AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus. METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated...AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus. METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated with restriction endonuclease sequences, was designed. T 4 DNA ligase catalyzed the joint of the KIT shRNA and the green fluorescent protein-containing PDC316-EGFP-U6 to form PDC316EGFP-U6-KIT. Homologous recombination of AdEGFPU6-KIT was performed with the AdMax system. Heterotopically transplanted GISTs were established in nude mice. AdEGFP-U6-KIT was intratumorally injected. The volume, inhibition ratio of tumor and CD117 expression of GIST graft tumor in nude mice were compared between test and control groups. RESULTS: The length of KIT shRNA was determined to be about 50bp by agarose electrophoresis. Gene se-quencing detected the designed KIT RNAi sequence in PDC316-EGFP-U6-KIT. After transfection with AdEGFPU6-KIT, 293 cells displayed green fluorescence. The physical and infective titers of AdEGFP-U6-KIT were 5 × 10 11 viral particles/mL and 5.67 × 10 7 plaque forming units/mL, respectively. The mean volume of the grafted tumor was significantly smaller in test mice than in control mice (75.3 ± 22.9 mm 3 vs 988.6 ± 30.5 mm 3 , t = -18.132, P < 0.05). The inhibition ratio of the tumors was 59.6% in the test group. CD117 positive expression was evident in two cases (20%) in the test group and 10 cases (100%) in the control group (χ 2 = 10.2083, P < 0.005). CONCLUSION: AdEGFP-U6-KIT is successfully constructed, and KIT RNAi mediated with Admax vector system can effectively inhibit the expression of the KIT gene and the growth of GIST in nude mice.展开更多
Keratocystic odontogenic tumors （KCOT） are benign, locally aggressive intraosseous tumors of odontogenic origin. KCOT have a higher stromal microvessel density （MVD） than dentigerous cysts （DC） and normal oral m...Keratocystic odontogenic tumors （KCOT） are benign, locally aggressive intraosseous tumors of odontogenic origin. KCOT have a higher stromal microvessel density （MVD） than dentigerous cysts （DC） and normal oral mucosa. To identify genes in the stroma of KCOT involved in tumor development and progression, RNA sequencing （RNA-Seq） was performed using samples from KCOT and primary stromal fibroblasts isolated from gingival tissues. Seven candidate genes that possess a function potentially related to KCOT progression were selected and their expression levels were confirmed by quantitative PCR, immunohistochemistry and enzyme-linked immunosorbent assay. Expression of lysyl oxidase-like 4 （LOXL4）, the only candidate gene that encodes a secreted protein, was enhanced at both the mRNA and protein levels in KCOT stromal tissues and primary KCOT stromal fibroblasts compared to control tissues and primary fibroblasts （P〈0.05）. In vitro, high expression of LOXL4 could enhance proliferation and migration of the human umbilical vein endothelial cells （HUVECs）. There was a significant, positive correlation between LOXL4 protein expression and MVD in stroma of KCOT and control tissues （r=0.882）. These data suggest that abnormal expression of LOXL4 of KCOT may enhance angiogenesis in KCOT, which may help to promote the locally aggressive biological behavior of KCOT.展开更多
AIM: To investigate the effect of microRNA-1(miR-1) on tumor endothelial cells(TECs) of human hepatocellular carcinoma(HCC).METHODS: MiR-1 specific short hairpin RNA(shRNA)was synthesized and cloned into a recombinant...AIM: To investigate the effect of microRNA-1(miR-1) on tumor endothelial cells(TECs) of human hepatocellular carcinoma(HCC).METHODS: MiR-1 specific short hairpin RNA(shRNA)was synthesized and cloned into a recombinant lentiviral vector. TECs were then infected by the miRNA-1-shRNA recombinant lentivirus. TECs were divided into three groups: a control(CON) group consisting of normal TECs without lentiviral infection, a negative control(NC) group consisting of normal TECs infected with a negative control virus, and a microdown(MD) group consisting of normal TECs infected with the miR-1-inhibition virus containing the target gene. Silencing of miR-1 expression was quantified via quantitative reverse transcription-polymerase chain reaction(qRT-PCR). The proliferation of TECs was detected using MTT(Thiazolyl Blue Tetrazolium Bromide) assay; the observations were continued for 5 d, and the optical density value at 490 nm was detected every day. Apoptosis was detected via flow cytometry using Annexin V-APC single staining. The migration and invasion of TECs were detected using transwell assays.RESULTS: Lentiviral miR-1 shRNA was successfully transduced into TECs, and specifically silenced the expression of miR-1. The results of qRT-PCR showed that the expression of miR-1 was significantly decreased in the MD group(2-ΔΔCt = 0.57 ± 0.14)compared with the CON group(2-ΔΔCt = 1) and the NC group(2-ΔΔCt = 1.05 ± 0.13)(P < 0.01). The results of MTT assay showed that the cell proliferation was all significantly inhibited in the MD group in the 5days compared with the CON and NC groups(P <0.01). The results of flow cytometry showed that the apoptosis was significantly increased in the MD group(6.32% ± 0.33%) compared with the CONgroup(2.03% ± 0.30%) and the NC group(2.18% ±0.15%)(P < 0.01). The ability of cell migration was significantly inhibited in the MD group(62.0 ± 5.48)compared with the CON group(99.8 ± 3.11) and the NC group(97.2 ± 3.70)(P < 0.01). The ability of invasion of TECs was also significantly inhibited in the MD group(29.8 ± 2.39) compared with the CON group(44.6 ± 3.36) and the NC group(44.4 ± 5.17)(P <0.01).CONCLUSION: MiR-1 might be a potential tumor activator. Inhibiting its expression could decrease proliferation, induce apoptosis, and inhibit the migration and invasion of TECs of human HCC.展开更多
基金Supported by Grants from the Chinese Ministry of Education Funds, No. 20070558266Guangdong Provincial Funds for Science and Technology, No. 2007B 031515004
文摘AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus. METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated with restriction endonuclease sequences, was designed. T 4 DNA ligase catalyzed the joint of the KIT shRNA and the green fluorescent protein-containing PDC316-EGFP-U6 to form PDC316EGFP-U6-KIT. Homologous recombination of AdEGFPU6-KIT was performed with the AdMax system. Heterotopically transplanted GISTs were established in nude mice. AdEGFP-U6-KIT was intratumorally injected. The volume, inhibition ratio of tumor and CD117 expression of GIST graft tumor in nude mice were compared between test and control groups. RESULTS: The length of KIT shRNA was determined to be about 50bp by agarose electrophoresis. Gene se-quencing detected the designed KIT RNAi sequence in PDC316-EGFP-U6-KIT. After transfection with AdEGFPU6-KIT, 293 cells displayed green fluorescence. The physical and infective titers of AdEGFP-U6-KIT were 5 × 10 11 viral particles/mL and 5.67 × 10 7 plaque forming units/mL, respectively. The mean volume of the grafted tumor was significantly smaller in test mice than in control mice (75.3 ± 22.9 mm 3 vs 988.6 ± 30.5 mm 3 , t = -18.132, P < 0.05). The inhibition ratio of the tumors was 59.6% in the test group. CD117 positive expression was evident in two cases (20%) in the test group and 10 cases (100%) in the control group (χ 2 = 10.2083, P < 0.005). CONCLUSION: AdEGFP-U6-KIT is successfully constructed, and KIT RNAi mediated with Admax vector system can effectively inhibit the expression of the KIT gene and the growth of GIST in nude mice.
基金supported by the National Natural Science Foundation of China (grant nos. 81030018, 30872900 and 30901680)the Doctoral Fund of Ministry of Education of China (grant no. 20120001110043)
文摘Keratocystic odontogenic tumors （KCOT） are benign, locally aggressive intraosseous tumors of odontogenic origin. KCOT have a higher stromal microvessel density （MVD） than dentigerous cysts （DC） and normal oral mucosa. To identify genes in the stroma of KCOT involved in tumor development and progression, RNA sequencing （RNA-Seq） was performed using samples from KCOT and primary stromal fibroblasts isolated from gingival tissues. Seven candidate genes that possess a function potentially related to KCOT progression were selected and their expression levels were confirmed by quantitative PCR, immunohistochemistry and enzyme-linked immunosorbent assay. Expression of lysyl oxidase-like 4 （LOXL4）, the only candidate gene that encodes a secreted protein, was enhanced at both the mRNA and protein levels in KCOT stromal tissues and primary KCOT stromal fibroblasts compared to control tissues and primary fibroblasts （P〈0.05）. In vitro, high expression of LOXL4 could enhance proliferation and migration of the human umbilical vein endothelial cells （HUVECs）. There was a significant, positive correlation between LOXL4 protein expression and MVD in stroma of KCOT and control tissues （r=0.882）. These data suggest that abnormal expression of LOXL4 of KCOT may enhance angiogenesis in KCOT, which may help to promote the locally aggressive biological behavior of KCOT.
基金Wuhan University,for excellent technical support
文摘AIM: To investigate the effect of microRNA-1(miR-1) on tumor endothelial cells(TECs) of human hepatocellular carcinoma(HCC).METHODS: MiR-1 specific short hairpin RNA(shRNA)was synthesized and cloned into a recombinant lentiviral vector. TECs were then infected by the miRNA-1-shRNA recombinant lentivirus. TECs were divided into three groups: a control(CON) group consisting of normal TECs without lentiviral infection, a negative control(NC) group consisting of normal TECs infected with a negative control virus, and a microdown(MD) group consisting of normal TECs infected with the miR-1-inhibition virus containing the target gene. Silencing of miR-1 expression was quantified via quantitative reverse transcription-polymerase chain reaction(qRT-PCR). The proliferation of TECs was detected using MTT(Thiazolyl Blue Tetrazolium Bromide) assay; the observations were continued for 5 d, and the optical density value at 490 nm was detected every day. Apoptosis was detected via flow cytometry using Annexin V-APC single staining. The migration and invasion of TECs were detected using transwell assays.RESULTS: Lentiviral miR-1 shRNA was successfully transduced into TECs, and specifically silenced the expression of miR-1. The results of qRT-PCR showed that the expression of miR-1 was significantly decreased in the MD group(2-ΔΔCt = 0.57 ± 0.14)compared with the CON group(2-ΔΔCt = 1) and the NC group(2-ΔΔCt = 1.05 ± 0.13)(P < 0.01). The results of MTT assay showed that the cell proliferation was all significantly inhibited in the MD group in the 5days compared with the CON and NC groups(P <0.01). The results of flow cytometry showed that the apoptosis was significantly increased in the MD group(6.32% ± 0.33%) compared with the CONgroup(2.03% ± 0.30%) and the NC group(2.18% ±0.15%)(P < 0.01). The ability of cell migration was significantly inhibited in the MD group(62.0 ± 5.48)compared with the CON group(99.8 ± 3.11) and the NC group(97.2 ± 3.70)(P < 0.01). The ability of invasion of TECs was also significantly inhibited in the MD group(29.8 ± 2.39) compared with the CON group(44.6 ± 3.36) and the NC group(44.4 ± 5.17)(P <0.01).CONCLUSION: MiR-1 might be a potential tumor activator. Inhibiting its expression could decrease proliferation, induce apoptosis, and inhibit the migration and invasion of TECs of human HCC.
