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To summarize the clinical diagnosis and treatment process and genetic test results and characteristics of one child with Angelman syndrome (AS) complicated with oculocutaneous albinism type 2 (OCA2), and to review the literature. "Angelman syndrome" "P gene" and "Oculocutaneous albinism type 2" were used as keywords to search at CNKI, Wanfang, and PubMed databases (from creation to December 2019). Then all the patients were analyzed. The patient in this study was a girl aged 1 year. After birth, she was found to present as white body, yellow hair, and nystagmus. She could raise her head at the age of 2 months and turn over at the age of 7 months. The head circumference was 42 cm and she could not sit alone or speak at present. Trio-based exome sequencing revealed that the patient carried a homozygous mutation of c.168del (p.Gln58ArgfsTer44) in the P gene, and her father was heterozygous and her mother was wild-type. The detection of copy number variation showed deletion on the maternal chromosome at 15q11.2-13.1 region (P gene located in this region) in the patient. Until December 2019, a total of 4 cases in the 4 literature had been reported. Adding our case here, the 5 cases were summarized and found that all the cases showed white skin, golden hair, and shallow iris after birth. Comprehensive developmental delay was found around 6 months of age after birth, and the language remained undeveloped in 2 cases till follow-up into childhood. Seizures occurred in 4 patients. Two cases had ataxia. All the 5 cases had acquired microcephaly. Two cases had a family history of albinism. Electroencephalogram monitoring was completed in 3 cases and the results were abnormal. Genetic tests showed that all the 5 cases had deletion on maternal chromosome at 15q11-13 region. Four cases carried mutation of P gene on paternal chromosome. And 1 case was clinically diagnosed as OCA2 without P gene test. AS combined with OCA2 is relatively rare. OCA2 is easily diagnosed based on the obvious clinical manifestations after birth. When combined with clinical manifestations such as neurodevelopmental delay, it might indicate the possibility of AS that is hardly diagnosed clinically at an early stage. Genetic tests can reveal the cross-genetic phenomenon of AS and OCA2 and the complex of them can be eventually diagnosed.
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Female , Humans , Infant , Albinism, Oculocutaneous/genetics , DNA Copy Number Variations , Membrane Transport Proteins/genetics , Molecular Biology , MutationABSTRACT
Objective:To investigate the effect of circ-PRKDC on lung cancer cell proliferation, apoptosis and radiosensitivity and its molecular mechanism.Methods:Normal lung epithelial cells BEAS-2B and lung cancer cell lines NCI-H1299, NCI-H2170, NCI-H1975 were cultured. NCI-H1299 cells were divided into the si-NC, si-PRKDC, pcDNA-NC, pcDNA-PRKDC, miR-NC, miR-505-3p, anti-miR-NC, anti-miR-505-3p, si-PRKDC+ anti-miR-NC and si-PRKDC+ anti-miR-505-3p groups. RT-qPCR was used to detect the expression levels of circ-PRKDC and miR-505-3p. Western blot was employed to measure the protein expression. MTT was used to detect cell proliferation. Flow cytometry was utilized to detect cell apoptosis. Plate clone formation assay was conducted to detect the cell radiosensitivity. Dual luciferase reporter assay was performed to analyze the targeting relationship between circ-PRKDC and miR-505-3p.Results:Compared with normal lung epithelial cells BEAS-2B, the expression levels of circ-PRKDC in the lung cancer cell lines NCI-H1299, NCI-H2170 and NCI-H1975 were significantly up-regulated (3.65, 3.10, 2.67 vs. 1.00, all P<0.05), whereas those of miR-505-3p were significantly down-regulated (0.42, 0.50, 0.54 vs. 1.02, all P<0.05). After low expression of circ-PRKDC, the expression level of CyclinD1 was significantly down-regulated (0.42 vs. 0.81, P<0.05), whereas those of Cleaved-caspase-3(0.71 vs. 0.33, P<0.05) and γ-H 2AX (0.89 vs. 0.46, P<0.05) were significantly up-regulated, the cell A value was significantly decreased (0.413 vs. 0.839, P<0.05), cell apoptosis rate was significantly increased (20.35 vs. 6.21, P<0.05), cell survival fraction was significantly decreased ( P<0.05), and β-catenin expression was significantly down-regulated (0.35 vs. 0.73, P<0.05). After high expression of miR-505-3p, the expression level of CyclinD1 was significantly down-regulated (0.34 vs. 0.83, P<0.05), those of Cleaved-caspase-3(0.65 vs. 0.32, P<0.05) and γ-H 2AX (0.96 vs. 0.45, P<0.05) were significantly up-regulated, the cell A value was significantly decreased (0.386 vs. 0.851, P<0.05), the apoptosis rate was significantly increased (16.38 vs. 6.20, P<0.05), and the cell survival fraction was significantly decreased ( P<0.05). Compared with miR-NC, the luciferase activity of miR-505-3p group transfected with circ-PRKDC wild-type reporter plasmid was significantly decreased (0.44 vs. 1.00, P<0.05). Down-regulation of miR-505-3p could reverse the effect of low expression of circ-PRKDC on the proliferation, apoptosis, radiosensitivity and β-catenin expression of NCI-H1299 cells. Conclusion:Low expression of circ-PRKDC may inhibit lung cancer cell proliferation, promote cell apoptosis and enhance cell radiosensitivity by up-regulating miR-505-3p, which is probably associated with the Wnt/β-catenin signaling pathway.
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Objective:To explore the mechanism of miR-205-5p/E2F1 signal axis in regulating the glioma U251, U87 radiotherapy resistance.Methods:X-ray gradual ascending and intermittent induction method was used to irradiate the glioma U251 cells to establish U251/TR, U87/TR radiation-resistant cell lines. Then, the morphology, migration, invasion and proliferation abilities of cells (U251/TR, U87/TR radiation-resistant cells and U251, U87 radiation-sensitive cells) were analyzed. Luciferase gene detection system and point mutation technique were employed to analyze the mechanism of miR-205-5p and E2F1 gene activity on U251 and U87 radiation-resistant cell lines.Results:Compared with the radiation-sensitive U251 cells, the radiation-resistant cells U251/TR, U87/TR showed increased proliferation activity, enhanced migration and invasion abilities and decreased apoptosis under X-ray irradiation. miR-205-5p mimics transfection could down-regulate the expression of E2F1 factor in U251/TR cells, inhibit cell proliferation, invasion and migration and increase the radiosensitivity of U251/TR cells. miR-205-5p mimics transfection combined with with E2F1 down-regulation exerted anti-tumor effect and decreased cell tolerance by suppressing the Wnt/β-catenin signaling pathway activity.Conclusions:The glioma radiation-resistant cell line U251/TR, U87/TR can be established by X-ray gradual ascending and intermittent induction method. The miR-205-5p/E2F1 signal axis exerts tumor-suppressing effect through the classical Wnt/β-catenin signaling pathway, which can be used as an therapeutic target to increase the radiosensitivity of glioma.
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Objective:To screen 17-AAG-M-induced differentially expressed miRNAs in human non-small cell lung cancer (NSCLC) A549 cells under X-ray and evaluate its effect on radio-sensitivity.Methods:A549 cells were treated with 17-AAG-M and 4 Gy. Total RNA was extracted for microarray screening. The expression of the miRNAs of interest in the tumor was observed by public database. The target miRNAs were analyzed by using GO and KEGG pathways, and verified by qPCR. The effect of target miRNAs on the survival rate and proliferation of A549 cells under X-ray was evaluated by MTT and clone formation assays. The radio-sensitivity of the target miRNAs was analyzed by the single-hit multi-target model formula.Results:20 differentially expressed miRNAs were screened. The down-regulated hsa-miR-30a-3p showed a close correlation with lung cancer in the database. It was involved with 50 biological processes including cell proliferation and affected the MAPK signaling pathway, cancer-related pathways and cell cycle, etc. Compared with the 17-AAG-M group, the relative expression level of hsa-miR-30a-3p under the action of 17-AAG-M and X-ray was down-regulated from 2.42 to 0.16. hsa-miR-30a-3p inhibited the survival rate of A549 cells (survival rate: 78.52%) and further decreased to 69.00% under X-ray. Up-regulation of hsa-miR-30a-3p expression inhibited the proliferation of tumor cells and increased the radio-sensitivity of A549 cells. The radio-sensitization ratio was 1.18. The above performance became more obvious under the action of 17-AAG-M.Conclusions:In A549 cells, hsa-miR-30a-3p is differentially expressed under the action of 17-AAG-M and X-ray. Moreover, up-regulation of the expression level of hsa-miR-30a-3p in A549 cells can reduce the viability and proliferation of tumor cells, and increase the radio-sensitivity of tumor cells. The inhibition effect of X-ray combined with 17-AAG-M upon tumors can be strengthened.
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Objection@#To investigate the effect of miR-32-5p on the radiosensitivity, migration and invasion of colorectal cancer cells and the underlying mechanism.@*Methods@#Human colorectal cancer SW480 cells and normal colonic epithelial NCM460 cells were cultured. The colorectal cancer cells were divided into the non-transfected and transfected groups (transfected with anti-miR-NC, anti-miR-32-5p, pcDNA, pcDNA-TOB1, anti-miR-32-5p+ si-NC and anti-miR-32-5p+ si-TOB1, respectively). The expression of miR-32-5p and TOB1 at the mRNA and protein levels was detected by RT-qPCR and Western blot. The radiosensitivity of the transfected cells was determined by colony formation assay. The migration and invasion ability of the transfected cells were detected by Transwell assay. Whether miR-32-5p targeted TOB1 was validated by dual luciferase reporter gene assay and Western blot.@*Results@#Compared with human colonic epithelial cells, the expression of miR-32-5p was significantly up-regulated, whereas the expression of TOB1 mRNA and protein was remarkably down-regulated in the colon cancer cells (all P<0.05). Compared with the anti-miR-NC, the quantity of cell migration and invasion was significantly decreased (both P<0.05) and the radiosensitivity ratio was 1.801 in the anti-miR-32-5p group. Compared with the pcDNA group, the quantity of cell migration and invasion was significantly decreased (both P<0.05) and the radiosensitivity ratio was 1.764 in the pcDNA-TOB1 group. Dual luciferase reporter gene assay and Western blot confirmed that miR-32-5p negatively regulated the expression of TOB1 protein. Compared with the anti-miR-32-5p+ si-NC group, the quantity of cell migration and invasion was significantly increased (both P<0.05) and the radiosensitivity ratio was 0.591 in the anti-miR-32-5p+ si-TOB1 group.@*Conclusions@#Inhibition of miR-32-5p expression can significantly enhance the radiosensitivity of colorectal cancer cells and suppress cell migration and invasion. The underlying mechanism might be related to the targeted up-regulation of TOB1 expression.
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Objective@#To investigate the effect of LncRNA OIP5-AS1 on radiosensitivity of non-small cell lung cancer (NSCLC) cells and its mechanism.@*Methods@#The radiation-resistant cell A549R was established by using A549 cells irradiated by X-ray 6Gy in 5 fractions. The expression levels of OIP5-AS1 and miR-34c-5p in A549 and A549R cells were detected by qRT-PCR. OIP5-AS1 inhibitor or miR-34c-5p mimetic was transfected into A549R cells, or OIP5-AS1 overexpression plasmid was transfected into A549 cells. Cell apoptosis was detected by flow cytometry. Cell radiosensitivity was analyzed by colony formation assay. The expression levels of p-Chk2 and p-ATM proteins were measured by Western blot. Dual luciferase assay was adopted to verify the relationship between OIP5-AS1 and miR-34c-5p.@*Results@#Compared with A549 cells, the expression of OIP5-AS1 was significantly up-regulated in A549R cells (1.97±0.11 vs.1.01±0.05, P<0.05), whereas the expression of miR-34c-5p was remarkably down-regulated (0.43±0.02 vs.1.02±0.06, P<0.05). The expression levels of p-Chk2 and p-ATM proteins in A549R cells in the silencing OIP5-AS1+ 6Gy group were significantly lower (0.43±0.03 vs.1.39±0.15, 0.51±0.0 5 vs.1.21± 0.11, both P<0.05), whereas the apoptotic rate was significantly higher than those in the silencing control+ 6Gy group [(13.29±1.25)% vs. (28.47±2.31)%, P<0.05)]. The expression levels of p-Chk2 and p-ATM proteins in A549 cells in overexpressing OIP5-AS1+ 6Gy group were significantly higher than those in overexpression control+ 6Gy group (1.23±0.13 vs.0.75±0.06, 1.08±0.11 vs.0.59±0.04, both P<0.05). Inhibiting miR-34c-5p expression reversed the effect of silencing OIP5-AS1 on survival fraction of A549R cells (SER=1.42). OIP5-AS1 negatively regulated the expression of miR-34c-5p.@*Conclusion@#Silencing OIP5-AS1 enhances the radiosensitivity of radiation-resistant A549 cells by up-regulating the expression of miR-34c-5p, providing a potential target for radiotherapy of NSCLC cells.
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Objective@#To investigate the effect of lncRNA GIHCG on the radiosensitivity of glioma cells and its mechanism.@*Methods@#The expression levels of GIHCG and miR-146a-3p in human brain normal glial cells HEB and glioma cell lines U251, A172, SHG139 and U87 were quantitatively measured by qRT-PCR assay. U251 and SHG139 cells were used for subsequent experiment. After silencing the expression of GIHCG or overexpressing miR-146a-3p in U251 and SHG139 cells, cell proliferation was detected by MTT assay, cell apoptosis was detected by flow cytometry, cell radiosensitivity was detected by colony formation assay and the expression levels of CDK1, CyclinD1, Bcl-2 and Bax proteins were measured by Western blot. The bioinformatics software predicted the presence of a binding site for GIHCG and miR-146a-3p. Dual luciferase reporter gene assay and qRT-PCR assay were adopted to verify the targeting relationship between GIHCG and miR-146a-3p.@*Results@#Compared with HEB cells, the expression of GIHCG was significantly up-regulated in glioma U87, U251, A172 and SHG139 cells (all P<0.05), whereas that of miR-146a-3p was remarkably down-regulated (P<0.05). Silencing GIHCG expression or overexpression of miR-146a-3p significantly decreased the U251 and SHG139 cell survival rate, survival fraction and the expression of CDK1, CyclinD1 and Bcl-2 proteins (all P<0.05), whereas considerably increased the apoptotic rate and expression of Bax protein (both P<0.05). GIHCG performed targeted negative regulation of miR-146a-3p expression in U251 and SHG139 cells and inhibition of miR-146a-3p expression reversed the effect of silencing GIHCG on proliferation, apoptosis and radiosensitivity of glioma cells.@*Conclusion@#Silencing GIHCG expression up-regulates the expression of miR-146a-3p, thereby enhancing the radiosensitivity of glioma cells.
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Objection To investigate the effect of miR-32-5p on the radiosensitivity,migration and invasion of colorectal cancer cells and the underlying mechanism.Methods Human colorectal cancer SW480 cells and normal colonic epithelial NCM460 cells were cultured.The colorectal cancer cells were divided into the non-transfected and transfected groups (transfected with anti-miR-NC,anti-miR-32-5p,pcDNA,pcDNA-TOB1,anti-miR-32-5p+si-NC and anti-miR-32-5p+si-TOB1,respectively).The expression of miR-32-5p and TOB1 at the mRNA and protein levels was detected by RT-qPCR and Western blot.The radiosensitivity of the transfected cells was determined by colony formation assay.The migration and invasion ability of the transfected cells were detected by Transwell assay.Whether miR-32-5p targeted TOB1 was validated by dual luciferase reporter gene assay and Western blot.Results Compared with human colonic epithelial cells,the expression of miR-32-5p was significantly up-regulated,whereas the expression of TOB1 mRNA and protein was remarkably down-regulated in the colon cancer cells (all P＜0.05).Compared with the anti-miR-NC,the quantity of cell migration and invasion was significantly decreased (both P＜0.05) and the radiosensitivity ratio was 1.801 in the anti-miR-32-5p group.Compared with the pcDNA group,the quantity of cell migration and invasion was significantly decreased (both P＜0.05) and the radiosensitivity ratio was 1.764 in the pcDNA-TOB1 group.Dual luciferase reporter gene assay and Western blot confirmed that miR-32-5p negatively regulated the expression of TOB1 protein.Compared with the anti-miR-32-5p+si-NC group,the quantity of cell migration and invasion was significantly increased (both P＜0.05) and the radiosensitivity ratio was 0.591 in the anti-miR-32-5p+si-TOB1 group.Conclusions Inhibition of miR-32-5p expression can significantly enhance the radiosensitivity of colorectal cancer cells and suppress cell migration and invasion.The underlying mechanism might be related to the targeted up-regulation of TOB1 expression.
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Objective To investigate the effect of LncRNA OIP5-AS1 on radiosensitivity of non-small cell lung cancer (NSCLC) cells and its mechanism.Methods The radiation-resistant cell A549R was established by using A549 cells irradiated by X-ray 6Gy in 5 fractions.The expression levels of OIP5-AS1 and miR-34c-5p in A549 and A549R cells were detected by qRT-PCR.OIP5-AS1 inhibitor or miR-34c-5p mimetic was transfected into A549R cells,or OIP5-AS1 overexpression plasmid was transfected into A549 cells.Cell apoptosis was detected by flow cytometry.Cell radiosensitivity was analyzed by colony formation assay.The expression levels of p-Chk2 and p-ATM proteins were measured by Western blot.Dual luciferase assay was adopted to verify the relationship between OIP5-AS1 and miR-34c-5p.Results Compared with A549 cells,the expression of OIP5-AS1 was significantly up-regulated in A549R cells (1.97±0.11 vs.1.01±0.05,P＜0.05),whereas the expression of miR-34c-5p was remarkably down-regulated (0.43±0.02 vs.1.02±0.06,P＜0.05).The expression levels of p-Chk2 and p-ATM proteins in A549R cells in the silencing OIP5-AS1 +6Gy group were significantly lower (0.43±0.03 vs.1.39±0.15,0.51 ±0.0 5 vs.1.21 ± 0.11,both P＜0.05),whereas the apoptotic rate was significantly higher than those in the silencing control + 6Gy group [(13.29± 1.25)％ vs.(28.47± 2.31)％,P＜0.05)].The expression levels of p-Chk2 and p-ATM proteins in A549 cells in overexpressing OIP5-AS1+6 Gy group were significantly higher than those in overexpression control+6 Gy group (1.23±0.13 vs.0.75±0.06,1.08±0.11 vs.0.59± 0.04,both P＜0.05).Inhibiting miR-34c-5p expression reversed the effect of silencing OIP5-AS1 on survival fraction of A549R cells (SER =1.42).OIP5-AS1 negatively regulated the expression of miR-34c-5p.Conclusion Silencing OIP5-AS1 enhances the radiosensitivity of radiation-resistant A549 cells by up-regulating the expression of miR-34c-5p,providing a potential target for radiotherapy of NSCLC cells.
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Objective To investigate the effect of lncRNA GIHCG on the radiosensitivity of glioma cells and its mechanism.Methods The expression levels of GIHCG and miR-146a-3p in human brain normal glial cells HEB and glioma cell lines U251,A172,SHG139 and U87 were quantitatively measured by qRT-PCR assay.U251 and SHG139 cells were used for subsequent experiment.After silencing the expression of GIHCG or overexpressing miR-146a-3p in U251 and SHG139 cells,cell proliferation was detected by MTI assay,cell apoptosis was detected by flow cytometry,cell radiosensitivity was detected by colony formation assay and the expression levels of CDK1,CyclinD1,Bcl-2 and Bax proteins were measured by Western blot.The bioinformatics software predicted the presence of a binding site for GIHCG and miR-146a-3p.Dual luciferase reporter gene assay and qRT-PCR assay were adopted to verify the targeting relationship between GIHCG and miR-146a-3p.Results Compared with HEB cells,the expression of GIHCG was significantly up-regulated in glioma U87,U251,A172 and SHG139 cells (all P＜0.05),whereas that of miR-146a-3p was remarkably down-regulated (P＜0.05).Silencing GIHCG expression or overexpression of miR-146a-3p significantly decreased the U251 and SHG139 cell survival rate,survival fraction and the expression of CDK1,CyclinDl and Bcl-2 proteins (all P＜0.05),whereas considerably increased the apoptotic rate and expression of Bax protein (both P＜0.05).GIHCG performed targeted negative regulation of miR-146a-3p expression in U251 and SHG139 cells and inhibition of miR-146a-3p expression reversed the effect of silencing GIHCG on proliferation,apoptosis and radiosensitivity of glioma cells.Conclusion Silencing GIHCG expression up-regulates the expression of miR-146a-3p,thereby enhancing the radiosensitivity of glioma cells.
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Objective To explore the effect of LncRNA HOXD-AS1 on the radiosensitivity of H460 cells by targeting the regulation of miR-454-3p expression. Methods H460 cells were irradiated with 0, 2, 4, 6 and 8 Gy, and the expression levels of HOXD-AS1 and miR-454-3p were detected by qRT-PCR. Cell apoptosis rate were measured by flow cytometry experiments. Cell cloning experiments were used to detect cell radiosensitivity. The protein expression levels of Caspase-3, Cyclin D1 and γ-H2AX were detected by Western blot. Results The expression of HOXD-AS1 in H460 cells was significantly increased after X-ray irradiation ( P<0.05) , while the expression of miR-454-3p was significantly decreased ( P<0.05) . Silencing HOXD-AS1 significantly promoted apoptosis, increased radiosensitivity of lung cancer cells, promoted Caspase-3, γ-H2AX protein expression, and inhibited Cyclin D1 expression. HOXD-AS1 could target the expression of miR-454-3p. Conclusion Silencing HOXD-AS1 can promote the apoptosis of lung cancer cells and inhibit cell survival by targeting miR-454-3p to increase the radiosensitivity of lung cancer cells.
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Objective@#To analyze variations of TYR and P genes among 14 patients with clinically diagnosed oculocutaneous albinism.@*Methods@#Potential variations of the TYR and P genes were detected by Sanger sequencing. Novel variations were predicted with bioinformatics software including SIFT and PolyPhen-2.@*Results@#No variation was found in the TYR gene, while 9 types of variations were found in the P gene among the 14 patients, which included c. 803-3C>G (7/26), c. 1327G>A (p.Val443Ile) (5/26), c. 632C>T (p.Pro211Leu) (4/26), c. 1832T>C (p.Leu611Pro) (3/26), c. 1349C>A (p.Thr450Lys) (2/26), c. 2363C>T (p.Ser788Leu) (2/26), c. 2228C>T (p.Pro743Leu) (1/26), c. 1525A>G(p.Thr509Ala) (1/26), and c. 1349C>T(p.Thr450Met) (1/26). Only 1 heterozygous variation was detected in 2 families. c. 2363C>T (p.Ser788Leu), c. 1832T>C (p.Leu611Pro) and c. 1525A>G (p.Thr509Ala) were not reported previously and predicted as "harmful" to the protein function.@*Conclusion@#The main type of ocular albinism is oculocutaneous albinism type Ⅱ in Liuzhou region, where the most common variations of the P gene were c. 803-3C>G and c. 1327G>A (p.Val443Ile). Above finding has enriched the variation spectrum of the P gene.
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Candida tropicalis uses alkanes and fatty acids to produce long chain dicarboxylic acids. However, the yield can be affected by β-oxidation in peroxisomes. Pxa1p was a membrane protein of Saccharomyces cerevisiae peroxisomes. Pxa1p and Pxa2p form a dimer that is involved in transporting of long chain fatty acids into peroxisomes, but the similar transporting system of Candida tropicalis has not yet been reported. In this study, a ctpxa1 gene deletion strain named C. tropicalis 1798-pxa1 was constructed by homologous single exchange method using PCR fragment. The expression of ctpxa1 gene in C. tropicalis 1798, C. tropicalis 1798-pxa1 was detected by semi-quantitative RT-PCR, and the ratio of gray value was 2.03, implying that the expression of ctpxa1 in C. tropicalis 1798-pxa1 was weakened. After 144 h fermentation, the dodecanedioic acid production of C. tropicalis 1798-pxa1 was increased 94.3% than the former strain, the maximum yield was 10.3 g/L.
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Objective To investigate whether miR?485?3p plays a role in regulation of radiosensitivity of gastric cancer cells by targeting TLR1. Methods Quantitative real?time PCR and Western blot were used to determine the expression of miR?485?3p and TLR1, respectively. The interaction between miR?485?3p and TLR1 was verified by target prediction software ( DIANA, TargetScan, and miRanda) and dual luciferase reporter assay. Gastric cancer MGC803 cells transfected with miR?485?3p mimic or TLR1 siRNA were exposed to irradiation. Apoptosis assay, colony formation assay, and MTT assay were used to evaluate the changes in radiosensitivity of gastric cancer cells. Dual luciferase reporter assay was used to determine the effects of miR?485?3p overexpression and TLR1 silencing on the activity of NF?κB. Western blot was used to study the effects of miR?485?3p overexpression and TLR1 silencing on NF?κB target genes. Results In gastric cancer cells exposed to radiation, the expression of miR?485?3p was downregulated and the expression of TLR1 was upregulated. TLR1 was predicted to be the target of miR?485?3p by target prediction software. Dual luciferase reporter assay further confirmed TLR1 as the direct target of miR?485?3p. miR?485?3p negatively regulated the expression of TLR1. The overexpression of miR?485?3p, as well as TLR1 silencing, increased the apoptosis rate of cells, reduced colony formation and cell proliferation, and enhanced the radiosensitivity of the cells. Both miR?485?3p overexpression and TLR1 silencing reduced the activity of NF?κB and downregulated the expression of multiple NF?κB target genes. Conclusions miR?485?3p enhances the radiosensitivity of gastric cancer cells probably by targeting TLR1 and regulating the NF?κB signaling pathway.
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Viral gene oncotherapy is emerging as a biotherapeutic cancer treatment modality based on targeted killing of cancer cells by viral genes. Newcastle disease virus (NDV) has the property to cause selective oncolysis of tumor cells sparing normal cells. NDV has a single stranded negative sense RNA genome, which is 15,186 nucleotide long and consists of six genes, which codes for eight proteins. NDV like other paramyxoviruses has the ability to generate multiple proteins from the P gene. P protein is encoded by an unedited transcript of the P gene, whereas the V and W protein are the results of RNA editing event in which one and two G residues are inserted at a conserved editing site within the P gene mRNA resulting in V and W transcripts, respectively. Although NDV is known to cause oncolysis by triggering apoptosis, the role of different viral proteins in selective oncolysis is still unclear. P gene edited products are known for its anti-apoptotic property in homologous host. In the present study, NDV P gene and its RNA edited products were amplified, cloned, sequenced and in vitro expression was done in HeLa cells. Further constructs were assayed for their apoptosis inducing ability in HeLa cells. Preliminary study suggested that P, V and W proteins are not apoptotic to HeLa cells.
Subject(s)
Amino Acid Sequence , Animals , Annexin A5/metabolism , Base Sequence , Chickens , Cloning, Molecular , Gene Expression Regulation, Viral , Genes, Viral/genetics , HeLa Cells , Humans , Molecular Sequence Data , Newcastle disease virus/genetics , Open Reading Frames/genetics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Reproducibility of Results , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Rabies is a major zoonotic disease that causes approximately 55,000 human deaths worldwide on an annual basis. The nucleocapsid protein and glycoprotein genes of the Korean rabies virus (RABV) have been subjected to molecular and phylogenetic analyses. Although the phosphoprotein (P) has several important functions in viral infection and pathogenicity, the genetic characterizations of the P of Korean RABV isolates have not yet been established. In the present study, we conducted genetic analyses of P genes of 24 RABV isolates circulating in the Republic of Korea (hereafter, Korea) from 2008 to 2011. This study revealed that the P genes of Korean RABVs are genetically similar to those of RABV strains of lyssavirus genotype I including V739 (dogs, Korea), NNV-RAB-H (humans, India), NeiMeng925 (raccoon dogs, China), and RU9.RD (raccoon dogs, Russia). Among Korean isolates, the RABV P genes showed low variability in the variable domains among Korean isolates; they had specific consensus sequences and amino acid substitutions capable of identifying geographic characteristics and retained specific sequences thought to be important for viral function. These results provide important genetic characteristics and epidemiological information pertaining to the P gene of the Korean RABV.
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Animals , Dogs , Humans , Amino Acid Substitution , Consensus Sequence , Genotype , Glycoproteins , Korea , Lyssavirus , Molecular Epidemiology , Nucleocapsid Proteins , Rabies , Rabies virus , Republic of KoreaABSTRACT
Objective: To construct lentivirus carrying both novel human pancreatic cancer gene S100P and green fluorescent protein (GFP) gene. Methods: The fragments containing all the exons of S100P were amplified by RT-PCR and were cloned into the lentivirus vectors labeled with GFP. The lentivirus was packaged and was used to transfect 293T cells together with pShuttle. The supernatant of virus-producing cells was harvested, concentrated, identified, and was used to infect 293T cells and pancreatic cancer cells. Fluorescent microscopy was used to observe the fluorescence in the 293T cells; and real time-PCR was used to examine the relative contents of S100P in pancreatic cancer cells. Results: Electrophoresis showed that the sequence of the RT-PCR product was consistent with the data of NCBI by DNA sequencing analysis. The lentivirus effectively transfected 293T cells. Strong green fluorescence was observed by fluorescent microscopy. The supernatant of lentivirus-transfected 293T cells effectively infected 293T cells and the relative content of S100P in the transfected pancreatic cancer cells was higher than that of control group. Conclusion: The lentivirus vector containing S100P-GFP recombinant gene have been successfully constructed, which provides a basis for further study of S100P function.
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The complete length of P gene from rabies virus was amplified by RT-PCR using a pair of specific primers designed according to the relevant sequences from GenBank. The PCR product was cloned into cloning expression vestor pGM-T to obtain the cloning expressed plasmid pGM-T-P. After double-digestion by NotI and EcoRI, the product was transferred into prokaryotic expression vetor pET-32a(+)to obtain the prokaryotically expressed plasmid pET-32a-P. The target gene was then expressed in the E.coli BL21(DE3) cell with IPTG induction. The highest expression of target protein was analysed by SDS-PAGE, and the good immunoreactivity to rabies virus antibodies was proved by Western-blot analysis. By using purified protein, the indirect ELISA assay for the detection of rabies virus antibodies in canine serum was applied after management of the optional working condition.
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OBJECTIVES: This study is aimed to test the association between the coding sequence functional polymorphism (I105 V) of glutathione S-transferase P gene (GSTP1) and schizophrenia. METHODS: Two hundred fourteen (214) patients with schizophrenia according to the DSM-IV criteria and one hundred ten (110) healthy controls were enrolled in this study. Patients and controls were biologically unrelated age and sex- matched native Koreans. Genotyping for the GSTP1 polymorphism was performed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Genotype and allele distributions of the GSTP1 polymorphism in patients with schizophrenia were not significantly different from those of the controls. Comparisons of clinical variables also were not different according to genotype and allele distribution. CONCLUSION: The present study suggests that the GSTP1 polymorphism may not confer susceptibility to development of schizophrenia, at least in the Korean population.
Subject(s)
Humans , Alleles , Clinical Coding , Diagnostic and Statistical Manual of Mental Disorders , Genotype , Glutathione Transferase , Glutathione , SchizophreniaABSTRACT
@#ObjectiveTo investigate effects of electroacupuncture(EA) of Zusanli(ST36) on changes of substance P(SP) mRNA expression in rat brain stem.Methods78 SD rats were evenly randomized into Zuanli-EA group, non-acupoint group, bondage groups(at the end of 2h,4h,6h,8h after 30 minute EA stimulation in each group) and control group. RT-PCR and image processing were used to study the change of SP mRNA level in brain stem.ResultsIn control groups,there was SP expression. An increase of SP mRNA expression was seen in Zusanli group and non-acupoint group comparing with control group at the end of 2h after 30min EA stimulation(P<0.05).There was difference between above two groups at the end of 6h and 8h(P<0.05).ConclusionEA stimulation elicited an accelerated expression of SP gene. Zusanli-EA stimulation can increased this expression, which may constitute one of the mechanisms for gastrointestinal motility and eletroacupuncture analgesia.