Effect of RNAi-mediated IGF1R gene silencing on growth,migration, and invasion of hepatocellular carcinoma cells

AIM: To investigate the effect of RNA interference (RNAi)-mediated insulin-like growth factor 1 receptor (IGF1R) gene silencing on the growth, migration, and invasion of hepatocellular carcinoma cells. METHODS: The most effective siRNA targeting IGF1R gene was designed and screened. After lentiviral expression vector pLVX-shRNA2-IGF1R carrying the most effective siRNA sequence was constructed, it was transfected into 293T cells and packed into pLVX-shRNA2-IGF1R lentivirus. Huh7 and Hep3B cells were infected with the pLVX-shRNA2-IGF1R lentivirus to screen the positive clone Huh7 cells and Hep3B cells with the lentivirus. These Huh7 cells and Hep3B cells were cultured to analyze the mRNA level of IGF1R, cell proliferation, cell cycle, cell apoptosis, cell migration/invasion, and the protein levels of IGF1R, Ki-67, p-AKT, p-ERK1, Gli1, β-catenin, cyclin D1, p21 and BCL-XL. RESULTS: The mRNA expression of IGF1R in Huh7 cells and Hep3B cells with pLVX-shRNA2-IGF1R lentivirus was significantly reduced. The proliferation of these cells was remarkably inhibited, and the number in G₁ phase was increased significantly. The percentages of apoptotic cells were increased markedly, and the number of cell migration/invasion was decreased markedly. The protein levels of IGF1R, Ki-67, p-AKT, p-ERK1, Gli1, β-catenin, cyclin D1, p21 and BCL-XL were decreased significantly compared with the blank control group and negative control group. CONCLUSION: The RNAi-mediated IGF1R gene silencing significantly suppresses the growth and the malignant biological characteristics of Huh7 cells and Hep3B cells, which may be involved in the reduced protein levels of the above genes induced by down-regulation of IGF1R expression.     

干旱胁迫对马铃薯类黄酮和类胡萝卜素合成关键酶基因表达的影响

利用马铃薯抗旱二倍体材料H145和对干旱敏感二倍体材料H214,通过半定量RT-PCR方法研究了干旱胁迫0、7、10、12、14 d时叶片和根系中3-二氢黄酮羟化酶（F3H）基因、黄酮合成酶（FLS）基因和β-胡萝卜素羟化酶1（HYD-1）基因的表达状况。结果表明：干旱胁迫过程中马铃薯品系H145 和H214 叶片和根系中F3H基因、FLS基因、HYD-1基因的表达量在轻度或中度干旱胁迫下有不同程度的增加,但品系H145在干旱的早期叶片中F3H基因表达量比根系里增加快,并且叶片中FLS基因和HYD-1基因受干旱诱导的表达持续时间比根系中长;品系H214叶片中F3H 基因受干旱诱导表达持续的时间比根系中长,在干旱的早期叶片里FLS 基因和HYD-1 基因表达量没有根系里增加快。说明F3H、FLS和HYD-1基因参与了马铃薯抗旱反应,但不同的品系这些基因起作用的方式可能不同。     

施肥水平对马铃薯块茎发育过程中PAs、GA3和JAs含量的影响

 为探讨肥力水平对马铃薯块茎发育影响的机理,以‘台湾红皮’（Cardinal）马铃薯为试验材料,采用反相高效液相色谱（HPLC）技术,研究了低、中、高肥力水平对块茎发育过程中多胺（PAs）、赤霉素（GA₃）和茉莉酸（JAs）含量的影响。研究结果表明：（1）腐胺（Put）、精胺（Spm）和亚精胺（Spd）含量在块茎发育过程中呈降低-升高-降低的变化趋势,但对不同肥力水平的响应有差异,中肥力水平下块茎发育中期（播种后60 ~ 68 d）PAs含量高于低肥力和高肥力水平含量,高肥力水平使Put含量降低,含量峰值出现时间提前,而使Spd和Spm峰值出现时间延后。Put/Spd呈现随肥力水平的提高,峰值不断降低,峰值出现时间提前的趋势。（2）低肥力水平下GA₃含量在中期高,中肥力水平下在中期含量最低。低肥力水平下JAs含量在块茎发育的各阶段都比较低,中肥力水平下在中期含量高于低肥力和高肥力水平。低、高肥力水平下GA₃/JAs有先升高后降低的变化趋势,与中肥力水平变化相反。（3）JAs与PAs的相关性较大,其中与Put为正相关,与Spm、Spd显著负相关,Spm和Spd极显著正相关。合理的肥力水平提高了块茎发育中期PAs、JAs的含量,降低了GA₃的含量,有利于块茎的发育和膨大。     

马铃薯根和叶空间分布的品种间差异及其对产量的影响

利用根量多、耐旱、抗倒伏的根优1号、根优4号及高产优质、根量少、易倒伏的粉吹雪为试材,在马铃薯地上部最大期调查了根和叶空间分布的品种间差异及其对产量的影响。结果表明：① 根优1号和根优4号分布在土壤0～120 cm处的总根长和30～120 cm处的根长都显著高于粉吹雪|② 根优1号和根优4号的根系主要分布在土壤深层（30～120 cm）,而粉吹雪的根系则主要分布在土壤耕作层（0～30 cm）|③ 根优1号叶面积分布最均匀,而根优4号则主要集中分布在中上层（40～80 cm）,特别是在60～80 cm（占总叶面积的41.7%）,而根优1号的吸光系数显著低于粉吹雪和根优4号,因此根优1号从地上部最大期到收获期的块茎干物质增加量和增加速度最大|④ 株高、主茎粗和比根长各品种间差异显著,根优1号的植株最高,主茎最粗,但比根长最小,粉吹雪的植株最矮,主茎最细,但比根长最大。     

Effect of shRNA-mediated survivin gene silencing on sensitivity of lymphoma cells to adriamycin

AIM: This study was designed to use RNA interference technique to down-regulate the expression of survivin gene in human Burkitts lymphoma cell line Daudi and to explore the effect on sensitivity of Daudi cells to adriamycin. METHODS: The survivin-shRNA expression vector was constructed and transfected into Daudi cells. Expression of survivin mRNA and protein were assessed by RT-PCR and Western blotting analysis, respectively. Apoptosis index of transfected Daudi cells was quantified by flow cytometry. The sensitivity of Daudi cells to adriamycin (ADR) before and after transfection was detected by MTT test. RESULTS: The mRNA and protein levels of survivin were down-regulated by 62.32% and 61.88%, respectively, compared to those in control-shRNA treated group and PBS treated group (P<0.05). Meanwhile, the apoptosis index was significantly increased (19.10%±2.15%), compared to that in control group (4.48%±1.54%) and PBS group (4.35%±1.37%, P<0.05). The 50% inhibition concentration (IC50) of ADM to Daudi cells was significantly decreased (0.25±0.43) μmol/L, compared to that in control group (0.87±0.21) μmol/L and PBS group (0.91±0.36) μmol/L, P<0.05. CONCLUSION: Down-regulation of survivin expression in Daudi cells by shRNA effectively induces apoptosis and increases the sensitivity of Daudi cells to ADR.     

Effect of silencing hHBrk1 on proliferation and migration of lung carcinoma cells

AIM: To observe the effect of hHBrk1 gene on proliferation and migration of lung carcinoma cells. METHODS: Recombinant plasmids harboring 19-nt-long small interfering RNA (siRNA) were constructed and tested to selectively downregulate hHBrk1 gene in human lung cancer 95D cell line in vitro by stable transfection with Lipofectamine 2000. The mRNA level of the cells transfected with siRNA plasmids were monitored by Northern blotting and RT-PCR. Growth curve and flow cytometry were applied to determine the cell proliferation and cell cycle. Ability of cell migration was measured by Trans-well system. RESULTS: hHBrk1 gene was silenced by targeting siRNA, and stable silencing cell model was constructed. No difference in proliferation and clone formation between hHBrk1 silencing cells and control cells was observed. The ability of migration was decreased in hHBrk1 silencing cells as compared with control cells. CONCLUSION: hHBrk1 may play an important role in migration of the lung cancer cells.     

Effect of Wip1 gene silencing on chemotherapy sensitivity of human colon cancer cells

AIM: To observe the inhibitory effect of siRNA targeting to Wip1 gene on the Wip1 gene expression in the colon cancer cells and to investigate the influence of Wip1 gene silencing on the chemotherapy sensitivity of colon cancer cells. METHODS: Wip1-811 siRNA targeting to Wip1 gene was transfected into RKO colon cancer cells with high expression of Wip1 gene. The mRNA expression of Wip1 was measured by real-time PCR. The protein level of Wip1 was detected by Western blotting. The viability of RKO colon cancer cells was measured by MTS assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Wip1-811 siRNA efficiently inhibited the expression of Wip1 at mRNA and protein levels. The enhanced chemotherapy sensitivity of RKO colon cancer cells was observed after inhibition of Wip1 gene expression. The viability of RKO colon cancer cells was decreased from (89.4±6.6)% to (74.7±3.9)% after treated with 5-fluorouracil (P<0.05) and decreased from (77.9±2.4)% to (66.7±2.9)% after treated with oxaliplatin (P<0.05). The cell apoptotic rate was increased from (7.7±0.5)% to (12.3±3.2)% and from (14.7±2.1)% to (34.0±2.1)% when RKO colon cancer cells were treated with 5-fluorouracil and oxaliplatin, respectively (P<0.05). CONCLUSION: Wip1 gene silencing enhances chemotherapy sensitivity of colon cancer cells.     

Effect of microtubule-destabilizing protein stathmin gene silencing on proliferation and apoptosis of nasopharyngeal carcinoma cell line 5-8F

AIM: To investigate the effects of stathmin gene silencing on nasopharyngeal carcinoma cell line 5-8F. METHODS: Double-strand siRNA targeting to stathmin gene was obtained by chemical synthesis and annealing, and was sub-cloned into the vector pGenesil-1.1. The plasmid was introduced into 5-8F cells by liposome-mediated transfection. The gene expression of stathmin, and the proliferation, morphology and apoptosis of the cells were analyzed by Western blotting, MTT assay and flow cytometry. RESULTS: The cell suppression rate in stathmin gene silencing group was (53.01?1.12)%, significantly higher than that in transfection reagent group and in negative control group. The cell apoptotic rate in stathmin gene silencing group was (8.75?0.67)%, also significantly higher than that in transfection reagent group and in negative control group (P<0.05). CONCLUSION: Silencing of stathmin gene in nasopharyngeal carcinoma cells inhibits the cell proliferation and induces cell apoptosis.     

Cloning,silencing, and prokaryotic expression of strawberry sucrose synthase gene FaSus1

The sucrose synthase (Sus) gene is regarded to be involved in strawberry fruit ripening; however, direct molecular and biochemical evidence is lacking. Here, the coding cDNA sequence of FaSus1 was cloned by RT-PCR and both a hairpin-mediated RNA interference pBI121 vector and a recombinant E. coli expression PET28a vector were constructed. These vectors were then used to transform strawberry fruit and for prokaryotic expression in strain BL21, respectively. The results showed that hairpin-mediated RNA silencing of FaSus1 led to a decrease in sucrose content and inhibited fruit ripening. Enzymatic activity analysis of FaSus1 showed that the reaction system contains 0.309 mg of recombinant FaSus1 protein, reaching 0.617 mg/mL. The cleavage activity of the enzyme was 0.25 U with a specific activity of 0.812 U/mg, whilst the synthetic activity of the enzyme was 0.057 U with a specific activity of 0.185 U/mg. This study provides physiological, molecular, and biochemical evidence to demonstrate that the FaSus1 has high sucrolysis activity and plays an important role in the regulation of strawberry fruit ripening.     

甘薯膨大素对慈菇生长发育的影响

施用甘薯膨胀大素可改善慈菇营养生长状况,促进慈菇球茎的膨大,球茎开始膨大时叶面喷施甘薯膨大素,可显著增加早期产量;定植时及球茎开始膨大时２次施用甘薯膨大素,早期产量增加不显著,总产量显著增加。     

嫁接对寒地棚室茄子根际土壤微生物的影响

以"托鲁巴姆"茄子为砧木,以"龙园棚茄一号"为接穗,研究了嫁接技术对连作棚室茄子根际土壤微生物的影响。结果表明:随着连作年限的增加,未嫁接茄子根际土壤中的细菌和放线菌的数量明显降低,而真菌数量则明显增加,采用"托鲁巴姆"为砧木嫁接后与未嫁接相比,土壤中的细菌和放线菌稍有降低,但基本上仍维持在较高水平,真菌略有增加,土壤中的微生物数量基本上维持在相对平衡的水平。     

Effects of HIF-1α silencing on proliferation of rat hepatoma cells

AIM：To study the effect of hypoxia-inducible factor 1α (HIF-1α) silencing on the proliferation of hepatoma cells under hypoxia. METHODS：Rat hepatoma cell line CBRH-7919 was used in this study. Hypoxia model was established by treating the cells with cobalt chloride (CoCl2). The expression of HIF-1α was silenced by small interfe-rence RNA. Real-time RT-PCR and Western blotting were used to detect the mRNA and/or protein expression of HIF-1α, vascular endothelial growth factor (VEGF), p21 and cyclin D1 in CBRH-7919 cells under hypoxia. The proliferation of CBRH-7919 cells was measured by the technique of 5-bromo-2’-deoxyuridine (BrdU) incorporation. RESULTS：The expression of HIF-1α and VEGF at mRNA and protein levels was significantly increased under hypoxia (P<0.05). Silencing of HIF-1α significantly inhibited the expression of HIF-1α, VEGF and cyclin D1 at mRNA and/or protein levels, while increased the protein expression of p21 (P<0.05). The BrdU-positive cells in HIF-1α siRNA transfection group were significantly less than those in control group. CONCLUSION：HIF-1α silencing significantly inhibits the proliferation of hepatoma cells under hypoxia.     

Effects of Smo gene silencing on cell activity and apoptosis of human cervical carcinoma HeLa cells

AIM: To investigate the effect of Hedgehog (Hh) signaling pathway on the viability and apoptosis of cervical carcinoma cells by shRNA technique to knock down Smoothened (Smo) gene. METHODS: Smo shRNA was used to transfect the cervical carcinoma HeLa cells. The expression of Smo and Gli1 at mRNA and protein levels in the HeLa cells was determined by RT-PCR and Western blot, respectively. The effect of Smo gene silencing on the growth of the cells was measured by MTT assay. The apoptosis and cell cycle were determined by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression of Smo and Gli1 were evenly reduced obviously after transfected with Smo shRNA for 72 h (P<0.05). The viability of HeLa cells transfected with Smo shRNA was significantly inhibited. The percentages of the cells in G₀/G₁ phase and early apoptosis rate were obviously higher in Smo shRNA transfection group than those in control group. CONCLUSION: Smo gene silencing effectively inhibits the cell growth and induces the apoptosis of human cervical carcinoma cells.     

Effects of epididymal P34H gene silencing on expression of P34H and activity of hyaluronidase in mouse sperm

AIM: To investigate the effects of P34H gene silencing on the expression of P34H and activity of hyaluronidase (HYD) in mouse sperm.METHODS: The recombinant plasmid series of P34H targeted short hairpin RNA (shRNA) were constructed by GV248 plasmids vector. These recombinant plasmids were transformed into DH5α competent cells, and the plasmids were taken from DNA sequencing analysis. The HEK293T cells were co-transfected with shRNA and lentiviral packaging plasmids. The 3 kinds of recombinant lentiviruses and negative control lentiviruses were used to inject into the mouse epididymis and the expression of P34H at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The location of P34H protein on the mouse spermatozoa was determined by indirect immunofluorescent staining using P34H antibody. The positive rate and activity intensity of HYD was detected by modified sodium hyaluronate-gelatin membrane. RESULTS: DNA sequencing analysis confirmed that the 3 P34H-shRNA sequences were successfully inserted into the lentiviral vectors. P34H expression in epididymis tissue was significantly decreased at both mRNA and protein levels compared with those of the non-transfected and normal control vectors (P<0.05). The GV-P34H-shRNA-1 played a significant role in reducing the percentage of P34H positive rate and the activity of HYD in mouse sperm. The silencing effect did not significantly differ between the non-transfected and normal control vectors. CONCLUSION: Silencing of P34H significantly inhibits the percentage of P34H positive rate and the activity of hyaluronidase in mouse sperm.     

温度和GA_3对桃果实采后乙烯合成及桃ETR1同源基因表达的影响

为明确桃果实采后成熟衰老期间乙烯的合成与桃ETR1同源基因之间的关系,进一步了解乙烯的转导和作用过程,设计合成了桃ETR1扩增引物RTetr1和RTetr2,用RT-PCR方法研究了温度和GA3处理对白凤桃(Amyg-daluspersicaL.)果实采后乙烯生成和桃ETR1同源基因表达的影响。结果表明,低温处理降低了果实乙烯释放量,低温+GA3处理不仅降低了果实乙烯释放量,还延缓乙烯释放高峰期。PCR分析表明,桃ETR1的cDNA合成受温度和GA3调节,随着乙烯合成能力的增强,ETR1同源基因的表达水平增加,在乙烯信号转导过程中呈正向表达模式。     

Effect of mineralocorticoid receptor gene silencing by RNA interference on proliferation and apoptosis of murine macrophage cell line RAW 264.7

AIM: To establish stable knockdown of mineralocorticoid receptor (MR) expression through short hairpin RNA (shRNA)-mediated silencing in murine RAW 264.7 macrophages. METHODS: Stable MR silencing in RAW 264.7 cells was achieved by recombinant shRNA plasmid targeting murine MR gene via liposome-mediated transfection, followed by G418 selection. The efficacies of plasmid transfection and MR silencing in G418-resistant cells were verified by immunofluorescent microcopy and real-time PCR, respectively. Proliferative activity of MR-silencing cell line was analyzed by CCK-8 assay. Cell cycle and apoptosis were evaluated by flow cytometry. RESULTS: MR gene expression was down-regulated by 70% compared with the negative control (NC) plasmid transfection. In addition, MR-silencing cells exhibited lower proliferative activity compared with NC and wide type RAW 264.7 cells (P<0.05), along with reduced proliferation index of 31.0%±1.3% (P<0.05), compared with the wide type cells (37.2%±0.5%) and the NC cells (37.5%±1.6%). In resting state, the apoptotic rate in wide type, NC and MR-silencing cells were 2.18%±0.36%, 6.65%±0.81% and 7.70%±1.34%, respectively, and no statistical difference was observed between NC and MR-silencing cells (P>0.05). CONCLUSION: MR gene silencing inhibits the proliferation of RAW 264.7 macrophages, but has no obvious effect on the apoptosis of the resting state cells.     

Effect of RNA interference on Polo-like kinase-1 in A549 cells

AIM: To investigate whether RNA interference(RNAi) induced by small interference RNA(siRNA) could suppress Polo-like kinase-1 (Plk 1) expression and its effects in A549 cells.METHODS: A recombinant plasmid containing siRNA targeting Plk1 (psiRNA-hH1-Plk1) was transfected into A549 cells with Lipofectamine 2000.Expressions of Plk1,cyclin B1 and p53 protein were detected by Western blotting.Cell proliferation was evaluated by direct cell counting,while cell cycle and apoptosis were examined by flow cytometry,and expression of α-tubulin was detected by immunofluorescence.RESULTS: The results demonstrated that sequence specific siRNA targeting Plk1 was capable of suppressing Plk1 expression,and reflecting in lower kinase activity in A549 cells.The level of Plk1 protein was reduced by at least 70% after 48 h of psiRNA-hH1-Plk1 treatment relative to controls.Expressions of cyclin B1 and p53 were increased greatly after Plk1 depletion,and cells showed absence of microtubule polymerization and spindle abnormalities in staining for α-tubulin.Growth inhibition,G₂/M arrest and apoptosis were observed in psiRNA-hH1-Plk1 transfected group.CONCLUSION: All these data suggest that siRNA targeted against human Plk1 may be a valuable tool in cancer therapy.     

马铃薯GLDH基因的克隆及序列分析

 以马铃薯品种‘Favorita’叶片中的cDNA为模板,采用RT-PCR、巢式PCR、3′RACE和5′RACE技术,获得了L–半乳糖酸–1,4–内酯脱氢酶（EC 1131213,GLDH）基因cDNA 2 563 bp的全长序列,命名为StGLDH（GenBank：FJ755844）。序列分析表明,该基因编码区长1 773 bp,编码590个氨基酸,与其他植物GLDH氨基酸序列具有很高的同源性,尤其与番茄、辣椒、烟草GLDH 氨基酸序列具有90.6% ~ 95.9%的同源性。荧光定量分析表明,该基因在马铃薯不同器官中均有表达,在幼叶和功能叶中表达量最高,在茎中表达量最低;除匍匐茎外,其它器官中抗坏血酸（ascorbic acid,AsA）含量与StGLDH的表达高度一致。     

Notch-1 gene silencing promotes phosphorylations of JNK1 and p53 in human breast cancer MCF-7 cells

AIM: To investigate the effect of Notch1 gene silencing on phosphorylations of JNK1 and p53 in human breast cancer MCF-7 cells.METHODS: shRNA-Notch1 eukaryotic expression plasmid was constructed and transfected into MCF-7 cells. The expression of Notch1 and Hes-1 was observed by Western blotting after transfction. Apoptosis and mitochondrial membrane potential were detected by flow cytometry. Western blotting was also used to determine the protein levels of p-JNK1, p-p53, PUMA, NOXA and cleaved caspase-3 after Notch1 silencing was performed in MCF-7 cells.RESULTS: Silencing of Notch1 significantly reduced the expression of Notch1 and Hes-1 in MCF-7 cells (P<0.01). In shNotch1 group, the number of apoptotic cells was much higher (P<0.01) and mitochondrial membrane potential was much lower (P<0.05) than those in shControl group. The protein levels of p-JNK1, p-p53, PUMA, NOXA and cleaved caspase-3 increased obviously after silencing of Notch1 was performed in MCF-7 cells (P<0.05).CONCLUSION: Notch1 silencing induces apoptosis of human breast cancer MCF-7 cells through promoting phosphorylations of JNK1 and p53, and increasing the production of PUMA, NOXA and cleaved caspase-3.     

Effects of JAG1 gene silencing on proliferation and apoptosis of human breast cancer MDA-MB-231 cells

AIM:To investigate the effects of Jagged 1 (JAG1) gene silencing on the proliferation and apoptosis of human breast cancer MDA-MB-231 cells. METHODS:The specific recombinant vector pRS-JAG1 was transfected into MDA-MB-231 cells with lipofectamine. The protein expression of JAG1 was observed by Western blotting after transfection. MTT assay was used to detect the effect of JAG1 gene silencing on the growth of the cells. The apoptosis and cell cycle were analyzed by flow cytometry. The protein levels of cyclin D1, p21CIP1/WAF1, p27KIP1, p-Rb, Bcl-2, Bax, Bcl-xL and cleaved caspase-3 were determined by Western blotting. RESULTS:Compared with control group, the expression level of JAG1 was reduced by pRS-JAG1 transfection for 72 h (P<0.05). The growth of MDA-MB-231 cells in shJAG1 group was significantly inhibited (P<0.05). The percentages of G 0/G 1-phase cells and early apoptotic rate were obviously higher in shJAG1 group than those in control group (P<0.05). The shRNA-mediated JAG1 silencing decreased the protein levels of cyclin D1, p-Rb, Bcl-2 and Bax, and increased the protein levels of p21CIP1/WAF1, p27KIP1, Bax and cleaved caspase-3 (P<0.05). CONCLUSION:JAG1 silencing effectively inhibits the proliferation and induces the apoptosis of human breast cancer cells, suggesting that JAG1 might serve as a therapeutic target for triple-negative breast cancer.