miRNA特异性抑制RhoC基因表达对卵巢癌细胞作用的研究
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摘要
微小RNA是一类长度为二十几个核苷酸的内源性非编码调控RNA,短发夹状miRNA可在细胞内直接转化为siRNA,通过序列特异性抑制或针对mRNA降解来调控基因表达,从而参与细胞发育、增殖及凋亡等一系列重要生物学进程。本研究应用RNAi技术沉默RhoC基因观察其抗肿瘤作用,探讨特异性抑制RhoC基因表达对卵巢癌细胞作用的影响。针对RhoC的mRNA序列设计合成编码miRNA的DNA模板,经过鉴定、测序证明并成功的构建了pcDNATM6.2-GW/EmGFP-RhoC-miRNA真核表达载体(以后简称RhoC-miRNA),并转染人卵巢癌SKOV3细胞,转染RhoC-miRNA重组质粒后能特异地降解细胞内RhoC基因转录的mRNA,从而抑制RhoC蛋白表达;细胞计数统计结果显示,特异性干扰组活细胞的增殖速率明显低于非特异性干扰组、空白对照组;采用划痕法检测细胞趋向运动能力,利用细胞穿越Matrigel基质实验检测细胞侵袭能力结果示:RhoC-miRNA载体有效地特异性地抑制SKOV3细胞增殖、趋向运动能力及侵袭能力,其机制为下调了卵巢癌细胞中MMP2蛋白表达。关闭RhoC基因后,SKOV3细胞对紫杉醇的敏感性显著提高,与单独使用紫杉醇相比,RhoC-miRNA重组质粒联合紫杉醇组中促进凋亡的基因Caspase-3明显增高,而抗凋亡蛋白Suvivin mRNA水平显著降低。
本实验成功构建了RhoC基因的干扰基因miRNA真核表达载体pcDNATM6.2- GW/EmGFP-RhoC-miRNA,可以在人卵巢癌细胞内长期“敲弱”(knock down)RhoC基因,通过抑制RhoC基因下游的细胞调控系统,抑制SKOV3细胞增殖、趋向运动能力及侵袭能力,间接恢复卵巢癌细胞对化疗的敏感性。这将为揭示卵巢癌转移的分子机制奠定基础,也将为卵巢癌的基因治疗开辟一条崭新的途径。
Ovarian carcinoma, one of the top three malignant tumors is threatening the life and health of women. The principle of ovarian carcinoma therapy is to remove the carcinoma by operation and followed by chemical therapy. There are great progress in ovarian carcinoma therapy, the survival rate of five years increased from 30 percent in 1970s to 50 percent at present, but 80 percent of the ovarian carcinoma patients will suffer recurrence and some of them will develop resistance to chemical therapy. So, it is very imperative to develop an effective means to treat ovarian carcinoma. With the development of molecular biology, molecule targetting therapy is promising to be a new way to treat ovarian carcinoma. RNA interference(RNAi) is a self protection phenomenon existing in eukaryotic creature, which can resist the invasion of foreign DNA or viral mRNA and degrade self mRNA with problems. Specific interference genes is a characteristics of RNAi. RNAi has successfully blocked the expression of certain gene in the cells of mice and human in vitro and fulfiled the gene knocking. Rho, that is Ras homologous, comprise three subfamily, RhoA and RhoC, which play important role in cell signal transduction and the are implicated in a variety of physiological functions associated with changes in the actin cytoskeleton, such as cell adhesion, motility, migration, and contraction, cell cycle modulation and gene transcription. There are research results which showed that RhoC was overexpressed in ovarian carcinoma, the overexpression was a sign of poor prognosis, invasion, metastasis and low survival. Therefore, RhoC gene played a very important role in the development and progressio of ovarian carcinoma, exploration of its functions would help to elucidate the mechanisms of ovarian carcinoma development and progression. The present study was to use specific RhoC-miRNA to transfect ovarian carcinoma SKOV3 cell line through liposome to explore its effects on the expression of RhoC gene and protein in SKOV3 cells and establish stably transfected ovarian carcinoma cell line.
Objective:To construct pcDNATM6.2-GW/EmGFP-RhoC-miRNA eukaryotic expression vector and analyse its expression in ovarian carcinoma SKOV3 cells. To explore the effects of RhoC silencing in ovarian carcinoma SKOV3 cell by RNAi on the cell migration and invasion.
Methods:1.Design and synthesize single-stranded oligo to form double-stranded DNA by annealing. 2.Ligate linear pcDNATM6.2-GW/EmGFP-miR with RhoC.3. Construct recombinant plasmidpcDNATM6.2-GW/EmGFP-RhoC-miRNA eukaryotic vector and amplify, screen and sequence the recombinant plasmid.4. Transfect SKOV3 cells with the recombinant plasmid by liopsome and screen the stably transfected SKOV3 cells.5. Test the genes and protein expression of RhoC in SKOV3 cells through RT-PCR and Western-blot.6. Test the proliferating ability of SKOV3 cells by MTT methods.7 .Test the migration ability by scratching method. 8. Test the invasive ability of SKOV3 by Matrigel matrix penetrating experiment.9. Test the expression level of MMP2 protein in the ovarian carcinoma tissue by immunohistochemistry methods. 10. Test the sensitivity changes of SKOV3 to zishanchun after it was interfered by MTT.11.Test the level of apoptotic gene caspase-3 and survivin mRNA in SKOV3 cells before and after transfection by RT-PCR.
Results:1.DNA oligo was successfully ligated to the vector through recombinant plasmid sequencing. 2.SKOV3 cells were seen green under fluorescent microscope 24 hours after the cells were transfected with recombinant plasmid, the transfected cells were round, stronger refraction, contracted not stretched.3.There was a significant decrease in the RhoC expression level in SKOV3 cells transfected with pcDNATM6.2-GW/EmGFP-RhoC-miRNA compared to blank control and non- specific transfection by Western-Blot.4.There was a significant decrease in RhoC-mRNA level in SKOV3 cells transfected with recombinant plasmid pcDNATM 6.2-GW/ EmGFP- RhoC- miRNAcompared to cells transfected with pcDNATM 6.2-GW/ EmGFP-miR-neg and SKOV3 cells by RT-PCR(P<0.05).5.The cell count showed that the live cells in SKOV3 cells transfected with specific recombinant plasmid was 78%, 56.8%, 59.8%, 46.6% that of blank control on 24h, 48h, 72h, and 96h respectively, and 77.8%, 63.2%, 57.7%, 47.6% that of non-specific transfection respectively. For specific transfection cells, there was a significant decrease in the number of live cells compared to non-specific transfected cells and blank control(P﹤0.001).6.There was a significant decrease in cell migration for specific transfection cells compared with blank control and non-specific transfection cells.7.There was a significant decrease in penetrating and invasive ability in specific transfection cells compared to blank control and non-specific transfection cells, which showed that specific transfection decreased the invasive ability of ovarian carcinoma cells.8.There was a significant decrease in the expression of MMP2 in specific transfection cells compared to blank control and non-specific transfection cells(P<0.05). 9. The sensitivity of SKOV3 cells to zishanchun was significantly increased after RhoC gene was blocked.10. The gene of caspase-3 was significantly elevated and suvivin reduced in combined zishanchun and RhoC-miRNA compared to individual zishanchun.
Conclusion:1.The eukaryotic expressing vector pcDNATM6.2-GW/EmGFP-RhoC- miRNA was successfully constructed. 2. The expression of RhoC protein in SKOV3 cells was effectively inhibited after they were transfected with the recombinant plasmid by liposome, which established a research platform for further investigation of RhoC function and development of gene therapy targetted at RhoC.3. There was significant difference in morphology and growing pattern between SKOV3 and transfected SKOV3.4. The growth of SKOV3 cells was significantly inhibited by RhoC-miRNA.
5.The migration and invasion ability of SKOV3 was significantly inhibited by RhoC-miRNA.6. The expression of RhoC-miRNA was enhanced by the expression of MMP2.7. The sensitivity of SKOV3 to zishanchun was increased after the cells were interfered with RNA-Rho, its possible mechanisms may be the upregulation of Caspase-3 and downregulation of Suvivin.
本实验成功构建了RhoC基因的干扰基因miRNA真核表达载体pcDNATM6.2- GW/EmGFP-RhoC-miRNA,可以在人卵巢癌细胞内长期“敲弱”(knock down)RhoC基因,通过抑制RhoC基因下游的细胞调控系统,抑制SKOV3细胞增殖、趋向运动能力及侵袭能力,间接恢复卵巢癌细胞对化疗的敏感性。这将为揭示卵巢癌转移的分子机制奠定基础,也将为卵巢癌的基因治疗开辟一条崭新的途径。
Ovarian carcinoma, one of the top three malignant tumors is threatening the life and health of women. The principle of ovarian carcinoma therapy is to remove the carcinoma by operation and followed by chemical therapy. There are great progress in ovarian carcinoma therapy, the survival rate of five years increased from 30 percent in 1970s to 50 percent at present, but 80 percent of the ovarian carcinoma patients will suffer recurrence and some of them will develop resistance to chemical therapy. So, it is very imperative to develop an effective means to treat ovarian carcinoma. With the development of molecular biology, molecule targetting therapy is promising to be a new way to treat ovarian carcinoma. RNA interference(RNAi) is a self protection phenomenon existing in eukaryotic creature, which can resist the invasion of foreign DNA or viral mRNA and degrade self mRNA with problems. Specific interference genes is a characteristics of RNAi. RNAi has successfully blocked the expression of certain gene in the cells of mice and human in vitro and fulfiled the gene knocking. Rho, that is Ras homologous, comprise three subfamily, RhoA and RhoC, which play important role in cell signal transduction and the are implicated in a variety of physiological functions associated with changes in the actin cytoskeleton, such as cell adhesion, motility, migration, and contraction, cell cycle modulation and gene transcription. There are research results which showed that RhoC was overexpressed in ovarian carcinoma, the overexpression was a sign of poor prognosis, invasion, metastasis and low survival. Therefore, RhoC gene played a very important role in the development and progressio of ovarian carcinoma, exploration of its functions would help to elucidate the mechanisms of ovarian carcinoma development and progression. The present study was to use specific RhoC-miRNA to transfect ovarian carcinoma SKOV3 cell line through liposome to explore its effects on the expression of RhoC gene and protein in SKOV3 cells and establish stably transfected ovarian carcinoma cell line.
Objective:To construct pcDNATM6.2-GW/EmGFP-RhoC-miRNA eukaryotic expression vector and analyse its expression in ovarian carcinoma SKOV3 cells. To explore the effects of RhoC silencing in ovarian carcinoma SKOV3 cell by RNAi on the cell migration and invasion.
Methods:1.Design and synthesize single-stranded oligo to form double-stranded DNA by annealing. 2.Ligate linear pcDNATM6.2-GW/EmGFP-miR with RhoC.3. Construct recombinant plasmidpcDNATM6.2-GW/EmGFP-RhoC-miRNA eukaryotic vector and amplify, screen and sequence the recombinant plasmid.4. Transfect SKOV3 cells with the recombinant plasmid by liopsome and screen the stably transfected SKOV3 cells.5. Test the genes and protein expression of RhoC in SKOV3 cells through RT-PCR and Western-blot.6. Test the proliferating ability of SKOV3 cells by MTT methods.7 .Test the migration ability by scratching method. 8. Test the invasive ability of SKOV3 by Matrigel matrix penetrating experiment.9. Test the expression level of MMP2 protein in the ovarian carcinoma tissue by immunohistochemistry methods. 10. Test the sensitivity changes of SKOV3 to zishanchun after it was interfered by MTT.11.Test the level of apoptotic gene caspase-3 and survivin mRNA in SKOV3 cells before and after transfection by RT-PCR.
Results:1.DNA oligo was successfully ligated to the vector through recombinant plasmid sequencing. 2.SKOV3 cells were seen green under fluorescent microscope 24 hours after the cells were transfected with recombinant plasmid, the transfected cells were round, stronger refraction, contracted not stretched.3.There was a significant decrease in the RhoC expression level in SKOV3 cells transfected with pcDNATM6.2-GW/EmGFP-RhoC-miRNA compared to blank control and non- specific transfection by Western-Blot.4.There was a significant decrease in RhoC-mRNA level in SKOV3 cells transfected with recombinant plasmid pcDNATM 6.2-GW/ EmGFP- RhoC- miRNAcompared to cells transfected with pcDNATM 6.2-GW/ EmGFP-miR-neg and SKOV3 cells by RT-PCR(P<0.05).5.The cell count showed that the live cells in SKOV3 cells transfected with specific recombinant plasmid was 78%, 56.8%, 59.8%, 46.6% that of blank control on 24h, 48h, 72h, and 96h respectively, and 77.8%, 63.2%, 57.7%, 47.6% that of non-specific transfection respectively. For specific transfection cells, there was a significant decrease in the number of live cells compared to non-specific transfected cells and blank control(P﹤0.001).6.There was a significant decrease in cell migration for specific transfection cells compared with blank control and non-specific transfection cells.7.There was a significant decrease in penetrating and invasive ability in specific transfection cells compared to blank control and non-specific transfection cells, which showed that specific transfection decreased the invasive ability of ovarian carcinoma cells.8.There was a significant decrease in the expression of MMP2 in specific transfection cells compared to blank control and non-specific transfection cells(P<0.05). 9. The sensitivity of SKOV3 cells to zishanchun was significantly increased after RhoC gene was blocked.10. The gene of caspase-3 was significantly elevated and suvivin reduced in combined zishanchun and RhoC-miRNA compared to individual zishanchun.
Conclusion:1.The eukaryotic expressing vector pcDNATM6.2-GW/EmGFP-RhoC- miRNA was successfully constructed. 2. The expression of RhoC protein in SKOV3 cells was effectively inhibited after they were transfected with the recombinant plasmid by liposome, which established a research platform for further investigation of RhoC function and development of gene therapy targetted at RhoC.3. There was significant difference in morphology and growing pattern between SKOV3 and transfected SKOV3.4. The growth of SKOV3 cells was significantly inhibited by RhoC-miRNA.
5.The migration and invasion ability of SKOV3 was significantly inhibited by RhoC-miRNA.6. The expression of RhoC-miRNA was enhanced by the expression of MMP2.7. The sensitivity of SKOV3 to zishanchun was increased after the cells were interfered with RNA-Rho, its possible mechanisms may be the upregulation of Caspase-3 and downregulation of Suvivin.
引文
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