microRNA参与脑肿瘤干细胞凋亡的研究
摘要
研究背景与目的
肿瘤干细胞(cancer stem cells)是存在于肿瘤中的一小部分具有干细胞性质的细胞群体,它具有自我更新的能力,是形成不同分化程度的肿瘤细胞和肿瘤不断扩大的源泉。到目前为止,人们已成功的从造血系统恶性肿瘤、乳腺癌和脑肿瘤患者组织中分离并培养出各自的肿瘤干细胞。这证明在肿瘤细胞群体中确实存在一类极少数的能使群体扩增的肿瘤干细胞。
肿瘤细胞具有异质化的特性,即由一个克隆来源的肿瘤细胞在生长过程中,形成在侵袭能力、生长速度、分化程度、对激素的反应、对抗癌药物的敏感性等方面有所不同的亚克隆。肿瘤细胞异质性的现象只有用肿瘤干细胞的理论才能解释,即肿瘤干细胞在不同选择压力下,向不同功能方向分化、成熟,造成肿瘤细胞的群体漂移,从而形成异质性。因此,肿瘤干细胞是肿瘤的根源,消灭了肿瘤干细胞就意味着消灭了肿瘤。长期以来,人们发现实体瘤的缺氧程度与肿瘤的不良预后相关。传统的肿瘤化疗主要针对迅速增殖的瘤细胞,TSC在细胞群中增殖缓慢,对化疗不敏感,TSC的膜表面蛋白受HIFs的调控,在低氧环境中能高效地排除药物,保护TSC不被杀灭,其对通过减少肿瘤细胞负载的治疗方法有抗性,而TSC在缺氧的环境中也能得到更好的保护。缺氧的细胞对放疗也不敏感,低氧是胶质母细胞瘤的普遍特征,研究表明胶质母细胞瘤干细胞放射抗性归结于细胞DNA修复能力的增强,提示在缺氧状态下,癌症干细胞的辐射抗性会显著提高。因此不论是化疗或是放疗,缺氧都是肿瘤治疗的重要靶点。
miRNA在肿瘤发生、发展、侵袭、转移中起重要作用,相关领域的研究已取得很多成果,为miRNA在肿瘤诊断和治疗中的应用奠定了基础。但miRNA在肿瘤中表达失调的机制等问题仍需要进一步研究。随着研究的深入,miRNA与肿瘤的关系必将得以阐明,miRNA在肿瘤防治中的应用也必将会有更广阔的前景,成为肿瘤治疗的新策略。
本研究应用miRNA芯片从miRNA水平分析U87肿瘤干细胞缺氧后的改变,发现U87肿瘤干细胞缺氧后特异表达的miRNA。通过合成特异miRNA mimic (miRNA寡核苷酸模拟物),转染人脑胶质瘤细胞U87肿瘤干细胞。运用细胞和分子生物学实验对其进行功能研究,为脑胶质瘤提供发病机制提供新的思路,也为脑胶质瘤的分子诊断和miRNA治疗奠定基础。
研究方法
1.将U87细胞接种于含B27、肝素、表皮生长因子、碱性成纤维生长因子的无FBS的DMEM/F12培养液中,在37℃、5%CO2饱和湿度培养箱中培养。待U87细胞增殖形成细胞球3～4d后,吸取上清培养液(含细胞球),重新吹打成单细胞悬液,按1：2或1：3比例传代。原代细胞球形成并达到100～200个细胞后,收集其细胞球,以2%多聚甲醛固定,室温15min；10%驴血清封闭10min,加入鼠抗人CD133一抗,4℃孵育过夜；加入Cy3标记的兔抗小鼠二抗,室温孵育2h,Hoechst33342染核。在显微镜下随机选择20个高倍视野进行阳性CSCs计数。缺氧状态下的细胞培养使用缺氧细胞培养箱,培养条件为37℃、5%CO2、1%O2。应用microRNA芯片芯片系统研究U87肿瘤干细胞具有缺氧相关的miRNAs。
2.以缺氧与正常的U87肿瘤干细胞的总RNA为模版,使用两部法荧光定量PCR方法,扩增目的基因,同时设U6rRNA为内参。反应结束后,以U6rRNA为内标,采用2-△△Ct方法来计算每种miRNA在的相对定量值。
3取对数生长期U87肿瘤干细胞,计数,无抗生素培养基重悬,按6孔板每孔接种3×105细胞,加培养基至2m1,培养16-24h后转染。合成特异miRNA mimic(miRNA寡核苷酸模拟物),转染人脑胶质瘤细胞U87肿瘤干细胞,RT-PCR检测hsa-let-7i表达量,Western Blot检测转染U87肿瘤干细胞Bcl-2Caspase9蛋白的表达,TUNEL检测转染hsa-let-7mimic U87肿瘤干细胞凋亡,流式细胞仪检测miRNA mimics诱导凋亡。
研究结果
1.应用microRNA芯片芯片系统研究U87肿瘤干细胞具有缺氧相关的miRNAs。
经Hy3荧光标记、纯化,进行芯片杂交,图像采集,数据进行归一化处理后,以两种细胞Hy3荧光标记信号强度的比值≤0.5或≥2为标准判定差异表达miRNA。结果发现,与对照组U87肿瘤干细胞相比,处理组的U87肿瘤干细胞中,上调表达的miRNA有10个,下调表达的miRNA有13个。处理组与对照组U87MG比较,细胞中下调表达的miRNAs为hsa-let-7i、hsa-miR-29c sv40-miR-S1-5p、hsa-miR-1290、hsa-miR-58、hsa-miR-625*、hsa-miR-1914、 hsa-miRPlus-G1246-3p、hsa-miR-4279、hsv2-miR-H7-3p、hsa-miR-3679-3p、 hsa-miR-3675-3p、hsa-miR-1246,上调表达的miRNAs是hsa-miR-143、 hsa-miR-124、hsa-miR-15a、hsa-miR-144、hsa-miR-9*、hsa-miR-33b、 hsa-miR-24-1*、hsv1-miR-H4*、hsa-miR-4297、hsa-miR-3613-3p,其中hsa-let-7i、 hsa-miR-143等多个有文献报道与细胞增殖、肿瘤发生发展等密切相关。
2. qRT-PCR方法对芯片结果进行检测
使用Agilent2100生物分析仪鉴定从干细胞中提取总RNA的质量,分别测定28S/18S峰值面积都在2：1左右,RNA的质量符合荧光定量分析的要求。选取4种miRNA进行qRT-PCR检测。反应结束后,以U6为内参,采用2-△△Ct方法来计算每种miRNA的在处理前后的相对定量值。hsa-miR-124上调10.82±1.17hsa-miR-143上调4.82±0.20hsa-let-7i下调2.04±0.08hsa-miR-29c下调1.96±0.01与芯片结果基本相同(芯片结果：hsa-miR-124上调11.79441663倍,hsa-miR-143上调4.748774253倍,hsa-let-7i下调2.087019倍,hsa-miR-29c下调2.0929982倍)
3. hsa-let-7i mimics对U87肿瘤干细胞的生物学影响
QRT-PCR检测显示转染hsa-let-7i mimics的U87肿瘤干细胞的hsa-let-7i的表达水平升高。流式细胞仪检测hsa-let-7i mimic转染U87肿瘤干细胞48小时后能诱导U87肿瘤干细胞的凋亡,各组间差异显著(F=464.229,P=0.000),且各处理组与对照组差异均有统计学意义(P<0.05)。Western blot结果显示：Bcl-2的蛋白水平降低达,Caspase3, Caspase9蛋白水平增强。Tunel检测显示DNA断裂的凋亡细胞标记有增强的荧光。说明转染hsa-let-7i mimics可以诱导U87肿瘤干细胞的凋亡。
结论
应用microRNA芯片芯片系统研究U87肿瘤干细胞具有缺氧相关的miRNAs,成功筛选到上调表达的miRNA有10个,下调表达的miRNA有13个。选取4种miRNA进行qRT-PCR检测。qRT-PCR结果与芯片结果基本相同,说明芯片结果可靠。文献挖掘发现hsa-let-7i与肿瘤干细胞联系密切。最后运用细胞和分子生物学实验对hsa-let-7i进行了功能研究,证实hsa-let-7i促进U87肿瘤干细胞凋亡。
Tumor stem cells (cancer stem cells) are a small subset of the tumor cell population with stem cell properties. They possess the potential for self-renewal. They are the cause of the formation of various degrees of differentiation seen in tumor cells and tumor expansion. Cancer stem cells have been successfully isolated from hematopoietic malignancies, breast cancer and brain tumor tissue. This proves the existence of stem cells in the tumor cell population can cause groups of tumor to amplify.
Tumor cells possess the characteristic of heterogeneity. Tumor cells from one clone can form subclones that differ in their growth process, their propensity to invade, growth rate, degree of differentiation, their hormone response and their variable sensitivity to anti-cancer drugs. The theory of cancer stem cells can explain the phenomenon of tumor cell heterogeneity, i.e., cancer stem cells differentiate in different directions based on function, they mature under different selection pressures resulting in the drift of tumor cell groups, thus resulting in heterogeneity. Therefore, since cancer stem cells are the source of the tumors, the eradication of cancer stem cells means the elimination of the tumor.
Biological characteristics of cancer stem cells (CSC/TSC)
1Self-renewal:the growth and proliferation of the tumor is guided by the tumor stem cells, cancer stem cells similar to adult stem cells that also have the characteristics of self-renewal and maintain the continued growth of the tumor through self-renewal. Another important reason for tumor growth is that an increasing number of tumor cells have self-renewal capacity caused by disorders in certain genes involved in the regulation mechanism in stem cells. In addition, several important signaling pathways that regulate normal self-renewal of stem cell including Notch and Wnt, NF-kB and Shh also play an important role in self-renewal in tumor stem cells.The proliferation of normal stem cells display self-stability in vivo. Their number in the proliferation process is maintained in a constant state. Cancer stem cells do not have self-stability and errors in gene duplication during proliferation cannot be repaired
2Differentiation potential:tumor stem cells originate from mature blocked normal stem cells and have the ability to generate progeny of different degrees of differentiation of cancer cells and form new tumors in vivo. Tumor cell differentiation is different in the same tumor tissue, where the degree of malignancy of tumor cells is low in mature differentiation, while poorly differentiated tumor cells are highly malignant.
3High tumorigenicity:a large number of trials have proved cancer stem cells have higher tumorigenic potential than non-tumor stem cells. Beier et al reported that in nude mice inoculated with22kinds of malignant glioma cells,11kinds containing CD133+cancer stem cell populations had significant growth and tumorigenicity,4kinds with CD133-cell populations grew into tumors slowly, while seven malignant glioma cell-derived cells did not grow. Singh et al reported that each mouse inoculated with100CD133+cancer stem cells resulted in the formation of a tumor6months after inoculation; at the same time the inoculation of100000CD133-non-tumor stem cells in mice did not form tumors. The results above support that cancer stem cells have a higher tumorigenic potential than non-tumor stem cells.
4Drug resistance:drug resistance is one of the characteristics of tumor stem cells. Many reasons cause tumor treatment failure, multidrug resistance (mutidrug, resistance, MDR) being one of the main reasons. The membrane of cancer stem cells express the proteins of the ABC transporter family that can transport and discharge metabolites, drugs, toxic substances, peptides, nucleotides and other substances, thus significantly decreasing the effectiveness of many chemotherapeutic drugs in inhibiting or killing tumor stem cells.
It has long been known that hypoxia in solid tumours is related to poor prognosis. Traditional chemotherapy is aimed at rapidly proliferating tumor cells. Proliferation of TSC in cell populations is slow, and thus insensitive to chemotherapy. TSC membrane protein regulated by HIFs, can be efficiently excluded from the drug in a hypoxic environment. TSC in a hypoxic environment can be better protected. Hypoxic cells are not sensitive to radiotherapy, hypoxia being a general feature of glioblastoma. Studies have shown that the radiation resistance of glioblastoma stem cells is attributed to the enhancement of DNA repair capacity suggesting that radiation resistance of stem cells will significantly improve under anaerobic conditions. Therefore, hypoxia is an important target for cancer treatment whether chemotherapy or radiotherapy. Clinical observations reveal that TSC treatment-resistance is the reason why tumors are difficult to eradicate. Clinical treatment targeting TSCs have received extensive attention. A recent study found that in brain tumor stem cells associated with tumor microvasculature, the TSC distribution and positioning of endothelial cells are closely related. Endothelial cells provide oxygen for the TSC. Neuroblastoma cell lines express of HIF-2a under5%O2. Neuroblastoma tissue rich in blood vessels still have significantly high HIF-2a expression that indicates that in vivo tumors rich in blood vessels still remain in hypoxic states possibly because of malformed vasculature. This view suggests that tumor microvascularized areas are likely to be the TSC niche. Targeting these vascular niches can more effectively remove the TSC.
MicroRNAs (miRNAs) are a group of single-stranded RNA (～22nt) encoded by the animals, plants, and the viral genome. They do not have an open reading frame (ORF) and do not encode protein but participate in a variety of important physiological and pathological processes in the body. They are able to target mRNA3-UTR of complementary base pairing (untranslated region) area. Thus, degradation or inhibiting its expression by silencing specific genes that regulate body growth, development and diseases especially has an important regulatory function on tumor occurrence and development. In recent years, miRNA has become a hot research field of molecular biology, genetics and clinical medicine. About one-third of the human gene encoding mRNA is presumably negatively regulated by microRNA.
MiRNA has the following characteristics:①specificity:different organizations of different cells have specific miRNA expression patterns and sequence features, which can be used as specific molecular markers of certain tissues or cells.②Sequential:miRNA composition is different at different developmental stages. Specific miRNA in a specific stage of specific cells determine the direction of cell differentiation and the differentiation phase, regulate the timing and direct the differentiation switch. For example miR-3to miR-7genes expressed in Drosophila in early embryogenesis, are not expressed in other phases. Drosophila begins to express miR-12, miR-21and miR-28from its larval stage, and these are maintained at a high level in the adult stage.③conservative:Different species, tissues, organs and cells with the same or similar miRNA molecules have similar regulatory functions. Three individual human embryonic cells miRNA (miR-302b, miR-302c, miR-302d) and mouse embryonic cells are same.miR-371and miR-302family is the same in both humans and mice.④miRNA targets:Mainly are time and space-specific transcriptional regulation genes and apoptosis regulatory genes. They also regulate specialized cell function and structure mediated through the balance between cell proliferation and apoptosis.
Abnormal miRNA expression is found in a variety of human tumors,some miRNA specific expression is found in stem cells. High expression of miRNA-17and miRNA-92in human lung cancer and B-cell lymphoma can promote tumor cell proliferation. Chen et al found that content of some complex miRNA in embryonic stem cells is less in the mature somatic tissues. A large number of known tumorigenic miRNAs expressed in some of the original cells but its expression gradually decreased with cell differentiation. Silber et al found miR-124and miR-137had a low expression in malignant gliomas but had a high expression in the differentiated mouse neural stem cells. These two types of miRNA over-expression in glioma oligodendrocytes and malignant glioma stem cells in mice can lead to morphological changes and loss of self-renewal and tumorigenicity. Scholars have proposed that the degree of differentiation of cells or tissues can be detected by specific miRNA markers and there is evidence showing that non-coding miRNA expression distinguishes between stem cells and differentiated mature cells. The important role of miRNA in cancer and some of the miRNA-specific expression in stem cells provide evidence that these miRNA help in the transformation from stem cells to malignant cells.
Spectrum changes and the identification of target mRNA through miRNA expression provides a new approach for cancer treatment strategy.There are several ways:they may have a carcinogenic effect through inhibition of miRNA; degradation of cancer-causing mRNA through complementary competition;3stimulate miRNA with a tumor suppressor role;4.external modification of miRNA expression. Although we know very little about the biological function of miRNA in tumorigenesis, and their relationship is poorly understood, people have begun to explore how miRNA may be applied in tumor treatment.
Cheng et al inhibited human miRNA by designing antisense oligonucleotides to interfere with the role of miRNA in cell growth and apoptosis. The synthetic antisense oligonucleotide complementary miRNA oncogene known as the anti-miRNA oligonucleotides (anti-miRNA oligonucleotide AMO) can effectively inhibit the activation of carcinogenic miRNA in tumors thereby delaying tumor growth. Inhibition of miRNA activity through successively administered specifically designed antisense oligonucleotides miRNA, such as of miRNA-155antisense oligonucleotides is more stable and less toxic than other cancer treatment method.AMO and cholesterol-conjugated anti-miRNA oligonucleotides (antagomirs) inhibit miRNA activity after injection in mice in various organs. Several studies have shown that the breakthrough achieved in the modified miRNA expression at some biological systems. By using miRNA, perfectly complementary antisense oligonucleotides can be designed for the specific downregulation of miRNA expression of the nucleotide sequence. This oligonucleotide is called an antagomir. Recently, antagomir treatment of cancer was proven effective. Tumor growth of neuroblastoma in nude mice can be aborted by antagomir-17-5p. Another more effective method is antisense oligonucleotides that play a similar miRNA sponge role.These sponges from the transgenic RNA, by a strong and specific miRNA competitive complementary combination leads to disinhibition of targets. The transfection of exogenous miRNA-agomir complementary to a specific cancer-causing mRNA can result in the corresponding mRNA inactivation. Stimulating tumor inhibiting miRNA provides another theoretical basis for the treatment of tumors. For example, in order to achieve the over-expression of let-7we transfect let-7on lung cancer cells resulting in inhibition of tumor cell growth.miRNA can affect the function of external mechanisms, and external mechanisms affect miRNA expression. Therefore, proteins such as DNA methyltransferase, group deacetylase inhibitor and drugs can affect miRNA expression. For example, methylation of the CpG site promoter and histone deacetylation lead to downregulation of miRNA-127levels in bladder cancer cells. Using these two kinds of drugs in combination can reduce DNA methylation and histone acetylation resulting increasing miRNA-127levels.miRNA-127plays a tumor suppressor function by blocking cell proliferation. All of these treatment strategies are based on the miRNA targeting.miRNA therapy or a combination of treatment with traditional medicines has good prospects. Due to the relationship between a variety of cellular functions and miRNA, their roles in various signaling pathways are still in research. This miRNA-related technology has not yet entered clinical application.
MiRNA plays an important role in tumorigenesis, development, invasion and metastasis. A lot of achievements have been made in related fields that have laid the foundation for the use of miRNAs in tumor diagnosis and treatment. Imbalance in mechanisms of MiRNA function in tumor expression still needs further study. With further research the relationship between miRNA and cancer will be clarified and the application of miRNA in cancer therapy will also have a broader outlook and become a new strategy for cancer treatment.
This study is divided into the following three parts:
Part I:(1)U87cells were seeded in humidified incubator in DMEM/F12medium containing10%FBS at37℃,5%CO2saturated humidity in incubator culture. Passage after5-7d according to the ratio of1:2or1:3.
(2)CSCs culture and subculture:U87cells were inoculated in FBS-free DMEM/F12medium with B27, heparin, epidermal growth factor, basic fibroblast growth factor at37℃,5%CO2. The supernatants (containing ball of cells) were drawn4days after U87cells form ball of cells and then blown into a single cell suspension and passaged according to the ratio of1:2or1:3.
(3)CSCs identification:The cell balls were collected when the original generation cells forming the ball had reached100-200cells, fixed with2%poly-formaldehyde for15min at room temperature;10%donkey serum for10min, add mouse anti-human CD133,4℃for overnight; Cy3-labeled rabbit anti-mouse secondary antibody was added and incubated at room temperature for2h, Hoechst33342dye nuclear. Positive CSCs count was conducted of randomly selected20fields of vision under a microscope.
(4)Cells were placed in the hypoxic state culture using the hypoxic cell incubator, the culture conditions were37℃,5%CO2,1%O2. Application of microRNA chip system was used to study miRNAs of U87tumor stem cells and their relation to hypoxia.
Part II:Application of real-time fluorescent quantitative RT-PCR method on four miRNA (hsa-miR-124hsa-miR-143hsa-let-7i hsa-miR-29c) and verification. This experiment used the total RNA of hypoxic and normal U87tumor stem cell as a template, using a two-step quantitative PCR to amplify the target gene.
U6rRNA was used as an internal standard.2-ΔΔCt method was used to calculate the relative quantitative value of each miRNA in different phases after the reaction.(Chip results:hsa-miR-124up11.79441663times, hsa-miR-143raised4.748774253times,hsa-let-7i down2.087019times,hsa-miR-29c down2.0929982times).That consistent with the microarray results, verifying the reliability the microarray results.
Part III:Tumor stem cells were taken while in the logarithmic phase U87and counted. They were resuspended without antibiotic medium, inoculated with3×105cells per well in6well plate, medium added to2ml16-24h after transfection.(1) The specificity of synthesis of miRNA.(miRNA oligonucleotide mimics), transfection of human U87tumor stem cells and RT-PCR detection of hsa-let-7i expression levels were carried out;(2) Western Blot. Detection of Bcl-2, Caspase3, Caspase9protein's expression in U87tumor stem cells was carried out (3) TUNEL detection of U87tumor stem cells apoptosis(4) flow cytometry detection of miRNA mimics apoptosis induction.Results:(1) After Transfection of hsa-let-7i miRNA levels of U87MG cells significantly increased.(2).Western blot results showed that: compared with the control group, Bcl-2protein levels was significantly reduced, Caspase3and Caspase9protein levels were significantly increased48hours after transfection of U87tumor stem cells.(3)The U87tumor stem cells showed apoptosis compared with control group by flow cytometry.(4).TUNEL detected the U87tumor stem cells, results showed that markers have enhanced fluorescence in apoptotic cell after increase of hsa-let-7i expression levels.
肿瘤干细胞(cancer stem cells)是存在于肿瘤中的一小部分具有干细胞性质的细胞群体,它具有自我更新的能力,是形成不同分化程度的肿瘤细胞和肿瘤不断扩大的源泉。到目前为止,人们已成功的从造血系统恶性肿瘤、乳腺癌和脑肿瘤患者组织中分离并培养出各自的肿瘤干细胞。这证明在肿瘤细胞群体中确实存在一类极少数的能使群体扩增的肿瘤干细胞。
肿瘤细胞具有异质化的特性,即由一个克隆来源的肿瘤细胞在生长过程中,形成在侵袭能力、生长速度、分化程度、对激素的反应、对抗癌药物的敏感性等方面有所不同的亚克隆。肿瘤细胞异质性的现象只有用肿瘤干细胞的理论才能解释,即肿瘤干细胞在不同选择压力下,向不同功能方向分化、成熟,造成肿瘤细胞的群体漂移,从而形成异质性。因此,肿瘤干细胞是肿瘤的根源,消灭了肿瘤干细胞就意味着消灭了肿瘤。长期以来,人们发现实体瘤的缺氧程度与肿瘤的不良预后相关。传统的肿瘤化疗主要针对迅速增殖的瘤细胞,TSC在细胞群中增殖缓慢,对化疗不敏感,TSC的膜表面蛋白受HIFs的调控,在低氧环境中能高效地排除药物,保护TSC不被杀灭,其对通过减少肿瘤细胞负载的治疗方法有抗性,而TSC在缺氧的环境中也能得到更好的保护。缺氧的细胞对放疗也不敏感,低氧是胶质母细胞瘤的普遍特征,研究表明胶质母细胞瘤干细胞放射抗性归结于细胞DNA修复能力的增强,提示在缺氧状态下,癌症干细胞的辐射抗性会显著提高。因此不论是化疗或是放疗,缺氧都是肿瘤治疗的重要靶点。
miRNA在肿瘤发生、发展、侵袭、转移中起重要作用,相关领域的研究已取得很多成果,为miRNA在肿瘤诊断和治疗中的应用奠定了基础。但miRNA在肿瘤中表达失调的机制等问题仍需要进一步研究。随着研究的深入,miRNA与肿瘤的关系必将得以阐明,miRNA在肿瘤防治中的应用也必将会有更广阔的前景,成为肿瘤治疗的新策略。
本研究应用miRNA芯片从miRNA水平分析U87肿瘤干细胞缺氧后的改变,发现U87肿瘤干细胞缺氧后特异表达的miRNA。通过合成特异miRNA mimic (miRNA寡核苷酸模拟物),转染人脑胶质瘤细胞U87肿瘤干细胞。运用细胞和分子生物学实验对其进行功能研究,为脑胶质瘤提供发病机制提供新的思路,也为脑胶质瘤的分子诊断和miRNA治疗奠定基础。
研究方法
1.将U87细胞接种于含B27、肝素、表皮生长因子、碱性成纤维生长因子的无FBS的DMEM/F12培养液中,在37℃、5%CO2饱和湿度培养箱中培养。待U87细胞增殖形成细胞球3～4d后,吸取上清培养液(含细胞球),重新吹打成单细胞悬液,按1：2或1：3比例传代。原代细胞球形成并达到100～200个细胞后,收集其细胞球,以2%多聚甲醛固定,室温15min；10%驴血清封闭10min,加入鼠抗人CD133一抗,4℃孵育过夜；加入Cy3标记的兔抗小鼠二抗,室温孵育2h,Hoechst33342染核。在显微镜下随机选择20个高倍视野进行阳性CSCs计数。缺氧状态下的细胞培养使用缺氧细胞培养箱,培养条件为37℃、5%CO2、1%O2。应用microRNA芯片芯片系统研究U87肿瘤干细胞具有缺氧相关的miRNAs。
2.以缺氧与正常的U87肿瘤干细胞的总RNA为模版,使用两部法荧光定量PCR方法,扩增目的基因,同时设U6rRNA为内参。反应结束后,以U6rRNA为内标,采用2-△△Ct方法来计算每种miRNA在的相对定量值。
3取对数生长期U87肿瘤干细胞,计数,无抗生素培养基重悬,按6孔板每孔接种3×105细胞,加培养基至2m1,培养16-24h后转染。合成特异miRNA mimic(miRNA寡核苷酸模拟物),转染人脑胶质瘤细胞U87肿瘤干细胞,RT-PCR检测hsa-let-7i表达量,Western Blot检测转染U87肿瘤干细胞Bcl-2Caspase9蛋白的表达,TUNEL检测转染hsa-let-7mimic U87肿瘤干细胞凋亡,流式细胞仪检测miRNA mimics诱导凋亡。
研究结果
1.应用microRNA芯片芯片系统研究U87肿瘤干细胞具有缺氧相关的miRNAs。
经Hy3荧光标记、纯化,进行芯片杂交,图像采集,数据进行归一化处理后,以两种细胞Hy3荧光标记信号强度的比值≤0.5或≥2为标准判定差异表达miRNA。结果发现,与对照组U87肿瘤干细胞相比,处理组的U87肿瘤干细胞中,上调表达的miRNA有10个,下调表达的miRNA有13个。处理组与对照组U87MG比较,细胞中下调表达的miRNAs为hsa-let-7i、hsa-miR-29c sv40-miR-S1-5p、hsa-miR-1290、hsa-miR-58、hsa-miR-625*、hsa-miR-1914、 hsa-miRPlus-G1246-3p、hsa-miR-4279、hsv2-miR-H7-3p、hsa-miR-3679-3p、 hsa-miR-3675-3p、hsa-miR-1246,上调表达的miRNAs是hsa-miR-143、 hsa-miR-124、hsa-miR-15a、hsa-miR-144、hsa-miR-9*、hsa-miR-33b、 hsa-miR-24-1*、hsv1-miR-H4*、hsa-miR-4297、hsa-miR-3613-3p,其中hsa-let-7i、 hsa-miR-143等多个有文献报道与细胞增殖、肿瘤发生发展等密切相关。
2. qRT-PCR方法对芯片结果进行检测
使用Agilent2100生物分析仪鉴定从干细胞中提取总RNA的质量,分别测定28S/18S峰值面积都在2：1左右,RNA的质量符合荧光定量分析的要求。选取4种miRNA进行qRT-PCR检测。反应结束后,以U6为内参,采用2-△△Ct方法来计算每种miRNA的在处理前后的相对定量值。hsa-miR-124上调10.82±1.17hsa-miR-143上调4.82±0.20hsa-let-7i下调2.04±0.08hsa-miR-29c下调1.96±0.01与芯片结果基本相同(芯片结果：hsa-miR-124上调11.79441663倍,hsa-miR-143上调4.748774253倍,hsa-let-7i下调2.087019倍,hsa-miR-29c下调2.0929982倍)
3. hsa-let-7i mimics对U87肿瘤干细胞的生物学影响
QRT-PCR检测显示转染hsa-let-7i mimics的U87肿瘤干细胞的hsa-let-7i的表达水平升高。流式细胞仪检测hsa-let-7i mimic转染U87肿瘤干细胞48小时后能诱导U87肿瘤干细胞的凋亡,各组间差异显著(F=464.229,P=0.000),且各处理组与对照组差异均有统计学意义(P<0.05)。Western blot结果显示：Bcl-2的蛋白水平降低达,Caspase3, Caspase9蛋白水平增强。Tunel检测显示DNA断裂的凋亡细胞标记有增强的荧光。说明转染hsa-let-7i mimics可以诱导U87肿瘤干细胞的凋亡。
结论
应用microRNA芯片芯片系统研究U87肿瘤干细胞具有缺氧相关的miRNAs,成功筛选到上调表达的miRNA有10个,下调表达的miRNA有13个。选取4种miRNA进行qRT-PCR检测。qRT-PCR结果与芯片结果基本相同,说明芯片结果可靠。文献挖掘发现hsa-let-7i与肿瘤干细胞联系密切。最后运用细胞和分子生物学实验对hsa-let-7i进行了功能研究,证实hsa-let-7i促进U87肿瘤干细胞凋亡。
Tumor stem cells (cancer stem cells) are a small subset of the tumor cell population with stem cell properties. They possess the potential for self-renewal. They are the cause of the formation of various degrees of differentiation seen in tumor cells and tumor expansion. Cancer stem cells have been successfully isolated from hematopoietic malignancies, breast cancer and brain tumor tissue. This proves the existence of stem cells in the tumor cell population can cause groups of tumor to amplify.
Tumor cells possess the characteristic of heterogeneity. Tumor cells from one clone can form subclones that differ in their growth process, their propensity to invade, growth rate, degree of differentiation, their hormone response and their variable sensitivity to anti-cancer drugs. The theory of cancer stem cells can explain the phenomenon of tumor cell heterogeneity, i.e., cancer stem cells differentiate in different directions based on function, they mature under different selection pressures resulting in the drift of tumor cell groups, thus resulting in heterogeneity. Therefore, since cancer stem cells are the source of the tumors, the eradication of cancer stem cells means the elimination of the tumor.
Biological characteristics of cancer stem cells (CSC/TSC)
1Self-renewal:the growth and proliferation of the tumor is guided by the tumor stem cells, cancer stem cells similar to adult stem cells that also have the characteristics of self-renewal and maintain the continued growth of the tumor through self-renewal. Another important reason for tumor growth is that an increasing number of tumor cells have self-renewal capacity caused by disorders in certain genes involved in the regulation mechanism in stem cells. In addition, several important signaling pathways that regulate normal self-renewal of stem cell including Notch and Wnt, NF-kB and Shh also play an important role in self-renewal in tumor stem cells.The proliferation of normal stem cells display self-stability in vivo. Their number in the proliferation process is maintained in a constant state. Cancer stem cells do not have self-stability and errors in gene duplication during proliferation cannot be repaired
2Differentiation potential:tumor stem cells originate from mature blocked normal stem cells and have the ability to generate progeny of different degrees of differentiation of cancer cells and form new tumors in vivo. Tumor cell differentiation is different in the same tumor tissue, where the degree of malignancy of tumor cells is low in mature differentiation, while poorly differentiated tumor cells are highly malignant.
3High tumorigenicity:a large number of trials have proved cancer stem cells have higher tumorigenic potential than non-tumor stem cells. Beier et al reported that in nude mice inoculated with22kinds of malignant glioma cells,11kinds containing CD133+cancer stem cell populations had significant growth and tumorigenicity,4kinds with CD133-cell populations grew into tumors slowly, while seven malignant glioma cell-derived cells did not grow. Singh et al reported that each mouse inoculated with100CD133+cancer stem cells resulted in the formation of a tumor6months after inoculation; at the same time the inoculation of100000CD133-non-tumor stem cells in mice did not form tumors. The results above support that cancer stem cells have a higher tumorigenic potential than non-tumor stem cells.
4Drug resistance:drug resistance is one of the characteristics of tumor stem cells. Many reasons cause tumor treatment failure, multidrug resistance (mutidrug, resistance, MDR) being one of the main reasons. The membrane of cancer stem cells express the proteins of the ABC transporter family that can transport and discharge metabolites, drugs, toxic substances, peptides, nucleotides and other substances, thus significantly decreasing the effectiveness of many chemotherapeutic drugs in inhibiting or killing tumor stem cells.
It has long been known that hypoxia in solid tumours is related to poor prognosis. Traditional chemotherapy is aimed at rapidly proliferating tumor cells. Proliferation of TSC in cell populations is slow, and thus insensitive to chemotherapy. TSC membrane protein regulated by HIFs, can be efficiently excluded from the drug in a hypoxic environment. TSC in a hypoxic environment can be better protected. Hypoxic cells are not sensitive to radiotherapy, hypoxia being a general feature of glioblastoma. Studies have shown that the radiation resistance of glioblastoma stem cells is attributed to the enhancement of DNA repair capacity suggesting that radiation resistance of stem cells will significantly improve under anaerobic conditions. Therefore, hypoxia is an important target for cancer treatment whether chemotherapy or radiotherapy. Clinical observations reveal that TSC treatment-resistance is the reason why tumors are difficult to eradicate. Clinical treatment targeting TSCs have received extensive attention. A recent study found that in brain tumor stem cells associated with tumor microvasculature, the TSC distribution and positioning of endothelial cells are closely related. Endothelial cells provide oxygen for the TSC. Neuroblastoma cell lines express of HIF-2a under5%O2. Neuroblastoma tissue rich in blood vessels still have significantly high HIF-2a expression that indicates that in vivo tumors rich in blood vessels still remain in hypoxic states possibly because of malformed vasculature. This view suggests that tumor microvascularized areas are likely to be the TSC niche. Targeting these vascular niches can more effectively remove the TSC.
MicroRNAs (miRNAs) are a group of single-stranded RNA (～22nt) encoded by the animals, plants, and the viral genome. They do not have an open reading frame (ORF) and do not encode protein but participate in a variety of important physiological and pathological processes in the body. They are able to target mRNA3-UTR of complementary base pairing (untranslated region) area. Thus, degradation or inhibiting its expression by silencing specific genes that regulate body growth, development and diseases especially has an important regulatory function on tumor occurrence and development. In recent years, miRNA has become a hot research field of molecular biology, genetics and clinical medicine. About one-third of the human gene encoding mRNA is presumably negatively regulated by microRNA.
MiRNA has the following characteristics:①specificity:different organizations of different cells have specific miRNA expression patterns and sequence features, which can be used as specific molecular markers of certain tissues or cells.②Sequential:miRNA composition is different at different developmental stages. Specific miRNA in a specific stage of specific cells determine the direction of cell differentiation and the differentiation phase, regulate the timing and direct the differentiation switch. For example miR-3to miR-7genes expressed in Drosophila in early embryogenesis, are not expressed in other phases. Drosophila begins to express miR-12, miR-21and miR-28from its larval stage, and these are maintained at a high level in the adult stage.③conservative:Different species, tissues, organs and cells with the same or similar miRNA molecules have similar regulatory functions. Three individual human embryonic cells miRNA (miR-302b, miR-302c, miR-302d) and mouse embryonic cells are same.miR-371and miR-302family is the same in both humans and mice.④miRNA targets:Mainly are time and space-specific transcriptional regulation genes and apoptosis regulatory genes. They also regulate specialized cell function and structure mediated through the balance between cell proliferation and apoptosis.
Abnormal miRNA expression is found in a variety of human tumors,some miRNA specific expression is found in stem cells. High expression of miRNA-17and miRNA-92in human lung cancer and B-cell lymphoma can promote tumor cell proliferation. Chen et al found that content of some complex miRNA in embryonic stem cells is less in the mature somatic tissues. A large number of known tumorigenic miRNAs expressed in some of the original cells but its expression gradually decreased with cell differentiation. Silber et al found miR-124and miR-137had a low expression in malignant gliomas but had a high expression in the differentiated mouse neural stem cells. These two types of miRNA over-expression in glioma oligodendrocytes and malignant glioma stem cells in mice can lead to morphological changes and loss of self-renewal and tumorigenicity. Scholars have proposed that the degree of differentiation of cells or tissues can be detected by specific miRNA markers and there is evidence showing that non-coding miRNA expression distinguishes between stem cells and differentiated mature cells. The important role of miRNA in cancer and some of the miRNA-specific expression in stem cells provide evidence that these miRNA help in the transformation from stem cells to malignant cells.
Spectrum changes and the identification of target mRNA through miRNA expression provides a new approach for cancer treatment strategy.There are several ways:they may have a carcinogenic effect through inhibition of miRNA; degradation of cancer-causing mRNA through complementary competition;3stimulate miRNA with a tumor suppressor role;4.external modification of miRNA expression. Although we know very little about the biological function of miRNA in tumorigenesis, and their relationship is poorly understood, people have begun to explore how miRNA may be applied in tumor treatment.
Cheng et al inhibited human miRNA by designing antisense oligonucleotides to interfere with the role of miRNA in cell growth and apoptosis. The synthetic antisense oligonucleotide complementary miRNA oncogene known as the anti-miRNA oligonucleotides (anti-miRNA oligonucleotide AMO) can effectively inhibit the activation of carcinogenic miRNA in tumors thereby delaying tumor growth. Inhibition of miRNA activity through successively administered specifically designed antisense oligonucleotides miRNA, such as of miRNA-155antisense oligonucleotides is more stable and less toxic than other cancer treatment method.AMO and cholesterol-conjugated anti-miRNA oligonucleotides (antagomirs) inhibit miRNA activity after injection in mice in various organs. Several studies have shown that the breakthrough achieved in the modified miRNA expression at some biological systems. By using miRNA, perfectly complementary antisense oligonucleotides can be designed for the specific downregulation of miRNA expression of the nucleotide sequence. This oligonucleotide is called an antagomir. Recently, antagomir treatment of cancer was proven effective. Tumor growth of neuroblastoma in nude mice can be aborted by antagomir-17-5p. Another more effective method is antisense oligonucleotides that play a similar miRNA sponge role.These sponges from the transgenic RNA, by a strong and specific miRNA competitive complementary combination leads to disinhibition of targets. The transfection of exogenous miRNA-agomir complementary to a specific cancer-causing mRNA can result in the corresponding mRNA inactivation. Stimulating tumor inhibiting miRNA provides another theoretical basis for the treatment of tumors. For example, in order to achieve the over-expression of let-7we transfect let-7on lung cancer cells resulting in inhibition of tumor cell growth.miRNA can affect the function of external mechanisms, and external mechanisms affect miRNA expression. Therefore, proteins such as DNA methyltransferase, group deacetylase inhibitor and drugs can affect miRNA expression. For example, methylation of the CpG site promoter and histone deacetylation lead to downregulation of miRNA-127levels in bladder cancer cells. Using these two kinds of drugs in combination can reduce DNA methylation and histone acetylation resulting increasing miRNA-127levels.miRNA-127plays a tumor suppressor function by blocking cell proliferation. All of these treatment strategies are based on the miRNA targeting.miRNA therapy or a combination of treatment with traditional medicines has good prospects. Due to the relationship between a variety of cellular functions and miRNA, their roles in various signaling pathways are still in research. This miRNA-related technology has not yet entered clinical application.
MiRNA plays an important role in tumorigenesis, development, invasion and metastasis. A lot of achievements have been made in related fields that have laid the foundation for the use of miRNAs in tumor diagnosis and treatment. Imbalance in mechanisms of MiRNA function in tumor expression still needs further study. With further research the relationship between miRNA and cancer will be clarified and the application of miRNA in cancer therapy will also have a broader outlook and become a new strategy for cancer treatment.
This study is divided into the following three parts:
Part I:(1)U87cells were seeded in humidified incubator in DMEM/F12medium containing10%FBS at37℃,5%CO2saturated humidity in incubator culture. Passage after5-7d according to the ratio of1:2or1:3.
(2)CSCs culture and subculture:U87cells were inoculated in FBS-free DMEM/F12medium with B27, heparin, epidermal growth factor, basic fibroblast growth factor at37℃,5%CO2. The supernatants (containing ball of cells) were drawn4days after U87cells form ball of cells and then blown into a single cell suspension and passaged according to the ratio of1:2or1:3.
(3)CSCs identification:The cell balls were collected when the original generation cells forming the ball had reached100-200cells, fixed with2%poly-formaldehyde for15min at room temperature;10%donkey serum for10min, add mouse anti-human CD133,4℃for overnight; Cy3-labeled rabbit anti-mouse secondary antibody was added and incubated at room temperature for2h, Hoechst33342dye nuclear. Positive CSCs count was conducted of randomly selected20fields of vision under a microscope.
(4)Cells were placed in the hypoxic state culture using the hypoxic cell incubator, the culture conditions were37℃,5%CO2,1%O2. Application of microRNA chip system was used to study miRNAs of U87tumor stem cells and their relation to hypoxia.
Part II:Application of real-time fluorescent quantitative RT-PCR method on four miRNA (hsa-miR-124hsa-miR-143hsa-let-7i hsa-miR-29c) and verification. This experiment used the total RNA of hypoxic and normal U87tumor stem cell as a template, using a two-step quantitative PCR to amplify the target gene.
U6rRNA was used as an internal standard.2-ΔΔCt method was used to calculate the relative quantitative value of each miRNA in different phases after the reaction.(Chip results:hsa-miR-124up11.79441663times, hsa-miR-143raised4.748774253times,hsa-let-7i down2.087019times,hsa-miR-29c down2.0929982times).That consistent with the microarray results, verifying the reliability the microarray results.
Part III:Tumor stem cells were taken while in the logarithmic phase U87and counted. They were resuspended without antibiotic medium, inoculated with3×105cells per well in6well plate, medium added to2ml16-24h after transfection.(1) The specificity of synthesis of miRNA.(miRNA oligonucleotide mimics), transfection of human U87tumor stem cells and RT-PCR detection of hsa-let-7i expression levels were carried out;(2) Western Blot. Detection of Bcl-2, Caspase3, Caspase9protein's expression in U87tumor stem cells was carried out (3) TUNEL detection of U87tumor stem cells apoptosis(4) flow cytometry detection of miRNA mimics apoptosis induction.Results:(1) After Transfection of hsa-let-7i miRNA levels of U87MG cells significantly increased.(2).Western blot results showed that: compared with the control group, Bcl-2protein levels was significantly reduced, Caspase3and Caspase9protein levels were significantly increased48hours after transfection of U87tumor stem cells.(3)The U87tumor stem cells showed apoptosis compared with control group by flow cytometry.(4).TUNEL detected the U87tumor stem cells, results showed that markers have enhanced fluorescence in apoptotic cell after increase of hsa-let-7i expression levels.
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