与卵巢癌侵袭相关的1ncRNA的初步筛选及MALAT1对卵巢癌细胞生物学行为的影响

发布时间：2018-05-28 02:03

本文选题：卵巢癌 + 长链非编码RNA　；参考：《北京协和医学院》2014年博士论文

【摘要】：研究背景卵巢癌是病死率最高的妇科恶性肿瘤,而早期诊断技术以及长期有效的治疗方案的缺乏是卵巢癌病死率居高不下的原因。因此,寻找新的肿瘤标记物和治疗靶标一直以来都是卵巢癌研究领域的热点。长链非编码RNA (long non-coding RNA, lncRNA)是一类长度大于200nt、不具备蛋白编码功能的RNA。与niRNA等小分子非编码RNA相比,lncRNA近几年才开始受到关注。然而,已有越来越多的研究显示1ncRNA能够在染色质修饰、转录或转录后等水平调控下游基因的表达,进而参与细胞增殖、凋亡及侵袭等多种生物学过程并与肿瘤等疾病的发生发展相关。近年来,基因芯片和高通量测序技术在lncRNA的研究中发挥了重要作用。 Matastasis-associated lung adenocarcinoma transcript1(MALATl)是一个长约8,000nt的非编码RNA,在正常人体组织中普遍表达。2003年,Ji等首次报道MALAT1在非小细胞肺癌中表达上调,并与患者的预后相关。此后,MALAT1又被证实与肝癌、宫颈癌、膀胱癌及胆囊癌等肿瘤的进展相关。然而,其在卵巢癌中的作用尚无研究涉及。本研究分为两个部分。在第一部分中,我们利用基因芯片比较了具有不同侵袭潜能的卵巢癌细胞系的lncRNA表达谱,证实了lncRNA在具有不同侵袭潜能的卵巢癌细胞系中存在差异表达。在第二部分中,我们检测了MALAT1表达下调对卵巢癌细胞生物学行为的影响,发现MALAT1表达下调能够明显抑制卵巢癌细胞的增殖和转移,且MALAT1表达下调可导致多个与细胞增殖、转移和/或凋亡等生物学过程相关的基因的差异表达。第一部分长链非编码RNA在具有不同侵袭潜能的卵巢癌细胞系中表达谱的变化目的比较具有不同侵袭潜能的卵巢癌细胞系的lncRNA表达谱,筛选可能与卵巢癌转移相关的lncRNA,为后续研究奠定基础。方法 1.Transwell侵袭实验验证人卵巢癌细胞系SKOV3及其亚系SKOV3.ip1的体外侵袭能力。 2.基因芯片检测两个细胞系lncRNA表达谱的差异。 3.选取9个差异表达的lncRNA进行qRT-PCR验证。结果 1.与其母系细胞相比,SKOV3.ip1细胞的体外侵袭能力明显增强。 2.芯片共检出4,956个lncRNA,其中上调及下调2倍及以上的lncRNA分别有583个及578个。 3.7个lncRNA的qRT-PCR结果与芯片结果相符。结论 lncRNA在具有不同侵袭潜能的卵巢癌细胞系中存在差异表达,这一结果提示某些lncRNA可能在卵巢癌转移中发挥一定的作用,但其具体功能尚需进一步的研究证实。第二部分下调长链非编码RNA MALAT1的表达抑制卵巢癌细胞增殖和转移目的检测长链非编码RNA MALAT1在卵巢癌细胞中的表达情况,并进一步探索MALAT1在卵巢癌细胞增殖及转移中发挥的作用. 方法 1.用qRT-PCR检测人卵巢癌细胞株SKOV3和人正常卵巢上皮细胞HOSE中MALAT1的表达情况。 2.构建稳定低表达MALAT1的细胞株SKOV3-MALA-T1-KD及其阴性对照SKOV3-NC。 3.细胞增殖实验和平板克隆形成实验检测MALAT1表达下调对卵巢癌细胞体外增殖能力的影响；细胞迁移和侵袭实验检测MALAT1表达下调对卵巢癌细胞体外迁移和侵袭能力的影响；流式细胞术检测MALAT1表达下调对卵巢癌细胞凋亡水平的影响。 4.将稳转细胞株SKOV3-MALAT1-KD及其阴性对照分别做裸鼠移植瘤模型,检测MALAT1表达下调对卵巢癌细胞体内成瘤能力的影响。 5.分别从SKOV3-MALAT1-KD和SKOV3-NC细胞中提取RNA用于基因表达谱芯片研究,比较两组细胞基因表达差异并从中选取20个差异表达的、与细胞增殖、转移和/或凋亡等生物学过程相关的基因进行qRT-PCR验证。结果 1. qRT-PCR结果显示MALAT1在人卵巢癌细胞株中表达水平明显上调。 2.成功构建稳转细胞株SKOV3-MALAT1-KD和SKOV3-NC,干扰效果达77%。 3.细胞增殖实验和克隆形成实验证实MALAT1表达下调可显著地抑制卵巢癌细胞的体外增殖能力(P0.01)；迁移和侵袭实验证实MALAT1表达下调可显著地抑制卵巢癌细胞的体外迁移和侵袭能力(P0.01)；与其阴性对照相比,MALAT1表达下调的细胞凋亡水平显著升高(P0.01)。 4.体内实验结果显示SKOV3-MALAT1-KD组移植瘤的体积明显小于阴性对照组的体积,差异具有统计学意义(P0.01)。 5.芯片结果显示MALAT1表达下调所导致的2倍以上差异表达的基因有921个；qRT-PCR结果显示其中19个基因确实存在差异表达。结论 MALAT1表达下调能够在体外抑制卵巢癌细胞的增殖和转移。此外,MALAT1表达下调还能够抑制卵巢癌细胞的体内成瘤能力。MALAT1表达下调可导致多个与细胞增殖、转移及凋亡等生物学过程相关的基因的差异表达。我们的研究结果显示MALAT1对卵巢癌有促进作用,或许能够成为新的卵巢癌治疗靶点。
[Abstract]:Research background
Ovarian cancer is the most fatal gynecologic malignant tumor, and the early diagnosis technology and the lack of long-term effective treatment are the reasons for the high mortality of ovarian cancer. Therefore, looking for new tumor markers and therapeutic targets has always been a hot spot in the field of ovarian cancer research.
Long chain non coding RNA (long non-coding RNA, lncRNA) is a class of non coded RNA, such as RNA. and niRNA, which has a length larger than 200nt and does not have the function of protein coding. LncRNA has attracted much attention in recent years. However, more and more studies have shown that 1ncRNA can be controlled by the level of chromatin modification, transcription or post transcription. A variety of biological processes, such as cell proliferation, apoptosis and invasion, are associated with the development of cancer and other diseases. In recent years, gene chip and high throughput sequencing technology have played an important role in the research of lncRNA.
Matastasis-associated lung adenocarcinoma transcript1 (MALATl) is a non coding RNA with a long approximately 8000nt, which is generally expressed in normal human tissues. Ji is reported for the first time that MALAT1 is up-regulated in non-small cell lung cancer and is associated with the prognosis of the patients. Thereafter, MALAT1 was confirmed with liver cancer, cervical, bladder and gallbladder cancer. It is related to tumor progression. However, its role in ovarian cancer has not been studied.
This study is divided into two parts. In the first part, we use gene chip to compare the lncRNA expression profiles of ovarian cancer cell lines with different invasive potential and confirm that lncRNA has differential expression in ovarian cancer cell lines with different invasive potential. In the second part, we detected the downregulation of MALAT1 expression to ovarian cancer cells. The effect of biological behavior shows that down regulation of MALAT1 expression can obviously inhibit the proliferation and metastasis of ovarian cancer cells, and the downregulation of MALAT1 can lead to the differential expression of many genes related to biological processes such as cell proliferation, metastasis and / or apoptosis.
Part 1 the expression profile of long chain non coding RNA in ovarian cancer cell lines with different invasive potential.
objective
LncRNA expression profiles of ovarian cancer cell lines with different invasive potential were compared, and lncRNA, which might be related to metastasis of ovarian cancer, was screened.
1.Transwell invasion test confirmed the invasion ability of human ovarian cancer cell line SKOV3 and its subunit SKOV3.ip1 in vitro.
2. gene chip was used to detect the difference of lncRNA expression profiles between two cell lines.
3. select 9 differentially expressed lncRNA for qRT-PCR verification.
Result
1. compared with its maternal cells, the invasion ability of SKOV3.ip1 cells increased significantly in vitro.
A total of 4956 lncRNA were detected in 2. chips, of which 583 and 578 were up and down 2 times and above lncRNA respectively.
The qRT-PCR results of the 3.7 lncRNA coincide with the results of the chip.
conclusion
The differential expression of lncRNA in ovarian cancer cell lines with different invasive potential suggests that some lncRNA may play a role in ovarian cancer metastasis, but its specific function needs further research.
The second part down regulated the expression of long non coding RNA MALAT1 to inhibit the proliferation and metastasis of ovarian cancer cells.
objective
To detect the expression of long chain non coding RNA MALAT1 in ovarian cancer cells, and further explore the role of MALAT1 in the proliferation and metastasis of ovarian cancer cells.
Method
1. detect the expression of MALAT1 in human ovarian cancer cell line SKOV3 and human normal ovarian epithelial cell HOSE by qRT-PCR.
2. construction of stable cell line SKOV3-MALA-T1-KD with low expression of MALAT1 and its negative control SKOV3-NC.
The effect of down regulation of MALAT1 expression on the proliferation of ovarian cancer cells in vitro was detected by 3. cell proliferation experiments and flat clones. Cell migration and invasion tests were used to detect the effect of down regulation of MALAT1 expression on the migration and invasion ability of ovarian cancer cells in vitro; flow cytometry was used to detect the down regulation of MALAT1 expression on the apoptosis level of ovarian cancer cells Influence.
4. SKOV3-MALAT1-KD and its negative control were used as the transplanted tumor model in nude mice respectively, and the effect of down regulation of MALAT1 expression on the tumorigenicity of ovarian cancer cells in vivo was detected.
5. RNA was extracted from SKOV3-MALAT1-KD and SKOV3-NC cells to study the gene expression spectrum chip, and compared the difference of gene expression in the two groups and selected 20 differentially expressed genes. The genes related to biological processes such as cell proliferation, metastasis and / or apoptosis were verified by qRT-PCR.
Result
1. qRT-PCR results showed that MALAT1 expression was significantly up-regulated in human ovarian cancer cell lines.
2. successfully constructed stable cell lines SKOV3-MALAT1-KD and SKOV3-NC, and the interference effect reached 77%.
3. cell proliferation and cloning experiments confirmed that down regulation of MALAT1 could significantly inhibit the proliferation ability of ovarian cancer cells in vitro (P0.01). Migration and invasion experiments confirmed that down regulation of MALAT1 expression significantly inhibited the migration and invasion ability of ovarian cancer cells in vitro (P0.01); compared with negative to photography, the expression of MALAT1 was down regulated. The level of apoptosis was significantly increased (P0.01).
4. in vivo experiments showed that the volume of transplanted tumor in group SKOV3-MALAT1-KD was significantly smaller than that in negative control group (P0.01).
The 5. chip results showed that there were 921 genes of 2 times more differential expression resulting from the downregulation of MALAT1, and the results of qRT-PCR showed that there were 19 differentially expressed genes.
conclusion
The down regulation of MALAT1 expression can inhibit the proliferation and metastasis of ovarian cancer cells in vitro. In addition, the down regulation of MALAT1 expression can also inhibit the down regulation of.MALAT1 expression in ovarian cancer cells, which can lead to the differential expression of many genes related to biological processes such as cell proliferation, metastasis and apoptosis. Our results show MALAT1 pairs Ovarian cancer may be a new target for ovarian cancer therapy.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.31

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