
[Abstract]:Hepatocellular carcinoma (HCC) is a common malignant tumor, mostly in adults. The occurrence, development and metastasis of HCC is a complicated process, which is regulated by many factors and genes.MicroRNAs miRNAs are a class of endogenous non-coding RNAs in the range of 19-24 nucleotides, which can inhibit translation or promote mRNA degradation by complementing with target gene mRNA3'#en0#, thus regulating the expression of related genes.A large number of studies have reported that miRNA is differentially expressed in many tumor cells and may play an important role in tumorigenesis and development.Our previous study in our laboratory found that Umbilical cord-derived mesenchymal stem stem cells (UC-MSCs) conditioned medium could inhibit the proliferation of HepG2 cells, and high throughput sequencing technique was used.The differential expression profiles of HepG2 miRNAs after co-culture on UC-MSCs conditioned medium were analyzed, and some possible related miRNAs were found.The expression levels of miR-3065-5p and miR-1307-5p were upregulated significantly after HepG2 treatment in UC-MSCs conditioned medium.First, we detected the difference of the expression of miR-3065-5p in three kinds of hepatoma cell lines (HepG2MHCC97-LHCC87-H) and normal liver cell line L02 by qRT-PCR, the results showed that the expression of MHCC97-MHCC87-H2MHCC97-L02miR-3065-5p was significantly down-regulated than that of normal hepatoma cell line L02miR-3065-5p.Then we synthesized miR-3065-5p plasmid in vitro and transfected into three kinds of hepatoma cells. The results showed that overexpression of miR-3065-5p could significantly inhibit the proliferation invasion and clone formation of three kinds of hepatoma cells.In addition, we predicted that SATB1 might be the target gene of miR-3065-5p by bioinformatics, and further tested the reliability of the prediction by double luciferase reporter gene experiment.The results showed that there were complementary binding sites between SATB1 and miR-3065-5p. QRT-PCR and Western blot showed that overexpression of miR-3065-5p could significantly inhibit the expression of SATB1.Finally, siRNA was synthesized in vitro to interfere the expression of SATB1 in hepatoma cells. The results showed that interfering with SATB1 could significantly inhibit the proliferation of HCC cells, and its effect was similar to that of miR-3065-5p.In the study of miR-1307-5p, we found that overexpression of miR-1307-5p could significantly inhibit the proliferation and invasion of hepatoma cells. In the screening and verification of target genes, we predicted that PIM3 might be the target gene of miR-1307-5p by bioinformatics software.But the effect of double luciferase reporter gene is not obvious, which indicates that PIM3 is not the target gene of miR-1307-5p, and its target gene needs further screening and verification.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 彭正奎;楊定華;李湘z，

本文編號：1703959