慢病毒介导的生长抑素siRNA对小鼠生长轴激素的影响
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摘要
本研究将具有高效感染和基因组整合特性的慢病毒载体和RNA干扰结合,研究其对哺乳动物细胞中SS的抑制作用,探索SS基因的沉默对细胞以及小鼠生长轴激素的影响。首先通过质粒转染BHK-21细胞,筛选出了最有效的靶向SS的shRNA(SS 433-451,sh2)。将筛选出的psh2包装并浓缩产生高滴度的病毒液(6×108 ifu/mL),感染BHK-21和MFC细胞。结果表明慢病毒对贴壁及难转染的悬浮细胞均获得了高效的siRNA基因转移和表达效率,并且对内源性SS的基因表达发挥了显著的抑制作用。揭示了SS对正常细胞和肿瘤细胞均有抑制增殖作用,其对正常细胞的抑制增殖作用是通过诱导细胞凋亡发生的。本研究为SS作为治疗药物的开发应用提供了理论依据。最后,基于下丘脑SS的主要功能是抑制垂体GH分泌,本研究采用小鼠脑定位注射的办法,在活体内探讨慢病毒介导SS-siRNA对下丘脑SS的表达下调及GH/IGF-1轴相关因子的变化。结果表明,慢病毒有效的感染了下丘脑SS神经元细胞,并高效表达siRNA,发挥其对目标基因SS稳定的沉默作用。同时,揭示了下丘脑SS对垂体GH的抑制作用,SSTR 2和5是其中起关键作用的SSTRs。本研究首次采用慢病毒介导SS-shRNA沉默了内源性SS的表达,为动物生长调控的研究打下了基础,为研究其他脑内激素的作用机制建立了新的方法和途径。
The endocrine GH/IGF-1 growth axis is important for animal growth in which Growth hormone (GH) is the key factor. The inherent pusatility of the pituitary GH release is governed by alternating episodes of stimulation by growth hormone releasing hormone (GHRH) and inhibition by somatostatin (SS) released from the arcuate and periventricular nuclei of the hypothalamus, respectively. Accumulating physiological evidence suggests that lowering SS in vivo can improve animal productivity. Following the gene knock-down technique, small hairpin RNA (shRNA) can efficiently mediate gene silencing in mammalian cells by guiding sequence-specific degradation of target mRNA sequences after being truncated to siRNA, becoming a powerful tool for the analysis of gene functions. In addition, lentivirus mediated shRNA delivery system has been reported recently on behalf its high transfection efficiency, especially in primary non-dividing cells. These considerations prompted us to investigate that lentivirus-mediated siRNA targeting of SS would offer deep understanding for the effects of SS on cell proliferation, and also the role of preventricular SS in the GH-IGF-1 axis and regulating the somatic growth.
First, shRNAs targeting mouse SS were screened in BHK-21 cells, which can secret SS, to select the most effective construct. Three complementary DNA oligomers (sh1, sh2, sh3) targeting SS 246-264, 433-451, 539-557 bp and a scrambled shRNA (sh0) of the entire shRNA sequence (sense-loop-antisense-terminator) were designed and synthesized for either SS sense or anti-sense target regions. After annealing, the four double-stranded DNAs were cloned into the pshRNA-copGFP lentivector. Meanwhile, the local structures of siRNA target sequences which would probably be important in determining the inhibition efficacy of siRNA were predictd and analyzed. Towards the validation of shRNA inhibition for SS, the levels of SS mRNA and protein in BHK-21 cells were quantitated by real time PCR,immunostaining and RIA techniques after transient transfection. The results showed that all the three siRNAs could effectively decrease SS mRNA and protein level in transfected cells, among which sh2 specific for SS 433-451 gene was the most effective (reduced 59.3% in mRNA level and 55.6% in protein level, respectively, P<0.05). Then, pseudotyped viruses (LV-sh2) were prepared using a sufficient amount of pVSV-G, pGAG, pREV and the optimized psh2 plasmids by cotransfection into 293T cells, and LV-sh0 and LV-GFP were also packaged as controls. High-titer virus stocks (~6×108 ifu/mL) obtained by ulatracentrifugation were achieved routinely measuring by infecting BHK-21 cells in ten-fold serial dilutions.
Several lines of evidence indicate that SS analogs could inhibit the proliferation of tumor cells, but the functions of the endogenous SS on the proliferation and apoptosis of normal cells are still largely unknown. In this research, the endogenous SS in both BHK-21 and MFC cells were knocked down to get deep understanding of SS functions using the pcDNA3.1-SS (pSS) transfected cells as the positive control. The high infection efficiency was observed under the fluorescence at 96 h post infection, and the shRNA gene was detected to integrate into the genomic DNA of infected cells by PCR analysis. SS mRNA and protein levels in LV-sh2 infected BHK-21 cells, were reduced 86.49% in mRNA level and 78.80% in protein level, respectively, P<0.05), both of whom are higher than the gene silencing efficiencies gained by plasmid transfection. In MFC cells, 49.55% of SS was inhibited (P<0.05). The above results mean that lentivirus transduction gained high gene transfer effiency not only in adherent cells, but also in suspension cells which is difficult for gene transfection. Cell growth curve measured using the MTT assay showed that higher levels of SS inhibit the cell growth ability significantly (9.63% in BHK-21 cells, P<0.05 and 8.14% in MFC cells, P<0.05). Reversely, lowering SS could stimulate the cell growth ability (17.33% in BHK-21 cells, P<0.05 and 19.27% in MFC cells, P<0.05), which identified that SS could inhibit the growth of the normal cells and the tumor cells. In addition, the apoptosis of cells detected by flow cytometry indicated that lower percents (24.30% of the control group, P<0.05) of apoptosis cells were observed relative to controlled BHK-21 cells, and higer percents were showed in pSS transfected cells (1.97 folds, P<0.05), which indicated that the antiproliferative function of SS is via the induction of the cell apoptosis. This research gets theoretical foundation for SS and its analogs used as potential drugs that function as an inhibitor in humans or animals.
On behalf of the effects of SS on GH regulation, promoting the animal growth and its productivety via lowering SS thus elevation GH and IGF-1 level becomes more and more meaningful. Reports showed that the main function of the hypothalamic SS is to inhibit GH secretion. We transduced the lentiviral shRNA constructs packaged in pseudoviral particles into the periventricular nucleus of the hypothalamus of male pre-adolescent ICR mice, the site of SS secretion, by stereotaxic injection to study the change of SS expression and its physiological effects on GH/IGF-1 axis. A substantial knockdown of the hypothalamic SS was detected 4 d post-transduction and remained until 30 d, with the lowest level of SS observed on 7 d in regions adjacent to the infusion site (decreased 47.5%, P<0.05) confirmed by fluorescence, immunohistochemistry and RIA, which indicated that lentivirus could infect the SS neuron cells highly, and also the expressed siRNA acted to inhibit the endogenous SS expression. In the pituitary, the exceeded GH protein level (20.95% on 7 d, P<0.05) was closely related to a loss of SS in the hypothalamus, which indicated that SS has an effects on GH secretion, but not its sythesis. Complex expression patterns of mRNA levels of SS receptors (SSTRs) were observed, SSTR 1 decreased 33.00% (P<0.05),while SSTR 2 and SSTR 5 increased 33.00% (P<0.05) and 204% (P<0.05), separately. No change was observed on GHRH and SSTR 3 and 4 mRNA level. The above results reveal that the inhibition effects of the hypothalamic SS for the pituitary GH secretion is mediated by SSTR 2 and SSTR 5. Finally, the serum SS, GH, and IGF-1 concentration remained unchanged, and also the body weight of the male adolescent mice, indicating most of serum SS not provided by the hypothalamic SS.
To our knowledge, this is the first report of targeted knockdown of SS in the hypothalamus gaining deep understanding on the functions of SS on GH/IGF-1 axis in adolescent mouse. Furthermore, it is a strong indication that lentivirus mediated shRNA would be a novel way to study gene function in vivo, especially for genes in specific area at distinct development stage.
The endocrine GH/IGF-1 growth axis is important for animal growth in which Growth hormone (GH) is the key factor. The inherent pusatility of the pituitary GH release is governed by alternating episodes of stimulation by growth hormone releasing hormone (GHRH) and inhibition by somatostatin (SS) released from the arcuate and periventricular nuclei of the hypothalamus, respectively. Accumulating physiological evidence suggests that lowering SS in vivo can improve animal productivity. Following the gene knock-down technique, small hairpin RNA (shRNA) can efficiently mediate gene silencing in mammalian cells by guiding sequence-specific degradation of target mRNA sequences after being truncated to siRNA, becoming a powerful tool for the analysis of gene functions. In addition, lentivirus mediated shRNA delivery system has been reported recently on behalf its high transfection efficiency, especially in primary non-dividing cells. These considerations prompted us to investigate that lentivirus-mediated siRNA targeting of SS would offer deep understanding for the effects of SS on cell proliferation, and also the role of preventricular SS in the GH-IGF-1 axis and regulating the somatic growth.
First, shRNAs targeting mouse SS were screened in BHK-21 cells, which can secret SS, to select the most effective construct. Three complementary DNA oligomers (sh1, sh2, sh3) targeting SS 246-264, 433-451, 539-557 bp and a scrambled shRNA (sh0) of the entire shRNA sequence (sense-loop-antisense-terminator) were designed and synthesized for either SS sense or anti-sense target regions. After annealing, the four double-stranded DNAs were cloned into the pshRNA-copGFP lentivector. Meanwhile, the local structures of siRNA target sequences which would probably be important in determining the inhibition efficacy of siRNA were predictd and analyzed. Towards the validation of shRNA inhibition for SS, the levels of SS mRNA and protein in BHK-21 cells were quantitated by real time PCR,immunostaining and RIA techniques after transient transfection. The results showed that all the three siRNAs could effectively decrease SS mRNA and protein level in transfected cells, among which sh2 specific for SS 433-451 gene was the most effective (reduced 59.3% in mRNA level and 55.6% in protein level, respectively, P<0.05). Then, pseudotyped viruses (LV-sh2) were prepared using a sufficient amount of pVSV-G, pGAG, pREV and the optimized psh2 plasmids by cotransfection into 293T cells, and LV-sh0 and LV-GFP were also packaged as controls. High-titer virus stocks (~6×108 ifu/mL) obtained by ulatracentrifugation were achieved routinely measuring by infecting BHK-21 cells in ten-fold serial dilutions.
Several lines of evidence indicate that SS analogs could inhibit the proliferation of tumor cells, but the functions of the endogenous SS on the proliferation and apoptosis of normal cells are still largely unknown. In this research, the endogenous SS in both BHK-21 and MFC cells were knocked down to get deep understanding of SS functions using the pcDNA3.1-SS (pSS) transfected cells as the positive control. The high infection efficiency was observed under the fluorescence at 96 h post infection, and the shRNA gene was detected to integrate into the genomic DNA of infected cells by PCR analysis. SS mRNA and protein levels in LV-sh2 infected BHK-21 cells, were reduced 86.49% in mRNA level and 78.80% in protein level, respectively, P<0.05), both of whom are higher than the gene silencing efficiencies gained by plasmid transfection. In MFC cells, 49.55% of SS was inhibited (P<0.05). The above results mean that lentivirus transduction gained high gene transfer effiency not only in adherent cells, but also in suspension cells which is difficult for gene transfection. Cell growth curve measured using the MTT assay showed that higher levels of SS inhibit the cell growth ability significantly (9.63% in BHK-21 cells, P<0.05 and 8.14% in MFC cells, P<0.05). Reversely, lowering SS could stimulate the cell growth ability (17.33% in BHK-21 cells, P<0.05 and 19.27% in MFC cells, P<0.05), which identified that SS could inhibit the growth of the normal cells and the tumor cells. In addition, the apoptosis of cells detected by flow cytometry indicated that lower percents (24.30% of the control group, P<0.05) of apoptosis cells were observed relative to controlled BHK-21 cells, and higer percents were showed in pSS transfected cells (1.97 folds, P<0.05), which indicated that the antiproliferative function of SS is via the induction of the cell apoptosis. This research gets theoretical foundation for SS and its analogs used as potential drugs that function as an inhibitor in humans or animals.
On behalf of the effects of SS on GH regulation, promoting the animal growth and its productivety via lowering SS thus elevation GH and IGF-1 level becomes more and more meaningful. Reports showed that the main function of the hypothalamic SS is to inhibit GH secretion. We transduced the lentiviral shRNA constructs packaged in pseudoviral particles into the periventricular nucleus of the hypothalamus of male pre-adolescent ICR mice, the site of SS secretion, by stereotaxic injection to study the change of SS expression and its physiological effects on GH/IGF-1 axis. A substantial knockdown of the hypothalamic SS was detected 4 d post-transduction and remained until 30 d, with the lowest level of SS observed on 7 d in regions adjacent to the infusion site (decreased 47.5%, P<0.05) confirmed by fluorescence, immunohistochemistry and RIA, which indicated that lentivirus could infect the SS neuron cells highly, and also the expressed siRNA acted to inhibit the endogenous SS expression. In the pituitary, the exceeded GH protein level (20.95% on 7 d, P<0.05) was closely related to a loss of SS in the hypothalamus, which indicated that SS has an effects on GH secretion, but not its sythesis. Complex expression patterns of mRNA levels of SS receptors (SSTRs) were observed, SSTR 1 decreased 33.00% (P<0.05),while SSTR 2 and SSTR 5 increased 33.00% (P<0.05) and 204% (P<0.05), separately. No change was observed on GHRH and SSTR 3 and 4 mRNA level. The above results reveal that the inhibition effects of the hypothalamic SS for the pituitary GH secretion is mediated by SSTR 2 and SSTR 5. Finally, the serum SS, GH, and IGF-1 concentration remained unchanged, and also the body weight of the male adolescent mice, indicating most of serum SS not provided by the hypothalamic SS.
To our knowledge, this is the first report of targeted knockdown of SS in the hypothalamus gaining deep understanding on the functions of SS on GH/IGF-1 axis in adolescent mouse. Furthermore, it is a strong indication that lentivirus mediated shRNA would be a novel way to study gene function in vivo, especially for genes in specific area at distinct development stage.
引文
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