技术领域technical field
本发明首次公开了一种利用RNAi干扰技术靶向卵巢癌中干细胞中Lin28和Oct4的抗肿瘤生物制剂。Lin28和Oct4均为维持干细胞的关键多潜能因子，基因表达沉默后，肿瘤干细胞趋向分化，从而降低自我更新和增殖能力，从而达到抑制肿瘤细胞的生长、转移。The present invention discloses for the first time an anti-tumor biological agent targeting Lin28 and Oct4 in stem cells in ovarian cancer by using RNAi interference technology. Both Lin28 and Oct4 are key pluripotent factors for maintaining stem cells. After gene expression is silenced, tumor stem cells tend to differentiate, thereby reducing the ability of self-renewal and proliferation, thereby inhibiting the growth and metastasis of tumor cells.
技术背景technical background
Lin28是在线虫的时段发育中发挥着关键作用、进化上高度保守的RNA结合蛋白。Lin28具有调节基因表达的多重作用。它能阻断miRNAlet-7的生物合成，也能调节其它miRNA的表达。同时它也结合一群特异性mRNA，包括IGF-2，Oct4以及细胞周期相关因子，从而调节它们的表达。Lin28和Oct4在未分化的胚胎干细胞中高度表达，而随着干细胞分化而表达减少，提示Lin28和Oct4在维持干细胞而保持在未分化的多潜能状态中起着关键作用。Lin28 is an evolutionarily highly conserved RNA-binding protein that plays a key role in the stage development of nematodes. Lin28 has multiple roles in regulating gene expression. It blocks the biosynthesis of miRNAlet-7 and also regulates the expression of other miRNAs. At the same time, it also binds a group of specific mRNAs, including IGF-2, Oct4, and cell cycle-related factors, thereby regulating their expression. Lin28 and Oct4 are highly expressed in undifferentiated embryonic stem cells and their expression decreases as stem cells differentiate, suggesting that Lin28 and Oct4 play a key role in maintaining stem cells in an undifferentiated pluripotent state.
卵巢癌是女性生殖系统常见的肿瘤，恶性程度高，由于早期症状不明显而常常在诊断时已经为晚期。我们利用高通量卵巢癌组织芯片研究发现：卵巢癌细胞中Lin28和Oct4的同步表达与临床分期明显相关，显示低分化状态，预后不良。Lin28和Oct4是干细胞内维持多分化潜能的分子，两种分子都表达的亚群细胞可能代表了卵巢癌干细胞。Lin28在组织中的表达的细胞和Oct4表达的细胞一致。用siRNA技术分别沉默两个分子的表达，对细胞的生存和凋亡没有产生明显的影响；但同时沉默两个分子的表达，卵巢癌细胞的生长增殖和存活能力显著降低。这提示靶向卵巢癌干细胞的治疗可能成为防治卵巢癌的新思路。Ovarian cancer is a common tumor in the female reproductive system, with a high degree of malignancy, and it is often diagnosed at an advanced stage due to the lack of obvious early symptoms. Using high-throughput ovarian cancer tissue microarray studies, we found that the synchronous expression of Lin28 and Oct4 in ovarian cancer cells was significantly correlated with clinical stage, showing a poorly differentiated state and a poor prognosis. Lin28 and Oct4 are molecules that maintain multi-differentiation potential in stem cells, and the subpopulation cells expressing both molecules may represent ovarian cancer stem cells. The cells expressing Lin28 in the tissues were consistent with the cells expressing Oct4. Using siRNA technology to silence the expression of the two molecules separately has no obvious effect on the survival and apoptosis of the cells; but silencing the expression of the two molecules at the same time significantly reduces the growth, proliferation and survival of ovarian cancer cells. This suggests that the treatment targeting ovarian cancer stem cells may become a new idea for the prevention and treatment of ovarian cancer.
因此，我们提出了一种通过siRNA沉默Lin28和Oct4的表达而靶向卵巢癌干细胞而抑制卵巢癌进展的一种抗肿瘤生物制剂。Therefore, we propose an antitumor biologic that targets ovarian cancer stem cells by siRNA silencing the expression of Lin28 and Oct4 to inhibit ovarian cancer progression.
发明内容Contents of the invention
1.本发明公开了一种特异性沉默Lin28和Oct4的siRNAs抗肿瘤生物制剂，能特异抑制卵巢癌中肿瘤干细胞而阻止卵巢癌的进展。1. The present invention discloses an siRNAs anti-tumor biological agent that specifically silences Lin28 and Oct4, which can specifically inhibit tumor stem cells in ovarian cancer and prevent the progression of ovarian cancer.
2.siLin28为(ON-TARGETplus SMARTpool，L-018411-01)为Dharmacon(Lafayette，CO，USA)产品在卵巢癌和畸胎瘤的细胞系中沉默Oct4的效率达95％以上。2. siLin28 (ON-TARGETplus SMARTpool, L-018411-01) is a product of Dharmacon (Lafayette, CO, USA), and the efficiency of silencing Oct4 in ovarian cancer and teratoma cell lines is over 95%.
3.siOct4(L-019591-00)为Dharmacon(Lafayette，CO，USA)，该试剂包含特异的针对Oct4的序列，在卵巢癌和畸胎瘤的细胞系中沉默Oct4的效率达95％以上。3. siOct4 (L-019591-00) is Dharmacon (Lafayette, CO, USA). This reagent contains a specific sequence for Oct4, and the efficiency of silencing Oct4 in ovarian cancer and teratoma cell lines is over 95%.
附图说明Description of drawings
图1同时沉默Lin28和Oct4的表达显著抑制卵巢癌细胞的生存和诱导细胞凋亡。a：siLin8和siOct4抑制Lin28和Oct4的mRNA水平；b：siLin8和siOct4抑制Lin28和Oct4的蛋白水平；c，d：单独抑制Lin28对细胞的生存和凋亡影响不明显；e：同时沉默Lin28和Oct4的表达显著抑制卵巢癌细胞的生存和诱导细胞凋亡Figure 1 Simultaneously silencing the expression of Lin28 and Oct4 significantly inhibited the survival and induced apoptosis of ovarian cancer cells. a: siLin8 and siOct4 inhibit the mRNA levels of Lin28 and Oct4; b: siLin8 and siOct4 inhibit the protein levels of Lin28 and Oct4; c, d: Inhibition of Lin28 alone has no obvious effect on cell survival and apoptosis; e: Simultaneous silencing of Lin28 and The expression of Oct4 significantly inhibits the survival and induces apoptosis of ovarian cancer cells
具体实施方式Detailed ways
在前期工作中，发明人发现siLin28和siOct4同时转染卵巢癌细胞，发现显著地抑制细胞的生长并诱导细胞的凋亡，而单独siLin28和siOct4分别转染而并没有相似的效果。Lin28和Oct4是多潜能因子，是干细胞marker，因此Lin28和Oct4共同表达的细胞可能为卵巢癌干细胞。同时抑制两者的表达，导致癌干细胞不能维持其干性，或分化受阻，因此抑制整个细胞群的增殖，并诱导细胞凋亡，因此，是一种极具潜力的靶向卵巢癌干细胞而抑制卵巢癌进展的抗肿瘤生物制剂。In previous work, the inventors found that siLin28 and siOct4 were simultaneously transfected into ovarian cancer cells, which significantly inhibited cell growth and induced cell apoptosis, while separate transfection of siLin28 and siOct4 had no similar effect. Lin28 and Oct4 are pluripotent factors and stem cell markers, so cells co-expressing Lin28 and Oct4 may be ovarian cancer stem cells. Simultaneously inhibiting the expression of the two will lead to the failure of cancer stem cells to maintain their stemness, or block differentiation, thus inhibiting the proliferation of the entire cell population and inducing apoptosis. Antineoplastic biologics for ovarian cancer progression.
实例抗肿瘤生物制剂抑制卵巢癌细胞的生长Example anti-tumor biologics inhibit the growth of ovarian cancer cells
为了验证该生物制剂对卵巢癌细胞的抑制生长和诱导凋亡效应，分别用不同的实验进行了验证。具体实验步骤如下：In order to verify the growth inhibitory and apoptosis-inducing effects of the biological agent on ovarian cancer cells, different experiments were used to verify it. The specific experimental steps are as follows:
1.siLin28和siOct4的转染1. Transfection of siLin28 and siOct4
1)转染前一天，在不包含抗生素的适量的培养基中接种卵巢癌细胞，转染时细胞的汇合度为30-50％。1) One day before transfection, inoculate ovarian cancer cells in an appropriate amount of culture medium that does not contain antibiotics, and the confluence of the cells at the time of transfection is 30-50%.
2)每一个转染样品都要按照如下的方法准备siRNA-Lipofectamine2000复合物。2) For each transfection sample, prepare the siRNA-Lipofectamine2000 complex according to the following method.
3)在适量的不包含血清的Opti-MEM I低血清培养基稀释siLin28和siOct4，轻轻混匀。3) Dilute siLin28 and siOct4 in an appropriate amount of Opti-MEM I low-serum medium that does not contain serum, and mix gently.
4)使用前轻轻混合Lipofectamine2000，然后稀释适量的试剂到Opti-MEM I低血清培养基，轻轻混匀后在室温下孵育5分钟。4) Gently mix Lipofectamine2000 before use, then dilute an appropriate amount of reagent into Opti-MEM I low serum medium, mix gently and incubate at room temperature for 5 minutes.
5)孵育5分钟后，混合稀释的siRNA和稀释的Lipofectamine2000，轻轻混合，在室温下孵育20分钟，复合物形成。5) After incubation for 5 minutes, mix the diluted siRNA and the diluted Lipofectamine2000, mix gently, and incubate at room temperature for 20 minutes to form a complex.
6)将siRNA-Lipofectamine2000复合物加入到每一个包含细胞和培养基的孔中。通过轻轻地前后摇动培养板混合。6) Add siRNA-Lipofectamine2000 complex to each well containing cells and medium. Mix by gently rocking the plate back and forth.
7)37℃，CO2培养箱孵育24-96小时，直到适合进行基因阻断分析。7) Incubate in a CO2 incubator at 37°C for 24-96 hours until it is suitable for gene blocking analysis.
8)结果分析：siLin28和siOct4的转染导致卵巢细胞株PA-1和IGROV1细胞中Lin28和Oct4水平分别下调90％和80％以上。8) Analysis of the results: the transfection of siLin28 and siOct4 resulted in down-regulation of Lin28 and Oct4 levels in ovarian cell lines PA-1 and IGROV1 cells by more than 90% and 80% respectively.
2.细胞活力检测2. Cell Viability Assay
1)接种转染siLin28/siOct4和siCon的两组细胞各3,000细胞/孔于96-well细胞培养板中，细胞液的终体积为100ul，37度培养过夜。1) Inoculate 3,000 cells/well of two groups of cells transfected with siLin28/siOct4 and siCon in a 96-well cell culture plate, the final volume of cell liquid is 100ul, and culture overnight at 37 degrees.
2)除去培基，加入20ul/孔Reagent试剂，振摇10s，37度孵育1-4h。2) Remove the medium and add 20ul/well Reagent reagent, shake for 10s, incubate at 37 degrees for 1-4h.
3)振摇10s，检测560/590nm吸光值。3) Shake for 10s, and measure the absorbance at 560/590nm.
4)结果分析：转染siLin28/siOct4抗肿瘤生物制剂的实验组的细胞生存活力在PA-1和IGROV1细胞分别下调～65和50％。4) Result analysis: the cell viability of the experimental group transfected with siLin28/siOct4 anti-tumor biological preparation was down-regulated by ~65% and 50% in PA-1 and IGROV1 cells, respectively.
3.细胞凋亡分析3. Apoptosis Analysis
1)接种转染siLin28/siOct4和siCon的两组细胞各3,000细胞/孔于96-well细胞培养板中，细胞液的终体积为100ul，37度培养48h。1) Inoculate 3,000 cells/well of two groups of cells transfected with siLin28/siOct4 and siCon in a 96-well cell culture plate, the final volume of cell liquid is 100ul, and culture at 37°C for 48h.
2)加100μlCaspase-3/7试剂，继续孵育1-4h。2) Add 100μl Caspase-3/7 reagent, continue to incubate for 1-4h.
3)振摇30min-1h。3) Shake for 30min-1h.
4)485±20nm波长检测吸光值。4) 485 ± 20nm wavelength detection absorbance value.
5)结果分析：转染siLin28/siOct4的细胞凋亡率是对照组的4-6倍。5) Result analysis: the apoptosis rate of cells transfected with siLin28/siOct4 was 4-6 times that of the control group.
以上研究表明，siLin28/siOct4能有效抑制卵巢癌细胞的增殖和诱导细胞凋亡，Lin28和siOct4是干细胞marker，其共同表达的细胞可能为卵巢癌干细胞，靶向卵巢癌干细胞有效抑制卵巢癌细胞的增殖并诱导细胞凋亡，成为一种极有潜力的新的抗肿瘤生物制剂。The above studies show that siLin28/siOct4 can effectively inhibit the proliferation and induce apoptosis of ovarian cancer cells. Lin28 and siOct4 are stem cell markers, and the cells co-expressed by them may be ovarian cancer stem cells. Targeting ovarian cancer stem cells can effectively inhibit the proliferation of ovarian cancer cells. Proliferate and induce apoptosis, and become a new anti-tumor biological agent with great potential.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310226198.0ACN104225616A (en) | 2013-06-08 | 2013-06-08 | Anti-tumor biological preparation targeting to ovarian cancer stem cells |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310226198.0ACN104225616A (en) | 2013-06-08 | 2013-06-08 | Anti-tumor biological preparation targeting to ovarian cancer stem cells |
| Publication Number | Publication Date |
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| CN104225616Atrue CN104225616A (en) | 2014-12-24 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310226198.0APendingCN104225616A (en) | 2013-06-08 | 2013-06-08 | Anti-tumor biological preparation targeting to ovarian cancer stem cells |
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