MiR-22对小细胞肺癌放疗敏感性的影响及相关机制研究

发布时间：2019-06-28 12:23

【摘要】：近年来肺癌的发病率与死亡率迅速上升,严重威胁人类的生命健康。小细胞肺癌约占肺癌的20%,其恶性程度高、生长迅速、转移快、对化疗和放疗敏感,初治缓解率高,但极易发生继发性耐药和放疗抵抗、预后差。随着立体定向放射外科、放射治疗设备和技术的飞速发展,放射治疗为肺癌患者提供了非常有效的治疗手段。然而,肿瘤细胞在放射治疗中后期往往产生不同程度的辐射耐受性,从而降低了放射治疗预期的疗效。mi RNA是一种内源性非编码小分子RNA,在肿瘤的形成、增殖、凋亡、化疗耐药以及放疗抵抗中起着重要的作用,因此,miRNA的研究对肿瘤的诊断及治疗具有重大意义。miRNA-22是一种肿瘤抑制因子,miRNA-22高表达能够显著抑制肿瘤细胞的增殖和侵袭。本研究的目的在于探究mi R-22对小细胞肺癌细胞系NCI-H446放疗敏感性的影响,并进一步挖掘相关的分子机制,为改善小细胞肺癌的放射治疗效果提供新思路。主要研究内容如下:1.RT-qPCR检测miR-22在人正常肺上皮细胞系BEAS-2B和小细胞肺癌细胞系NCI-H446中的mRNA表达水平。结果表明,miR-22在小细胞肺癌细胞系NCI-H446的表达显著降低。2.利用miR-22模拟物(mimics)及其对照(nc),瞬时转染小细胞肺癌细胞系NCI-H446,建立miR-22过表达的细胞系。利用mi R-22抑制物(inhibitors)及其对照(inhibitors nc),瞬时转染小细胞肺癌细胞系NCI-H446,建立mi R-22敲低的细胞系。选择载体pLKO.1构建miR-22过表达质粒,采用慢病毒转染的方法,将成功构建的重组质粒及其空载质粒分别转染小细胞肺癌细胞系NCI-H446,建立miR-22过表达的稳转株。利用RT-qPCR技术分别检测阳性细胞株,结果与预期相符,表明miR-22过表达和敲低的细胞系以及miR-22过表达稳转株构建成功。3.采用0Gy、2Gy、4Gy的γ射线照射miR-22不同表达水平的小细胞肺癌细胞NCI-H446,通过MTS实验、集落形成实验和Ki-67抗体检测miR-22对小细胞肺癌细胞增殖的影响。结果表明,在γ射线不同剂量照射条件下,miR-22过表达显著抑制细胞增殖,而miR-22敲低显著促进细胞增殖,并且随着照射剂量的增加趋势更加明显。采用APC Annexin V/PI双染法,并借助流式细胞仪检测miR-22对小细胞肺癌细胞凋亡的影响。结果表明,miR-22过表达能够显著促进细胞凋亡。利用碘化丙啶(propidium iodide,PI)染色,并借助流式细胞仪进行细胞周期实验,发现miR-22对小细胞肺癌细胞NCI-H446的周期几乎无影响。利用划痕实验检测细胞迁移,发现miR-22过表达能够显著抑制小细胞肺癌细胞NCI-H446的迁移能力。4.通过生物信息学数据库Target Scan和Pictar预测miR-22的靶基因,寻找共同的预测靶基因,通过NCBI搜索和文献检索,选择与DNA损伤修复相关的基因WRNIP1进行验证。采用RT-qPCR、Western blot和双荧光素酶报告基因实验,验证WRNIP1与mi R-22的靶向关系。结果表明,WRNIP1是miR-22的直接靶基因,miR-22对WRNIP1具有负向调控作用。5.选择miR-22过表达的稳转株及其空载对照进行高通量转录组测序,寻找与细胞增殖、迁移和凋亡相关的差异表达基因(KLK8、PC、SCUBE1、STC1和GPM6A),进行RT-qPCR验证。结果显示,在miR-22过表达稳转株中,KLK8表达下调,其它四个基因均表达上调,符合理论预期。因此,推测miR-22抑制NCI-H446细胞增殖和迁移可能与KLK8、PC和SCUBE1基因相关,miR-22促进NCI-H446细胞凋亡可能与STC1和GPM6A基因相关。初步阐释了miR-22增加小细胞肺癌放疗敏感性的分子机制。
[Abstract]:In recent years, the incidence and mortality of lung cancer have risen rapidly, which is a serious threat to the health of human life. The small-cell lung cancer accounts for 20% of the lung cancer, the malignant degree of the small-cell lung cancer is high, the growth is rapid, the transfer is fast, the chemotherapy and the radiotherapy are sensitive, the initial treatment response rate is high, the secondary drug resistance and the radiotherapy resistance are easy to occur, and the prognosis is poor. With the rapid development of stereotactic radiosurgery, radiotherapy equipment and technology, radiation therapy provides a very effective means of treatment for patients with lung cancer. However, the tumor cells tend to produce different degrees of radiation resistance in the middle and later stages of radiation therapy, thus reducing the expected therapeutic effect of radiation therapy. Mi-RNA is an endogenous non-coding small-molecule RNA, plays an important role in the formation, proliferation, apoptosis, chemotherapy resistance and radiotherapy resistance of the tumor, and therefore, the research of miRNA is of great significance to the diagnosis and treatment of the tumor. MiRNA-22 is a tumor suppressor, and the high expression of miRNA-22 can significantly inhibit the proliferation and invasion of tumor cells. The purpose of this study was to explore the effect of mi R-22 on the sensitivity of NCI-H446 radiotherapy for small cell lung cancer cell lines, and to further explore relevant molecular mechanisms to provide a new way to improve the therapeutic effect of small cell lung cancer. The main contents of this study are as follows:1. RT-qPCR is used to detect the mRNA expression level of miR-22 in human normal lung epithelial cell line (BEAS-2B) and small-cell lung cancer cell line (NCI-H446). The results showed that the expression of miR-22 in small cell lung cancer cell line NCI-H446 was significantly decreased. The miR-22 mimetics and its control (nc) were used to transiently transfect the small cell lung cancer cell line NCI-H446 to establish a miR-22 overexpression cell line. Small cell lung cancer cell line NCI-H446 was transiently transfected with the mi R-22 inhibitor and its controls, and a cell line with a low mi R-22 was established. The expression plasmid of miR-22 was constructed by selection of vector pLKO.1, and the recombinant plasmid and its no-load plasmid were transfected into small-cell lung cancer cell line NCI-H446, respectively, and the expression of miR-22 was established. The positive cell line was detected by RT-qPCR. The results showed that the expression of miR-22 and the knockdown of the cell line and the expression of miR-22 were successful. The effects of miR-22 on the proliferation of small-cell lung cancer cells were detected by MTS assay, colony formation assay and Ki-67 antibody. The results showed that the expression of miR-22 significantly inhibited the proliferation of the cells under different doses of X-ray, and the knockdown of miR-22 significantly promoted the cell proliferation, and the trend of the increase of the irradiation dose was more obvious. The effect of miR-22 on the apoptosis of small cell lung cancer cells was detected by using the APC Annexin V/ PI double staining method. The results show that the overexpression of miR-22 can promote the apoptosis of cells. It was found that miR-22 had little effect on the cycle of NCI-H446 in small-cell lung cancer cells by using propidium iodide (PI) and cell cycle experiment by flow cytometry. Using the scratch test to detect cell migration, it was found that the overexpression of miR-22 could significantly inhibit the migration of small-cell lung cancer cells NCI-H446. The target gene of miR-22 was predicted by Bioinformatics database Target Scan and Pictar, and a common predicted target gene was found, and a gene WRNIP1 associated with DNA damage repair was selected by NCBI search and literature search. The targeting relationship between WRNIP1 and mi R-22 was verified by RT-qPCR, Western blot and double-luciferase reporter gene. The results show that WRNIP1 is a direct target gene of miR-22, and miR-22 has a negative control effect on WRNIP1. A high-throughput transcriptome sequencing was performed on a stable and no-load control of miR-22 overexpression to find differential expression genes associated with cell proliferation, migration and apoptosis (KLK8, PC, SCUBE1, STC1, and GM6A) for RT-qPCR validation. The results showed that the expression of KLK8 was down-regulated in the expression of miR-22 and the expression of KLK8 was up-regulated and the expression of KLK8 was up-regulated. Therefore, it is presumed that miR-22 inhibits the proliferation and migration of NCI-H446 cells and may be related to KLK8, PC and SCUBE1 genes, and miR-22 promotes the apoptosis of NCI-H446 cells, which may be related to the STC1 and GM6A genes. The molecular mechanism of the sensitivity of miR-22 to the radiotherapy sensitivity of small cell lung cancer is explained.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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