miR-21对骨肉瘤及其肿瘤干细胞调控作用的实验研究

发布时间：2018-05-18 04:25

本文选题：miR-21 + 骨肉瘤　；参考：《山东大学》2016年博士论文

【摘要】：研究背景:骨肉瘤是对青、少年危害最大的原发的恶性骨肿瘤,以往以手术切除(截肢)治疗为主。患者不但要承受因肢体残缺造成的肉体和精神的严重创伤,而且其5年生存率不超过20%,对于患者及其家庭来说都是一个灾难。即使现在采用各种保肢手术辅助术前、术后的化疗,可以大大提高五年生存率,并很大程度的保留患者的肢体功能,从而明显提高患者的生活质量,但仍有近四分之一到三分之一[1](国内文献报道为40%—50%[2])的患者生存期不超过5年。如何提高化疗的有效率,减少骨肉瘤的复发和转移,是目前临床中对骨肉瘤治疗的研究重点。近来,很多肿瘤学的研究把重点放在了肿瘤干细胞的领域[3]。肿瘤干细胞具有无限增值,自我更新和多向分化的能力,而且肿瘤干细胞多处于静止期,对化疗不敏感[4]。经化疗之后,肿瘤体内干细胞比例明显提高,是产生化疗耐药性和肿瘤复发的主要原因。骨肉瘤保肢手术联合辅助术前、术后化疗的综合治疗后,骨肉瘤的复发与转移是由于骨肉瘤干细胞的存在的原因。目前,骨肉瘤的诊断和治疗发展处于一个瓶颈期,如何有效杀伤肿瘤内的干细胞,减少肿瘤体内干细胞的比例,是提高治疗效果的有效途径。CD133是一种跨膜糖蛋白,先后被发现于造血干细胞和正常干细胞[5,6],后来又被发现于大多数肿瘤干细胞膜,现作为广谱的标记物被应用于肿瘤干细胞的分辨与分离。目前,CD133是筛选骨肉瘤中肿瘤干细胞的常规公认的关键性标识[7,8],所以,我们在本实验中亦把CD133基因的表达作为肿瘤细胞呈现干细胞特性的标志。MicroRNA(miRNA)是一族非编码的小分子RNA(长度平均约为22个核苷酸),在基因转录后水平发挥重要的调控作用,通常引起翻译抑制、目标退化和基因沉默,对人的生命过程具有重要意义。其中,许多miRNA作为原癌基因或是抑癌基因,在肿瘤的发生、发展和转移中扮演重要的角色。miR-21作为致癌的miRNA,在不同的恶性肿瘤的侵袭、血管浸润和转移中起重要作用[9-11]。有研究证实miR-21在骨肉瘤细胞中有高表达,以及miR-21促进骨肉瘤的生长和转移。同时,研究证实miR-21在多种恶性肿瘤的干细胞中亦有异常表达,并通过蛋白或基因的相互作用来调节恶性肿瘤干细胞的功能[12];另外,有研究证实miR-21的抑制剂(反义寡核苷酸链)可以提高某些肿瘤细胞对化疗药物的敏感性[13-15]。VEGF是对血管内皮细胞具有特异性的肝素结合的生长因子,是人体内主要的促血管生成因素。在肿瘤生长时,在多种促癌因素作用下肿瘤组织内VEGF及其受体的表达激增,肿瘤组织内大量不规则的新生血管生成,为肿瘤组织生长提供充足养分,同时为肿瘤的侵袭和转移提供通路,参与到肿瘤的生长、浸润和转移各个过程中[16]。研究证实VEGF在骨肉瘤的发生发展和转移中发挥重要作用;而在其他恶性肿瘤中,miR-21通过增强VEGF表达,从而促进肿瘤的生长和转移[17,18]。因此,在我们关于miR-21在骨肉瘤的发生、发展、转移和肿瘤干细胞的调控作用的研究中,引入VEGF,一是可以作为对照来分析miR-21的作用,另外可以分析miR-21与VEGF在对骨肉瘤干细胞的调控作用的相关性。目的:本课题主要研究阻断miR-21和VEGF作用后不同骨肉瘤细胞VEGF基因和/或CD133基因表达的变化,以及对骨肉瘤细胞生长的影响,证实miR-21对于骨肉瘤及其干细胞有调节作用。并结合VEGF对骨肉瘤及其干细胞特性的调节作用进行对照分析,旨在初步探讨miR-21对骨肉瘤及其干细胞特性的影响及其可能的作用机制及规律,以期找出在临床中能有效抑制骨肉瘤干细胞的发生、发展,减少骨肉瘤肿瘤干细胞比例的治疗措施。方法:1.细胞及细胞培养:选择人骨肉瘤CRL-1543 and CRL-1423细胞系作为实验细胞,按照ATCC推荐的细胞培养液和培养条件及操作步骤进行培养。2.实验一 Realtime-PCR 实验2.1对两个细胞系的种植培养分3个实验组,分别为Oh(初始)对照组,8h阻断组,24h阻断组,每组包括2孔1543细胞和2孔1423细胞,按照前面所述的细胞培养方法进行培养。2.2对两个细胞系的处理将三组细胞在C02细胞培养箱中培养24小时后,所种植细胞完全贴壁后收获初始对照组细胞。然后,在另两组细胞的2孔中分别加入miR-21(has-mir-21-5p)反义寡核苷酸链和VEGF受体抑制剂,然后,将两个抑制组细胞的培养板放回培养箱继续培养,分别于miR-21 inhibitor转染和VEGF受体抑制剂处理后8小时和24小时收集细胞,作为8小时抑制组和24小时抑制组。对各实验组的各个细胞样品分别提取RNA,然后反转录制备cDNA,进行real time-PCR实验,检测各样本的VEGF和CD133基因的表达。3.实验二细胞免疫组化实验3.1对两个细胞系的种植培养取两个8孔chamber slider(腔室载玻片),分别标记为CD133和VEGF。每个chamber slider均培养两排细胞,上面一排植入1543细胞,下面一排植入1423细胞,每个培养孔内植入10000个细胞,并加入相应的培养液0.5ml,放在孵化器中培养24小时,待细胞完全贴壁。3.2对两个细胞系的处理然后将每排四个培养孔的细胞中第一个孔作为空白对照,第二个孔作为阴性对照,第二个孔的作为VEGF(-)加入VEGF受体抑制剂,第四个孔作为miR-21(-)转染miR-21(has-mir-21-5p)阻断剂。将处理过的细胞再放入培养箱中培养24h,然后进行免疫组化染色实验,检测各细胞样品中VEGF基因和CD133基因的表达。4.实验三细胞划痕实验4.1对两个细胞系的种植培养分三个实验组,分别为空白对照组,miR-21阻断组,miR-21阻断+VEGF抑制组。每组包括2孔1543细胞和2孔1423细胞,按照前面所述的细胞培养方法进行培养。4.2对两个细胞系的处理所有细胞在培养箱中培养24小时,待细胞完全贴壁。然后,在第一组四个培养孔内不加任何阻断剂,第二组四个培养孔内加入miR-21的阻断剂,第三组四个培养孔内加入miR-21阻断剂和VEGF受体的抑制剂。然后,将十二孔细胞培养板放回培养箱中培养细胞,待细胞基本布满培养孔底部,在每个培养孔底部用p200 Pipet tip,经过圆心垂直底面标记线划一道线,用倒置显微镜观察并在划痕上与六条标记线的交点处留取照片;然后继续在培养箱中培养,于4h,8h,24h,48h分别留取照片,测量这6个点位置划痕两侧边缘间的距离,取平均值作为这个培养孔内细胞在这个时间点的划痕两侧边缘间距离,每个细胞系的每组有两个重复试验样品,所以再取平均值即为该细胞系该组细胞该时间点划痕两边缘间的距离,分别计算各个时间点两边缘间距离缩小的比例,分析各个细胞增生和长入的能力的差别。结果1.real-time PCR 实验结果:1.1各细胞系VEGF基因和CD133基因表达的总体变化:抑制VEGF后,1543细胞和1423细胞的VEGF的表达有所减低(p0.05);而CD133的表达均有明显增高(p0.05)。阻断mmiR-21后,1543细胞的VEGF基因和CD133基因的表达均有所下降(p0.05);而1423细胞的VEGF基因和CD133基因的表达均有显著提高(p0.05)。1.2各细胞系VEGF基因和CD133基因表达随时间变化的规律:(1)抑制VEGF后,1543细胞对VEGF和CD133基因的表达均是先有所升高,随着时间的进展表达逐渐降低,VEGF基因的表达到24h时已经低于初始对照组;在阻断miR-21后,1543细胞对VEGF和CD133基因的表达均有所下降,而且随着时间的进展表达无明显变化。(2)1423细胞在被抑制VEGF后CD133基因的表达和阻断miRNA-21后VEGF和CD133基因的表达,在两个时间点的变化差别均比较显著。其中,抑制VEGF后CD133基因的表达在8小时达到初始对照组的4.13倍,而在24小时则急剧降低到初始对照组的0.79。2.细胞免疫组化实验结果:2.1 1543 细胞为 VEGFlow/CD133high细胞,而 1423 细胞为 VEGFhi8h/CD133low细胞。2.2 1543细胞在抑制VEGF后表现为VEGF基因表达有所下降,而CD133基因表达有所上升;在阻断miR-21后表现为VEGF和CD133基因表达均下调。2.3 1423细胞在抑制VEGF后也表现为VEGF基因表达有所下降,而CD133基因表达有所上升;但在阻断miR-21后表现为VEGF和CD133基因表达均明显上升。3.细胞划痕实验结果:3.1 1543细胞增殖速度明显快于1423细胞。3.2 1543细胞两个实验组(抑制miR-21组和同时抑制miR-21和VEGF组)细胞均较对照组生长速度减慢(p0.05),两个实验组细胞的生长速度之间无明显差别。3.3 1423细胞经miR-21抑制剂处理后较对照组细胞生长速度减慢(p0.05),而添加两种抑制剂(miR-21和VEGF受体)的细胞生长速度最慢,差别具有统计学意义(p0.05)。结论1.阻断miR-21对于VEGF基因低表达的骨肉瘤细胞有较明显的调控作用,而抑制VEGF对于VEGF基因高表达的骨肉瘤细胞有较明显的调控作用。2.抑制VEGF后两种骨肉瘤细胞株CD133基因表达均上调,VEGF可能在骨肉瘤细胞中抑制CD133基因的表达。3.miR-21在VEGF低表达的骨肉瘤中有促进骨肉瘤干细胞的发生和增殖,从而增加肿瘤体内肿瘤干细胞比例的作用。在这一部分细胞中阻断miR-21能更显著的降低骨肉瘤细胞干细胞标志物CD133的表达,可能具有减少肿瘤干细胞的作用。4.对于VEGF高表达的1423细胞,抑制VEGF可以双向调控CD133基因的表达,表现为短时间的促进后长时间的抑制。意义:我们的研究发现在低表达VEGF的骨肉瘤细胞中转染miR-21的反义寡核苷酸链,可以显著下调骨肉瘤细胞VEGF和CD133的表达。VEGF是促进血管新生的关键生长因子,在肿瘤转移和血管新生中发挥关键作用;而CD133是重要的干细胞标志物,反映了肿瘤细胞的干细胞特性。这两个基因表达下降,提示肿瘤细胞促进血管新生的能力和干细胞的比例下降。这提示抑制miR-21的表达可能发挥抑制肿瘤的生长和血管新生以及转移,同时减少肿瘤干细胞比例的作用,可以有效提高化疗效果。而VEGF受体抑制剂能有效的抑制高表达VEGF的骨肉瘤细胞的生长、侵袭和转移,减少瘤体内肿瘤干细胞比例,从而提高肿瘤对化疗的敏感性。综上所述,我们的研究为不同分子特征的骨肉瘤的治疗提供了有价值的基础研究信息,为指导临床用药和治疗提供了帮助。基于我们的研究结果,可以对不同分子特征(VEGF高表达或VEGF低表达)的骨肉瘤使用相应的治疗药物,有助于实现精准用药和精准治疗。
[Abstract]:Background: osteosarcoma is a primary malignant bone tumor that is most harmful to young people. Surgical excision (amputation) is the main treatment in the past. Patients not only have to suffer severe physical and mental trauma caused by limb disability, but their 5 year survival rate is not more than 20%. It is a disaster for the patients and their families. Even now, it is a disaster. Preoperative chemotherapy can greatly improve the five year survival rate and greatly retain the patient's limb function, thus significantly improving the patient's quality of life, but there are still nearly 1/4 to 1/3 [1] (the domestic literature is reported to 40% - 50%[2]) for less than 5 years of survival. How to improve the chemotherapy Efficiency, reducing the recurrence and metastasis of osteosarcoma is the focus of the current clinical research on osteosarcoma. Recently, many oncology studies have focused on the infinite value added, self renewal and multidirectional differentiation of [3]. tumor stem cells in the field of cancer stem cells, and the tumor stem cells are mostly at stationary phase and are not sensitive to chemotherapy. After chemotherapy, the proportion of stem cells in the tumor is significantly increased, which is the main cause of chemotherapeutic resistance and tumor recurrence. The recurrence and metastasis of osteosarcoma is due to the existence of osteosarcoma stem cells after the combined surgery of osteosarcoma and postoperative chemotherapy combined with adjuvant chemotherapy, and the diagnosis and treatment of osteosarcoma at present. The exhibition is in a bottleneck period. How to kill the stem cells in the tumor effectively and reduce the proportion of the stem cells in the tumor is an effective way to improve the effect of the treatment..CD133 is a kind of transmembrane glycoprotein, which has been found in hematopoietic stem cells and normal stem cells, [5,6], and later found in most of the tumor stem cell membranes, which are now widely used as a broad spectrum marker. It is applied to the resolution and separation of cancer stem cells. Currently, CD133 is a commonly recognized key marker [7,8] for screening tumor stem cells in osteosarcoma. Therefore, in this experiment, the expression of CD133 gene expression as a marker of stem cell characteristics of tumor cells (miRNA) is a group of non coded small molecules RNA (miRNA). About 22 nucleotides, which play an important regulatory role at the post transcriptional level, usually cause translation inhibition, target degradation and gene silencing, which are important for human life process. Many of them play an important role in the development and metastasis of cancer as the proto oncogene or tumor suppressor gene,.MiR-21 as an important role in the development and metastasis of cancer. The miRNA of cancer plays an important role in the invasion, invasion and metastasis of different malignant tumors. [9-11]. studies have confirmed that miR-21 has high expression in osteosarcoma cells, and miR-21 promotes the growth and metastasis of osteosarcoma. Meanwhile, research has confirmed that miR-21 has abnormal expression in the stem cells of a variety of malignant tumors and through protein or gene. Interaction to regulate the functional [12] of malignant tumor stem cells; in addition, studies have shown that the inhibitor of miR-21 (antisense oligonucleotide chain) can improve the sensitivity of some tumor cells to chemotherapeutic drugs [13-15].VEGF is a specific heparin binding growth factor for vascular endothelial cells, and is the major vascular generation in the human body. Factors. In the growth of tumor, the expression of VEGF and its receptor in tumor tissue is increasing under the action of a variety of cancer promoting factors. There are a lot of irregular neovascularization in the tumor tissue, providing sufficient nutrients for tumor tissue growth, and providing access to tumor invasion and metastasis, and [16 in the process of tumor growth, infiltration and metastasis. Studies have confirmed that VEGF plays an important role in the development and metastasis of osteosarcoma, and in other malignant tumors, miR-21 promotes the growth and transfer of [17,18]. by enhancing VEGF expression, thus introducing VEGF in our study of the regulatory role of miR-21 in the occurrence, development, metastasis and tumor stem cells of osteosarcoma. One can be used as a contrast to analyze the role of miR-21 and to analyze the correlation between miR-21 and VEGF in the regulation of osteosarcoma stem cells. Objective: this topic mainly studies the alteration of the VEGF gene and / or CD133 gene expression in different osteosarcoma cells after blocking the action of miR-21 and VEGF, and the effect on the growth of osteosarcoma cells. MiR-21 has a regulatory effect on osteosarcoma and its stem cells. Combined with the regulation of VEGF on osteosarcoma and its stem cell characteristics, the effect of miR-21 on the characteristics of osteosarcoma and its stem cells and its possible mechanism and rules are discussed in order to find out the effective inhibition of osteosarcoma stem cells in the clinic. Methods: 1. cells and cell culture: the selection of human osteosarcoma CRL-1543 and CRL-1423 cell line as the experimental cell, the cell culture solution recommended by the ATCC, the culture conditions and the operation steps for the cultivation of.2. to test the planting of a Realtime-PCR experiment and the cultivation of two cell lines. The culture was divided into 3 experimental groups, which were Oh (initial) control group, 8h block group and 24h blockage group. Each group included 2 holes 1543 cells and 2 hole 1423 cells. In accordance with the cell culture method mentioned above, the two cell lines were cultured and three cells were cultured for 24 hours in the C02 cell culture box, and the cells were harvested and harvested at the beginning of the harvest. Then, miR-21 (has-mir-21-5p) antisense oligonucleotide chain and VEGF receptor inhibitor were added to the 2 cells of the other two groups. Then, the culture plates of two inhibitory groups were returned to culture box to continue to be cultured, and cells were collected for 8 hours and 24 hours after miR-21 inhibitor transfection and VEGF receptor inhibitor treatment, respectively. For 8 hour inhibition group and 24 hour inhibition group, RNA was extracted from each cell sample of each experiment group, then cDNA was reversed, real time-PCR experiment was carried out, and VEGF and CD133 genes were detected in each sample,.3. experiment two cell immuno histochemistry experiment 3.1 and two 8 hole chamber slider (chamber loading) were taken for two cell lines. Two rows of cells were cultured for each chamber slider, which were labeled as CD133 and VEGF. respectively. The top row was implanted with 1543 cells, the lower one was implanted 1423 cells, 10000 cells were implanted in each culture hole, and the corresponding culture medium 0.5ml was added to the incubator for 24 hours, and the cells completely adhered to the wall.3.2 to two cell lines. The first hole in each row of four cells was taken as a blank control, second holes were used as negative control, second holes were used as VEGF (-) to add VEGF receptor inhibitor and fourth holes as miR-21 (-) transfected miR-21 (has-mir-21-5p) blocker. The treated cells were then placed in the incubator and cultured for 24h, and then immunohistochemical staining was performed. The experiment was to detect the expression of VEGF gene and CD133 gene in each cell sample.4. experiment three cell scratch test 4.1 to two cell lines in three experimental groups, which were blank control group, miR-21 blockage group and miR-21 blocking +VEGF inhibition group. Each group consisted of 2 holes 1543 cells and 2 holes 1423 cells, according to the cell culture mentioned above. All the two cell lines treated by.4.2 were cultured for 24 hours in the culture box, and the cells were completely adhered to the wall. Then, no blockers were added in the first four culture holes, the second groups and four culture holes were added to the blocking agent of miR-21, and the third groups were added to the inhibitor and the inhibitor of the miR-21 blocker and the VEGF receptor in the four culture holes. After that, the twelve cell culture plate was put back in the culture box, and the cells were basically covered with the bottom of the culture hole. P200 Pipet tip was used at the bottom of each culture hole. The line was marked through the vertical bottom of the center. The photo was observed with the inverted microscope and the intersection of the six marking lines on the scratch, and then continued to be cultured in the incubator. 4h, 8h, 24h, and 48h were taken respectively to measure the distance between the two sides of the scratches at the 6 points, and the average value was taken as the distance between the two sides of the scratch in this time point of the cell. Each cell line had two repeated test samples, and the refetching average was the time point of the cell line. The distance between the two sides of the scratch was calculated and the distance between the two edges of each time point was reduced, and the differences in the ability to proliferate and grow in each cell were analyzed. Results 1.real-time PCR experimental results: 1.1 the overall changes in the expression of VEGF and CD133 genes in each cell line: after the inhibition of VEGF, the expression of VEGF in 1543 cells and 1423 cells was reduced. Low (P0.05), and the expression of CD133 increased significantly (P0.05). After blocking mmiR-21, the expression of VEGF and CD133 genes in 1543 cells decreased (P0.05), and the expression of VEGF and CD133 genes in 1423 cells increased significantly (P0.05).1.2 cell lines and the regularity of the expression of the gene expression with time: (1) The expression of VEGF and CD133 genes increased first, and the expression of VEGF and CD133 decreased with time. The expression of VEGF gene was lower than that of the initial control group. After blocking miR-21, the expression of VEGF and CD133 genes in the 1543 cells decreased, and there was no obvious change with the development of time. (2) 1423 cells. The expression of CD133 gene after inhibition of VEGF and the expression of VEGF and CD133 gene after blocking miRNA-21 were significantly different at two time points. Among them, the expression of CD133 gene was 4.13 times as high as that of the initial control group at 8 hours after the inhibition of VEGF, and at 24 hours it decreased sharply to the 0.79.2. cell immunohistochemistry experiment of the initial control group. Results: 2.11543 cells were VEGFlow/CD133high cells, while 1423 cells were VEGFhi8h/CD133low cells.2.2 1543 cells showed a decrease in the expression of VEGF gene after inhibition of VEGF, and the expression of CD133 gene increased. After blocking miR-21, the expression of VEGF and CD133 gene expressed in.2.3 1423 cells were also shown as VE after VEGF. The expression of GF gene decreased, but the expression of CD133 gene increased, but the expression of VEGF and CD133 gene expression increased obviously after blocking miR-21. The proliferation rate of 3.11543 cells was significantly faster than that of the two experimental groups of the 1423 cell.3.2 1543 cells (inhibiting the miR-21 group and simultaneously inhibiting the miR-21 and VEGF groups). The growth speed of the group was slow (P0.05). There was no significant difference between the growth speed of the cells in the two experimental groups. The growth speed of.3.3 1423 cells was slower than that of the control group (P0.05), while the growth speed of the cells added with two inhibitors (miR-21 and VEGF receptor) was the slowest, and the difference was statistically significant (P0.05). Conclusion 1. blocked MI. R-21 has obvious regulation effect on osteosarcoma cells with low expression of VEGF gene, while inhibition of VEGF has a significant regulatory effect on osteosarcoma cells with high expression of VEGF gene, CD133 gene expression of two osteosarcoma cell lines is up regulation after.2. inhibition VEGF. VEGF may inhibit the expression of CD133 gene in osteosarcoma cells,.3.miR-21 in VEGF The low expression of osteosarcoma promotes the occurrence and proliferation of osteosarcoma stem cells and increases the proportion of tumor stem cells in the tumor. In this part of the cells, blocking miR-21 can significantly reduce the expression of CD133, a stem cell marker of osteosarcoma cells, which may have the effect of reducing the effect of.4. on the high expression of VEGF by 14 23 cells, inhibition of VEGF can regulate the expression of CD133 gene in two direction, showing a short period of inhibition. Significance: our study found that the transfection of miR-21 antisense oligonucleotide chain in the osteosarcoma cells with low expression of VEGF can significantly reduce the expression of VEGF and CD133 in osteosarcoma cells, which is the key to promoting angiogenesis. Growth factors play a key role in tumor metastasis and angiogenesis, and CD133 is an important marker of stem cells, reflecting the stem cell characteristics of tumor cells. These two genes are reduced, suggesting that the tumor cell's ability to promote angiogenesis and the proportion of stem cells decrease. This suggests that inhibition of the expression of miR-21 may play a role in inhibiting tumor. The growth and angiogenesis and metastasis, as well as reducing the proportion of tumor stem cells, can effectively improve the effect of chemotherapy. VEGF
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R738.1

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