PGCsiRNA-AQP1重组质粒诱导RNAi抑制喉癌生长的实验研究
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摘要
喉癌是常见恶性肿瘤之一,现有治疗方法均不能满足临床需要。随着生物技术的发展,生物疗法已成为肿瘤综合治疗的重要组成部分。手术、放化疗及生物治疗间相互配合,已在多种肿瘤的治疗中取得了显著的疗效。水通道蛋白(AQP1)在肿瘤组织细胞增殖,血管生成和细胞迁移中发挥重要作用中高度表达。因此我们设想应用RNAi技术沉默AQP1蛋白的表达对癌细胞实施“饥渴”手段来阻抑喉癌的生长,发展和转移。期望寻找一个新的靶点,探讨一种有应用前景的生物疗法,对临床综合治疗喉癌将具有重要意义。
本研究首先观察AQP1在喉癌组织中的表达与喉癌发生,发展和转移中的相关性及其意义。在此基础上针对AQP1基因序列,构建PGCsiRNA-AQP1重组质粒:选择阳性脂质体2000作为体外转染试剂将重组质粒转染喉癌Hep-2细胞诱导RNAi进行体外研究;选择中性脂质体DOPC介导重组质粒转入荷瘤小鼠瘤体内诱导RNAi进行体内疗效学研究。结果证明:AQP1在喉癌中的高表达与喉癌的发生,发展和转移密切相关。并且成功构建了PGCsiRNA- AQP1重组质粒有效沉默了AQP1的表达。该重组质粒不仅在体外能够有效抑制Hep-2喉癌细胞的生长,运动和迁移能力,诱导其凋亡。而且由DOPC介导能够在BALB/C小鼠体内诱导RNAi沉默AQP1的表达,抑制喉癌组织的微血管形成,阻抑喉癌生长和发展。该研究为临床进一步探讨基因疗法提供了一个新的思路和实验依据。
Background and Objective:
Laryngeal carcinoma(LC) is one of the common malignant tumors in Otorhinolaryngology.The existing methods are not enough to cure the tumor. Malignant tumors often accompanied with the recurrence or even developed metastasis carcinoma. It has been become a tough problem . With the development of biotechnology, gene therapy has become an important component in the comprehensive treatment of tumor.Surgery operation combination with radiotherapy , chemotherapy or biological therapy have been achieved remarkable results in the treatment of tumors. Therefore,it will has a significant effect for the comprehensive treatment to explore a biological therapy to control the growth, recurrence and metastasis of laryngeal carcinoma.
Water is indispensable for the survival of cells. For a long time,it is generally agreed that the transmembrane transport of water molecules through cell membrane by simple proliferation. A large number of studies have proved that though water molecules can be through cell membrane by simple proliferation, but very slowly, far from to meet the needs of life activity. The rapid transm- embrane transport of water molecules is depending on the aquaporin(AQP) in the cell membrane. There are more than 10 kinds of aquaporins have been found expressing on the cells in different tissues and organs. They play a important role on maintaining the normal life activity. The expression or function of aquaporins(AQPs) have essential effectivenesses on the transmembrane transport of water molecules. At present, studies have found that AQP1 not only have a close relationgship with the cell proliferation,but also play a important role on the angiogenesis and cell migration of tumor tissue. Therefore, we intend to intervene the expresion of AQP1 to inhibit the growth and development of laryngeal carcinoma. We expect to explore a feasible means of gene therapy for laryngeal carcinoma.
RNA interference(RNAi) technology is an emerging gene intercept technology. This technology has the advantages that simplicity, specificity and efficiency. RNAi technology highlight the obvious advantage on the research and treatment of tumor.It opened a new target for gene therapy. However,the main obstacle of gene therapy at present is that the anti-tumor gene can not be transfered to the tumor tissue specifically. So,it is very important to explore a efficient and specific gene-oriented system for the gene therapy of malignant tumor.
M.D.Anderson Cancer Center in Texas recently report that the diameter of tumor blood vessels is 78-100nm while only 2nm in normal blood vessels. So,we take neutral liposome DOPC which diameter is 65-125nm holding RNAi can pass the tumor blood vessels. There was more advantage compare with charge moleculars. At the same time, laryngeal carcinoma has the characteristic that we can inject directly.
Therefore, we constructed the recombinant plasmid PGCsiRNA-AQP1 which mediated by neutral liposomes DOPC to silence the expression of AQP1 by induced RNAi in vivo to inhibit the growth and development of laryngeal carcinoma. We expect to explore a feasible means of gene therapy for laryngeal carcinoma.
Methods:
Firstly, we identified the expression of AQP1 in laryngeal carcinoma then analyzed the relationship with the occurrence,development and metastasis of laryngeal carcinoma. Secondly,according to AQP1 gene sequence we constructed the recombinant plasmid PGCsiRNA-AQP1 employing gene recombination technology.We observed the expression of AQP1 by immunohistochemistry. We detected the inhibitory rate of AQP1 at the level of mRNA and protein by RT-PCR and Western blot respectively. Thirdly, in vitro study,we choosed cationic liposome Lipofectamine TM2000 as transfection reagent transfected recombinant plasmid into laryngeal cancinoma Hep-2 cells. (1) We examined the survival and apoptosis rate of cells growth by MTT and flow cytometry respectively;(2) Studied the adhesion,movement and invasion ability of cells by cell matrix adhesion experiment,Transwell chamber and Matrigel matrix invasion assay,respectively. Fourthly,we selected neutral liposome DOPC mediated recombinant plasmid into tumor-bearing mice induced RNAi then studyed in vivo. (1) We constructed BALB / C mice models transplanted by laryngeal cancinoma Hep-2 cells. We observed the pathology,tumor size and inhibition rate; (2)We detected the expression of AQP1 and angiogenesis number in tumors by Immunohistochemistry then made the correlation analysis.
Results:
1.The expression of AQP1 was significantly increased in laryngeal cancinoma. It has a close relationship with the occurrence, development and metastasis of laryngeal cancinoma. 2. We constructed recombinant plasmid PGCsiRNARNA-AQP1 successfully. The effective inhibition rate of AQP1 protein and mRNA is 60.7% and 65.1% respectively. 3.Study on the recombinant plasmid PGCsiRNARNA-AQP1d in vitro: (1) MTT assay results: The survival rate of Hep-2 cells reduced with the time passing. The survival rate was significantly lower than negative control group and blank control group at the same time. Flow cytometry results: The apoptosis rate of Hep-2 cells increased with the time passing. The cell apoptosis rate was significantly higher than negative control group and blank control group at the same time. (2) Matrix adhesion results: The adhesion rate of Hep-2 cells with extracellular matrix after cultivated 48h in experimental group is not different significantly from negative control group and blank control group( all P>0.05). Cell movement and invasion ability of the experimental results:The number of penetrating cells after transfected 48h was significantly less than negative control group and blank control group (P <0. 01). There was no significant difference in negative control group and blank control group (P>0. 05). 4.We constructed BALB / C mice models of laryngeal carcinoma successfully. (1) HE staining results: Cell putrescence,nuclear pyknosis and apoptotic bodies can be seen in tissues of experimental group. But we can see the opposition in the other two groups. Tumor volume and inhibit rate: The tumor volume in control groups growed rapidly. The growth speed of tumor volume in experimental group was significantly slower than control groups. In tail vein injection group and around tumor injection group,the total volumes of tumor specimens in experimental group were both smaller than negative control group and blank control group. There was significant difference in experimental group compared with negative control group and blank control group(P <0.01). In tail vein injection group and around tumor injection group,the inhibition rate is 52.4% and 53.5% respectively. (2)The AQP1 positive cells and microvessel density (IMDV) after treating 28 days: In tail vein injection group and around tumor injection group, the total volumes of tumor specimens in recombinant plasmids experimental group were both smaller than negative control group and blank control group. The differences were all significantly(P<0.01). The IMDV in tail vein injection group was less than around tumor injection group. But there was not significant difference(P>0.05).
Conclusion:
1.The expression of AQP1 and IMVD increasd significantly in laryngeal cancinoma. We found that there was positive correlation between AQP1 with IMVD. Besides, we also found that the expression of AQP1 has a close relationship with the lymphnodes metastase and histopathology classification. Acetazolamide is a inhibitor of aquaporin. It inhibited the development and induced the apoptosis of laryngeal cancinoma Hep-2 cells by decreasing the expressiom of AQP1.
2.We constructed the recombinant plasmid PGCsiRNARNA-AQP1 successfully and silenced the expression of AQP1 effectively.
3. In vitro study,recombinant plasmid PGCsiRNARNA-AQP1 inhibited the growth,movement and transfer capability of laryngeal cancinoma Hep-2 cells and induced the apoptosis.
4.We constructed BALB / C mice models of laryngeal carcinoma successfully. The recombinant plasmid mediated by DOPC silenced the expression of AQP1 by induced RNAi. It inhibited the formation of microvascular and the growth of laryngeal carcinoma.
本研究首先观察AQP1在喉癌组织中的表达与喉癌发生,发展和转移中的相关性及其意义。在此基础上针对AQP1基因序列,构建PGCsiRNA-AQP1重组质粒:选择阳性脂质体2000作为体外转染试剂将重组质粒转染喉癌Hep-2细胞诱导RNAi进行体外研究;选择中性脂质体DOPC介导重组质粒转入荷瘤小鼠瘤体内诱导RNAi进行体内疗效学研究。结果证明:AQP1在喉癌中的高表达与喉癌的发生,发展和转移密切相关。并且成功构建了PGCsiRNA- AQP1重组质粒有效沉默了AQP1的表达。该重组质粒不仅在体外能够有效抑制Hep-2喉癌细胞的生长,运动和迁移能力,诱导其凋亡。而且由DOPC介导能够在BALB/C小鼠体内诱导RNAi沉默AQP1的表达,抑制喉癌组织的微血管形成,阻抑喉癌生长和发展。该研究为临床进一步探讨基因疗法提供了一个新的思路和实验依据。
Background and Objective:
Laryngeal carcinoma(LC) is one of the common malignant tumors in Otorhinolaryngology.The existing methods are not enough to cure the tumor. Malignant tumors often accompanied with the recurrence or even developed metastasis carcinoma. It has been become a tough problem . With the development of biotechnology, gene therapy has become an important component in the comprehensive treatment of tumor.Surgery operation combination with radiotherapy , chemotherapy or biological therapy have been achieved remarkable results in the treatment of tumors. Therefore,it will has a significant effect for the comprehensive treatment to explore a biological therapy to control the growth, recurrence and metastasis of laryngeal carcinoma.
Water is indispensable for the survival of cells. For a long time,it is generally agreed that the transmembrane transport of water molecules through cell membrane by simple proliferation. A large number of studies have proved that though water molecules can be through cell membrane by simple proliferation, but very slowly, far from to meet the needs of life activity. The rapid transm- embrane transport of water molecules is depending on the aquaporin(AQP) in the cell membrane. There are more than 10 kinds of aquaporins have been found expressing on the cells in different tissues and organs. They play a important role on maintaining the normal life activity. The expression or function of aquaporins(AQPs) have essential effectivenesses on the transmembrane transport of water molecules. At present, studies have found that AQP1 not only have a close relationgship with the cell proliferation,but also play a important role on the angiogenesis and cell migration of tumor tissue. Therefore, we intend to intervene the expresion of AQP1 to inhibit the growth and development of laryngeal carcinoma. We expect to explore a feasible means of gene therapy for laryngeal carcinoma.
RNA interference(RNAi) technology is an emerging gene intercept technology. This technology has the advantages that simplicity, specificity and efficiency. RNAi technology highlight the obvious advantage on the research and treatment of tumor.It opened a new target for gene therapy. However,the main obstacle of gene therapy at present is that the anti-tumor gene can not be transfered to the tumor tissue specifically. So,it is very important to explore a efficient and specific gene-oriented system for the gene therapy of malignant tumor.
M.D.Anderson Cancer Center in Texas recently report that the diameter of tumor blood vessels is 78-100nm while only 2nm in normal blood vessels. So,we take neutral liposome DOPC which diameter is 65-125nm holding RNAi can pass the tumor blood vessels. There was more advantage compare with charge moleculars. At the same time, laryngeal carcinoma has the characteristic that we can inject directly.
Therefore, we constructed the recombinant plasmid PGCsiRNA-AQP1 which mediated by neutral liposomes DOPC to silence the expression of AQP1 by induced RNAi in vivo to inhibit the growth and development of laryngeal carcinoma. We expect to explore a feasible means of gene therapy for laryngeal carcinoma.
Methods:
Firstly, we identified the expression of AQP1 in laryngeal carcinoma then analyzed the relationship with the occurrence,development and metastasis of laryngeal carcinoma. Secondly,according to AQP1 gene sequence we constructed the recombinant plasmid PGCsiRNA-AQP1 employing gene recombination technology.We observed the expression of AQP1 by immunohistochemistry. We detected the inhibitory rate of AQP1 at the level of mRNA and protein by RT-PCR and Western blot respectively. Thirdly, in vitro study,we choosed cationic liposome Lipofectamine TM2000 as transfection reagent transfected recombinant plasmid into laryngeal cancinoma Hep-2 cells. (1) We examined the survival and apoptosis rate of cells growth by MTT and flow cytometry respectively;(2) Studied the adhesion,movement and invasion ability of cells by cell matrix adhesion experiment,Transwell chamber and Matrigel matrix invasion assay,respectively. Fourthly,we selected neutral liposome DOPC mediated recombinant plasmid into tumor-bearing mice induced RNAi then studyed in vivo. (1) We constructed BALB / C mice models transplanted by laryngeal cancinoma Hep-2 cells. We observed the pathology,tumor size and inhibition rate; (2)We detected the expression of AQP1 and angiogenesis number in tumors by Immunohistochemistry then made the correlation analysis.
Results:
1.The expression of AQP1 was significantly increased in laryngeal cancinoma. It has a close relationship with the occurrence, development and metastasis of laryngeal cancinoma. 2. We constructed recombinant plasmid PGCsiRNARNA-AQP1 successfully. The effective inhibition rate of AQP1 protein and mRNA is 60.7% and 65.1% respectively. 3.Study on the recombinant plasmid PGCsiRNARNA-AQP1d in vitro: (1) MTT assay results: The survival rate of Hep-2 cells reduced with the time passing. The survival rate was significantly lower than negative control group and blank control group at the same time. Flow cytometry results: The apoptosis rate of Hep-2 cells increased with the time passing. The cell apoptosis rate was significantly higher than negative control group and blank control group at the same time. (2) Matrix adhesion results: The adhesion rate of Hep-2 cells with extracellular matrix after cultivated 48h in experimental group is not different significantly from negative control group and blank control group( all P>0.05). Cell movement and invasion ability of the experimental results:The number of penetrating cells after transfected 48h was significantly less than negative control group and blank control group (P <0. 01). There was no significant difference in negative control group and blank control group (P>0. 05). 4.We constructed BALB / C mice models of laryngeal carcinoma successfully. (1) HE staining results: Cell putrescence,nuclear pyknosis and apoptotic bodies can be seen in tissues of experimental group. But we can see the opposition in the other two groups. Tumor volume and inhibit rate: The tumor volume in control groups growed rapidly. The growth speed of tumor volume in experimental group was significantly slower than control groups. In tail vein injection group and around tumor injection group,the total volumes of tumor specimens in experimental group were both smaller than negative control group and blank control group. There was significant difference in experimental group compared with negative control group and blank control group(P <0.01). In tail vein injection group and around tumor injection group,the inhibition rate is 52.4% and 53.5% respectively. (2)The AQP1 positive cells and microvessel density (IMDV) after treating 28 days: In tail vein injection group and around tumor injection group, the total volumes of tumor specimens in recombinant plasmids experimental group were both smaller than negative control group and blank control group. The differences were all significantly(P<0.01). The IMDV in tail vein injection group was less than around tumor injection group. But there was not significant difference(P>0.05).
Conclusion:
1.The expression of AQP1 and IMVD increasd significantly in laryngeal cancinoma. We found that there was positive correlation between AQP1 with IMVD. Besides, we also found that the expression of AQP1 has a close relationship with the lymphnodes metastase and histopathology classification. Acetazolamide is a inhibitor of aquaporin. It inhibited the development and induced the apoptosis of laryngeal cancinoma Hep-2 cells by decreasing the expressiom of AQP1.
2.We constructed the recombinant plasmid PGCsiRNARNA-AQP1 successfully and silenced the expression of AQP1 effectively.
3. In vitro study,recombinant plasmid PGCsiRNARNA-AQP1 inhibited the growth,movement and transfer capability of laryngeal cancinoma Hep-2 cells and induced the apoptosis.
4.We constructed BALB / C mice models of laryngeal carcinoma successfully. The recombinant plasmid mediated by DOPC silenced the expression of AQP1 by induced RNAi. It inhibited the formation of microvascular and the growth of laryngeal carcinoma.
引文
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