Effects of lncRNA FEZF1-AS1 on proliferation,migration and invasion through regulating EZH2 of lung interstitial cells / 基础医学与临床

Objective To investigate the effects of long non-coding RNA FEZ family zinc finger 1 antisense RNA 1(lncRNA FEZF1-AS1)on enhancer of zeste homolog 2(EZH2)in regulation of proliferation,migration,invasion and epithelial-mesenchymal transition(EMT)of pulmonary interstitial cells and its mechanism.Methods The A549 cells human lung adenocarcinoma cell line were divided into control group and model group[model cells were induced into lung interstitial cells after being treated with transforming growth factor β1(TGF-β1)20 ng/mL for 48 h].The protein expression of E-cadherin,N-cadherin and vimentin in each group was detected by Western blot.The expression of lncRNA FEZF1-AS1 and EZH2 in the two groups was detected by RT-qPCR.Cells in the trans-fection group were divided into si NC group,lncRNA FEZF1-AS1+OE vector group and si lncRNA FEZF1-AS1+OE EZH2 group.Cell proliferation was examined by CCK-8 method,cell migration was detected by cell scratch,and cell invasion was detected by Transwell assays.The protein expression of E-cadherin,N-cadherin,vimentin and EZH2 in each group was detected by Western blot.The direct binding effect of FEZF1-AS1 and EZH2 was deter-mined by RNA immuno-precipitation(RIP).Results Compared with the control group,the protein expression level of E-cadherin in the model group was significantly decreased(P＜0.05),and the protein expression of N-cadherin and vimentin was significantly increased(P＜0.05).Compared with the control group,the expression level of lncRNA FEZF1-AS1 and EZH2 genes was significantly increased in the model group(P＜0.05).Compared with si NC group,the proliferation,migration and invasion ability of si lncRNA FEZF1-AS1+OE vector group were decreased,the ex-pression of E-cadherin protein was increased while the expression of N-cadherin,vimentin and EZH2 was decreased(P＜0.05).Compared with si lncRNA FEZF1-AS1+OE vector group,the proliferation,invasion and migration of si lncRNA FEZF1-AS1+OE EZH2 group were increased(P＜0.05).E-cadherin expression was decreased,while N-cad-herin,vimentin and EZH2 expressions were increased(P＜0.05).RIP experiment further confirmed that lncRNA FEZF1-AS1 had direct binding effect with EZH2.Conclusions LncRNA FEZF1-AS1 can promote the proliferation,invasion,metastasis and EMT process of pulmonary fibrosis cells by regulating EZH2.

Relationship between the Expression of LncRNA CTBP1-AS2 and miR-140-5p Levels in Nasopharyngeal Carcinoma Tissues and Radiotherapeutic Effect and Prognosis / 现代检验医学杂志

Objective To investigate the relationship between the expression of long non-coding RNA C-terminal binding protein 1 antisense RNA2(LncRNA CTBP1-AS2)and microRNA-140-5p(miR-140-5p)levels in nasopharyngeal carcinoma tissues and the radiotherapeutic effect and prognosis.Methods A total of 222 nasopharyngeal carcinoma patients diagnosed in Nantong Cancer Hospital from March 2018 to March 2020 were collected as the nasopharyngeal carcinoma group.The clinical data of these patients were recorded,the radiotherapeutic effect and prognosis were evaluated,and they were grouped into the survival group(n=194)and the death group(n=28).Meanwhile,another 219 patients with nasopharyngeal inflammation were collected as the control group.Correlation between LncRNA CTBP1-AS2 and miR-140-5p expression levels in nasopharyngeal carcinoma patients was calculated using Pearson correlation analysis.Kaplan-Meier survival curve was applied to analyze the relationship between the expression levels of LncRNA CTBP1-AS2 and miR-140-5p in nasopharyngeal carcinoma tissue and prognosis.Multivariate analysis was conducted on the prognosis of nasopharyngeal carcinoma patients using Cox proportional risk regression model.Results The expression level of LncRNA CTBP1-AS2 in the tissues of patients in nasopharyngeal carcinoma group(2.25±0.46)was higher than that in the control group(1.02±0.22),while the expression level of miR-140-5p(0.67±0.19)was lower than that in the control group(1.01±0.23),and the differences were statistically significant(t=35.742,16.934,all P<0.001).There was a negative correlation between LncRNA CTBP1-AS2 and miR-140-5p expression levels in nasopharyngeal carcinoma patients(r=-0.624,P<0.001).The total effective rate(74.11%)and 3-year survival rate(77.68%)of nasopharyngeal carcinoma patients with high expression of LncRNA CTBP1-AS2 after radiotherapy were lower than those with low expression(93.64%,97.27%),and the differences were statistically significant(χ2=15.578,19.331,all P<0.001).The total effective rate(93.58%)and 3-year survival rate(96.33%)of patients with high expression of miR-140-5p after radiotherapy were higher than those of patients with low expression(74.34%,78.76%),and the differences were statistically significant(χ2=15.119,15.538,all P<0.001).The magnetic resonance amide proton transfer(APT)value(2.10±0.26),the proportion of patients with radiotherapy failure(85.71%),high expression of LncRNA CTBP1-AS2(89.29%),and low expression of miR-140-5p(85.71%)in the death group were higher than those in the survival group(1.82±0.31,6.19%,44.85%,45.88%),and the differences were statistically significant(t/χ2=4.551,108.127,19.331,15.538,all P<0.001).The level of LncRNA CTBP1-AS2 was a risk factor for mortality within 3 years in nasopharyngeal carcinoma patients(HR=2.762,95%CI:1.510～5.051,P=0.001),while the level of miR-140-5p was a protective factor for mortality within 3 years in nasopharyngeal carcinoma patients(HR=0.817,95%CI:0.718～0.930,P=0.002).Conclusion LncRNA CTBP1-AS2 was highly expressed,while miR-140-5p was lowly expressed in nasopharyngeal carcinoma tissue,indicating the two may be closely related to the radiotherapeutic effect and prognosis.

LncRNA GNAS-AS1 participates in the proliferation and migration of gastric cancer cells by regulating the miR-449a/Notch1 axis / 实用医学杂志

Objective To explore the impacts of long non-coding RNA(LncRNA)GNAS antisense RNA1(GNAS-AS1)on the proliferation and migration of gastric cancer(GC)cells by regulating the miR-449a/Notch1 axis.Method Tumor tissue and adjacent tissue samples were collected from 30 patients diagnosed with GC at Sichuan Provincial People's Hospital from September 2013 to September 2017;GC cells AGS were randomly divided into Control group,si-NC group,si-GNAS-AS1 group,si-GNAS-AS1+inhibitor NC group,and si-GNAS-AS1+miR-449a inhibitor group.Real-time fluorescence quantitative PCR method was applied to detect the expres-sion of GNAS-AS1,miR-449a,and Notch1 mRNA;MTT experiments and plate cloning experiments were applied to detect the proliferation;wound healing test was applied to detect cell migration;Transwell experiment was applied to detect cell invasion.Western Blot was applied to detect the expression of Notch1,E-cadherin,Vimentin,and N-cadherin proteins.Double Luciferase reporter gene experiment was applied to verify the relationship between GNAS-AS1 and miR-449a,between miR-449a and Notch1,respectively.Results Compared with adjacent tissues,the expression of GNAS-AS1 and Notch1 mRNA in tumor tissue was increased,the expression of miR-449a was reduced(P<0.05).Compared with the Control group and si-NC group,the expression of GNAS-AS1,OD490 value,number of clones formed,scratch healing rate,number of cell invasions,and the expression of Notch1,Vimentin,and N-cadherin proteins in AGS cells in the si-GNAS-AS1 group reduced,the expression of miR-449a and E-cadherin protein increased(P<0.05).Compared with the si-GNAS-AS1 group and the si-GNAS-AS1+inhibitor NC group,the OD490 value,scratch healing rate,number of cell invasions,Notch1,Vimentin,and N-cadherin expression in the si-GNAS-AS1+miR-449a inhibitor group increased,the expression of miR-449a and E-cadherin protein reduced(P<0.05).GNAS-AS1 targeted and negatively regulated miR-449a expression,while miR-449a targeted and negatively regulated Notch1 expression.Conclusion Silencing GNAS-AS1 may inhibit the expression of Notch1 protein by up-regulating miR-449a,thereby inhibiting the proliferation,migration,and invasion pro-cesses of GC cells.

The effect of lncRNA FEZF1-AS1 targeting regulation of miR-200c-3p on biological behaviors of human lung fibroblasts / 天津医药

Objective To investigate the effect of FEZ family zinc finger 1-antisense RNA 1(LncRNA FEZF1-AS1)targeting regulation of miR-200c-3p expression on biological behaviors of human lung fibroblasts(HLF).Methods Transforming growth factor β1(TGF-β1)was used to induce the transformation of HLF into myofibroblasts,which were divided into the Blank group and the model group(HLF+TGF-β1 group).According to different transfection plasmid,cells were divided into the Blank group,the TGF-β1+Si LncRNA FEZF1-AS1 NC group and the TGF-β1+Si LncRNA FEZF1-AS1 group.The protein expressions of α-SMA,Collagen Ⅰ and Vimentin were detected by Western blot assay.The expressions of LncRNA FEZF1-AS1 and miR-200c-3p were detected by quantitative real-time PCR(qRT-PCR).Cell proliferation ability was detected by CCK-8 method,migration ability was detected by cell scratch experiment and invasion ability was detected by Transwell assay.The targeting relationship between FEZF1-AS1 and miR-200c-3p was detected by dual-luciferase reporter assay.Results Compared with the Blank group,protein expressions of α-SMA,Collagen Ⅰ,Vimentin and the expression of LncRNA FEZF1-AS1 were increased in the HLF+TGF-β1 group(P＜0.05),and the expression of miR-200c-3p was decreased(P＜0.05).Compared with the TGF-β1+Si LncRNA FEZF1-AS1 NC group,cell proliferation,migration,invasion ability,LncRNA FEZF1-AS1 expression,protein expressions of α-SMA,Collagen Ⅰ and Vimentin were decreased in the TGF-β1+Si LncRNA FEZF1-AS1 group(P＜0.05),and the expression of miR-200c-3p was increased(P＜0.05).There were binding sites between miR-200c-3p and FEZF1-AS1 gene sequence.Conclusion LncRNA FEZF1-AS1 promotes the formation and progression of idiopathic pulmonary interstitial fibrosis by inhibiting miR-200c-3p.

Long non-coding RNA GATA6-AS1 inhibits proliferation,apoptosis,and metastasis of 5-fluorouracil resistant gastric cancer cells SGC7901/5-FU by regulating GATA6 / 肿瘤

Objective:To investigate the effects of regulation of GATA6 expression by long non-coding RNA(lncRNA)GATA6 antisense RNA 1(GATA6-AS1)on the proliferation,apoptosis,and metastasis of 5-fluorouracil(5-FU)resistant gastric cancer(GC)cells SGC7901/5-FU,and to explore the underlying mechanisms. Methods:Parental SGC7901 cells and SGC7901/5-FU cells were used as controls(treated with blank culture medium).Empty vector pcDNA3.1(control for GATA6-AS1 overexpression),recombinant vector pcDNA3.1-GATA6-AS1(for GATA6-AS1 overexpression),shNC(negative control for GATA6-AS1 silencing)or shGATA6-AS1(for GATA6-AS1 silencing)were transfected into SGC7901/5-FU cells by Lipofectamine 2000 to overexpress or silent GATA-AS1.The mRNA expression of GATA6-AS1 and GATA6 in cells of different treatment groups were examined by real-time fluorescence quantitative PCR.After 5-FU treatment,the proliferation,migration,invasion and apoptosis were analyzed by CCK-8 assay,wound-healing assay,Transwell assay,and flow cytometry(FCM)assay,respectively.The protein expression levels of Bax,Bcl-2,Caspase 3 and GATA6 in different treatment groups were examined by Western blotting.The stability of GATA6 mRNA was evaluated after α-amanitin treatment.The relationship between GATA6-AS1 and GATA6 was studied by RNA pull-down experiment.Furthermore,the SGC7901 cell transplantation tumor model was established using Balb/c nude mice,and the grouping was the same as in vitro experiments with 12 mice in each group.The tumor growth was recored and the tumor inhibition rate was calculated after 5-FU treatment.The histopathological changes of tumor tissue in each group was assessed by HE staining.The protein expression of proliferating cell nuclear antigen(PCNA)in tumor tissues of nude mice in each group was detected by Western blotting. Results:Compared with parental SGC7901 cells(control group),the expression of GATA6-AS1 and GATA6 mRNA was significantly downregulated in SGC7901/5-FU cells(P＜0.05);the proliferation,migration and invasion of SGC7901/5-FU cells was significantly decreased while the apoptosis of SGC7901/5-FU cells was significantly decreased(P＜0.05);the protein expression levels of GATA6,Bax and Caspase 3 were significantly decreased while that of Bcl-2 was significantly increased(P＜0.05).Compared with SGC7901/5-FU-pcDNA group,the above changes showed opposite trends in SGC7901/5-FU cells overexpressing GATA6-AS1.Compared with SGC7901/5-FU-shNC group,the expression of GATA6-AS1 and GATA6 mRNA was significantly downregulated in GATA6-AS1-silencing SGC7901/5-FU cells(P＜0.05);the proliferation,migration and invasion of GATA6-AS1-silencing SGC7901/5-FU cells was significantly decreased while apoptosis of GATA6-AS1-silencing SGC7901/5-FU cells was significantly decreased(P＜0.05);the protein expression levels of GATA6,Bax and Caspase 3 were significantly decreased while that of Bcl-2 was significantly increased(P＜0.05).GATA6-AS1 can increase the stability of GATA6 mRNA and positively regulate the expression of GATA6.In vivo characterization showed that,compared with mice transplanted with parental SGC7901 cells,the tumor volume and PCNA protein expression in tumor tissues were significantly increased in mice transplanted with drug resistant SGC7901/5-FU cells after 5-FU treatment(P＜0.05).Mice in SGC7901/5-FU-GATA6-AS1 group showed significantly reduced tumor volume and PCNA protein expression compared with the SGC7901/5-FU-pcDNA group(P＜0.05),and pathological analysis of the tumor tissues revealed milder development of GC in SGC7901/5-FU-GATA6-AS1 group.Whereas,mice in SGC7901/5-FU-shGATA6-AS1 group showed significantly increased tumor volume and PCNA protein expression compared with the SGC7901/5-FU-shNC group(P＜0.05),and histopathological analysis of the tumor tissues revealed severer development of GC in SGC7901/5-FU-shGATA6-ASl group. Conclusion:Overexpression of GATA6-AS1 can promote the expression of GATA6,inhibit the growth of GC cells,and thus reverse the resistance of SGC7901/5-FU cells to 5-FU.

Pharmacokinetic analysis of mouse short interspersed nuclear element B1 antisense RNA / 中国生物制品学杂志

@#Objective To investigate the biological distribution of B1 antisense RNA(Blas RNA) of mouse short interspersed nuclear element in blood and tissues of normal mice after vein injection and detect the cell uptake efficiency of B1 as RNA using cultured normal mouse embryo cells after transfection.Methods Six 8～12-week-old BALB/c mice,three males and three females,were injected with 20 μg Blas RNA via tail vein,and blood samples were collected at different times after injection.54 BALB/c mice of 8～12-weeks were injected with 20 μg Blas RNA via tail vein,of which six mice,three males and three females,were euthanized at different times after injection,and various tissues,including the heart,liver,spleen,lung,kidney and thymus were harvested.Blas RNA was transfected into cultured mouse embryonic cells,and a certain amount of cells were taken at different time after transfection.The biological distribution of B1as RNA in mouse blood and different tissues and the persistence of Blas RNA in cultured embryo cells were detected by RT-qPCR.30naturally senescent BALB/c mice(≥ 14 months old) were divided into three groups:treatment group(20 μg Blas RNA injected via tail vein,once a week),irrelevant RNA control group(20 μg LacZ3F3R RNA injected via tail vein,once a week) and saline control group(injected with the same volume of saline),with 10 mice in each group,and a young control group(normal young 8～12-week-old BALB/c mice,five males and five females) was set.Four weeks after administration,mice in each group were euthanized,the liver tissues were taken,and the expression levels of aging-related genes(Sirtl,p21,p16~(Ink4a),p15~(Ink4b),p19~(Arf)) were detected by RT-qPCR.Results After tail vein injection,Blas RNA was available in the blood of mice for approximately 30 min,persisted for approximately 2～4 h in most detected tissues and persisted for approximately 48 h in lungs.The efficiency of cellular uptake of Blas RNA was approximately 400 molecules per mouse embryo culture cell 45 min after transfection with B1as RNA.Compared with the saline control group,Blas RNA treatment significantly down-regulated the mRN A expression of p21,p16~(In4a),p15~(In4b) and p19~(Arf) genes(t=10.01,4.461,4.420 and 5.309 respectively,each P <0.05),and significantly up-regulated the mRNA expression of Sirt1 gene(t=4.579,P <0.05).Conclusion Blas RNA was efficiently taken up by cells after transfection.After intravenous injection,Blas RNA stayed in the blood and tissues for a certain period of time and regulated the expression of aging-related genes in aged mice so as to make them approach to the expression level of young normal mice.

Long non-coding RNA alpha-2-macroglobulin antisense RNA 1 regulating oxidized low density lipoprotein-induced human brain microvascular endothelial cell damage by targeting microRNA-106b-5p / 解剖学报

Objective To investigate the effect of long non-coding RNA (lncRNA) alpha-2-macroglobulin antisense RNA 1 (A2M-AS1) targeting microRNA (miR) -106b-5p on oxidized low-density lipoprotein (ox-LDL) -induced injury of human brain microvascular endothelial cells. Methods Human brain microvascular endothelial cells (ox-LDL group) were induced by ox-LDL, normal cultured cells were control group (Ctrl); A2M-AS1 overexpression (pcDNAA2M-AS1 group), empty vector (pcDNA group), miR-106b-5p inhibitor (anti-miR-106b-5p group), negative control (anti-miR-NC group), pcDNA-A2M-AS1 with control mimic NC (miR-NC group), pcDNA-A2M-AS1 with miR-106b-5p mimic (miR-106b-5p mimics group) were transfected into cells and treated with ox-LDL, n = 9. Real-time PCR was used to detect the expression levels of A2M-AS1 and miR-106b-5p; Kits were used to detect malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT)); Flow cytometry and TUNEL detected apoptosis; Dual luciferase reporter gene assay detected A2M-AS1 and miR-106b-5p targeting; Western blotting detected Bcl-2 and Bax protein expression. Results Compared with the Ctrl group, the expression level of A2M-AS1 in the ox-LDL group decreased, and the activity of SOD and CAT and the protein level of Bcl-2 decreased (P<0.05), while the expression level of miR-106b-5p and the level of MDA increased (P<0.05), and the rate of apoptosis and the protein level of Bax increased (P<0.05). Overexpressing A2M-AS1 or interfering with miR-106b-5p decreased the MDA level, apoptosis rate and Bax protein level after ox-LDL-induced cells, and increased SOD, CAT activity and Bcl-2 protein level (P<0.05). A2M-AS1 targeted miR-106b-5p; upregulation of miR-106b-5p reversed the effect of overexpressed lncRNA A2M-AS1 on ox-LDL-induced injury of human brain microvascular endothelial cells (P < 0.05). Conclusion A2M-AS1 attenuates ox-LDL-induced injury of human brain microvascular endothelial cells by targeting miR-106b-5p.

Expression and clinical significance of LncRNA HIF1A-AS1 in proliferative diabetic retinopathy / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the expression and diagnostic value of long non-coding RNA(LncRNA)hypoxia-inducible factor 1 alpha antisense RNA 1(HIF1A-AS1)in serum of patients with proliferative diabetic retinopathy(PDR).METHODS: A total of 160 patients with diabetic retinopathy(DR)admitted to our hospital from July 2019 to July 2021 were selected as the research objects. According to the degree of disease, they were divided into PDR group(80 cases)and nonproliferative diabetic retinopathy(NPDR)group(80 cases). At the same time, 100 healthy cases in our hospital were selected as the control group. Detect and compare serum triglyceride(TG), total cholesterol(TC), high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein cholesterol(LDL-C), fasting blood glucose(FBG)and the level of glycosylated hemoglobin A1c(HbA1c); The expression level of LncRNA HIF1A-AS1 in serum was detected by real-time fluorescence quantitative PCR(qRT-PCR)method; Logistic regression was used to analyze the risk factors that affected the occurrence of PDR; Receiver operating characteristic curve(ROC)was used to analyze the clinical value of LncRNA HIF1A-AS1 level in the diagnosis of PDR. RESULTS: The expression level of LncRNA HIF1A-AS1 in the serum of the patients in the PDR group was significantly higher than that in the NPDR group and the control group, and the NPDR group was higher than the control group(P&#x003C;0.05); The course of disease, HbA1c, TC, TG, LDL-C, FBG levels in the PDR group and the NPDR group were significantly higher than those of the control group, the HDL-C level in the PDR group was significantly lower than that in the control group(P&#x003C;0.05); The level of LncRNA HIF1A-AS1 was positively correlated with the course of disease, HbA1c, TC, TG, LDL-C and FBG(P&#x003C;0.05), and negatively correlated with HDL-C(P&#x003C;0.05); Logistic regression analysis showed that the LncRNA HIF1A-AS1, course of disease, FBG, HbA1c, TC, TG, LDL-C were all risk factors for PDR(P&#x003C;0.05); ROC results showed that the area under the curve(AUC)of the LncRNA HIF1A-AS1 level predicting PDR was 0.766(95%CI: 0.692～0.829), the corresponding sensitivity was 66.25% and the specificity was 78.75%.CONCLUSION: The level of LncRNA HIF1A-AS1 in the serum of PDR patients is up-regulated, it is a risk factor for the occurrence of PDR and it can be used as a potential serological indicator for predicting the occurrence of PDR.

Expression of lncRNA TGFB2-AS1 in the placenta of preeclampsia and its relationship with placental spiral artery recasting / 中华内分泌外科杂志

Objective:To investigate the relationship between the expression level of long non-coding RNA transforming growth factor β2-antisense RNA1 (lncRNA TGFB2-AS1) and placental spiral artery recasting in the placenta of preeclampsia.Methods:A total of 108 pregnant women with severe preeclampsia who were hospitalized in Zaozhuang Maternal and Child Health Hospital and delivered by cesarean section from Oct. 2019 to Jun. 2021 were selected as the research objects, and they were divided into the late-onset preeclampsia group (late-onset severe preeclampsia pregnant women, 56 cases) and early-onset preeclampsia group (early-onset severe preeclampsia pregnant women, 52 cases) ; at the same time, 58 normal pregnant women were selected as the normal pregnancy group. The general data of pregnant women were collected, such as age, systolic blood pressure and diastolic blood pressure. Real-time fluorescent quantitative PCR (qRT-PCR) method was used to detect the expression level of lncRNA TGFB2-AS1 in placental tissues, a scanning electron microscope was used to measure the lumen area and wall thickness of spiral arteries. Pearson correlation analysis method was used to analyze the correlation between the level of lncRNA TGFB2-AS1 in the placenta tissue and the thickness of the spiral artery wall and the area of the lumen of pregnant women with early-onset and late-onset severe preeclampsia.Results:The tube wall thickness [ (119.69±8.31) μm], systolic blood pressure [ (162.86±4.94) mmHg], diastolic blood pressure [ (103.09±2.35) mmHg], and 24-hour urine protein [ (2.17±0.31) g/24 h] in the early preeclampsia group were higher than those in the late preeclampsia group [ (101.04±5.78) μm, (146.95±6.43) mmHg, (92.13±4.74) mmHg, (1.62±0.23) g/24 h] and the normal pregnancy group [ (99.82±5.56) μm, (116.42±9.31) mmHg, (74.25±6.74) mmHg, (0.06±0.02) g/24 h], the placental tissue lncRNA TGFB2-AS1 level (0.62±0.16), lumen area [ (133.74±20.16) μm 2], gestational week of delivery [ (32.15±1.74) weeks], weight of the newborns [ (2.25±0.26) g] were lower than those in the late-onset preeclampsia group [ (0.99±0.21), (185.49±22.75) μm 2, (36.14±1.59) weeks, (3.37±0.32) g] and the normal pregnancy group [ (1.02±0.23), (186.42±23.71) μm 2, (38.19±1.56) weeks, (3.42±0.37) g] ( P<0.05). The systolic blood pressure, diastolic blood pressure, and 24-hour urine protein in the late preeclampsia group were higher than those in the normal pregnancy group, gestational week of delivery was lower than the normal pregnancy group ( P<0.05). Placental tissue lncRNA TGFB2-AS1 of pregnant women with early-onset severe preeclampsia was positively correlated with the lumen area, and negatively correlated with the thickness of the tube wall ( P<0.05). There was no correlation between lncRNA TGFB2-AS1 and the lumen area and wall thickness in the placental tissue of pregnant women with late-onset severe preeclampsia ( P>0.05) . Conclusion:The lncRNA TGFB2-AS1 expression in the placenta tissue of pregnant women with early-onset severe preeclampsia is abnormally low, which may be related to the insufficient recasting of the placental spiral artery.

TRIM52 antisense RNA 1 alleviates injury of brain cortex neurons induced by hypoxia and re-oxygenation in rats through regulating miR-28-5p expression / 中华神经医学杂志

Objective:To investigate the effect of TRIM52 antisense RNA 1 (TRIM52-AS1) on injury of brain cortex neurons induced by hypoxia/re-oxygenation (H/R) and its possible mechanism in rats.Methods:(1) The cortical neurons were cultured in vitro and divided into control group and model group. In the model group, H/R-induced cell injury models were prepared. Real-time quantitative PCR (RT-qPCR) was used to detect the expressions of TRIM52-AS1 and micro RNA (miR)-28-5p in neurons of the two groups. (2) The cortical neurons were divided into control group, model group, small interfering (si)-TRIM52-AS1 transfection group, and nonsense sequence transfection group. Cells in the model group were prepared for H/R-induced cell damage models. After cells in the latter two groups were transfected with si-TRIM52-AS1 or its nonsense control sequence for 6 h, they were prepared for H/R-induced cell damage models. RT-qPCR, CCK-8, and flow cytometry were used to detect the TRIM52-AS1 expression, and proliferation and apoptosis of neurons in the 4 groups; enzyme-linked immunosorbent assay (ELISA) and Western blotting were used to detect the lactate dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD) levels and protein expressions of Cyclin D1, Bcl-2 and Bax. (3) The cortical neurons were divided into miR-28-5p transfection group and nonsense sequence transfection group. After the cells were respectively transfected with miR-28-5p or its nonsense control sequence for 6 h, they were prepared for H/R-induced cell damage models. RT-qPCR, CCK-8, and flow cytometry were used to detect the TRIM52-AS1 expression, and proliferation and apoptosis of neurons in the two groups; ELISA and Western blotting were used to detect the LDH, MDA and SOD levels and protein expressions of Cyclin D1, Bcl-2 and Bax. (4) The cortical neurons were divided into wild-type TRIM52-AS1+miR-28-5p mimic transfection group, wild-type TRIM52-AS1+miR-28-5p nonsense control sequence transfection group, mutant TRIM52-AS1+miR-28-5p mimic transfection group, and mutant TRIM52-AS1+miR-28-5p nonsense control sequence transfection group. After the cells were co-transfected for 6 h, the culture medium was replaced with fresh medium and they were cultured for another 12 h, and then, the luciferase activity of cells in each group was detected by dual luciferase reporter gene experiment. (5) Cortical neurons were divided into nonsense sequence transfection group, miR-28-5p inhibitor transfection group, nonsense sequence+si-TRIM52-AS1 transfection group, and miR-28-5p inhibitor+si-TRIM52-AS1 transfection group, and these cells were transfected with miR-28-5p inhibitor nonsense control sequence, miR-28-5p inhibitor, miR-28-5p inhibitor nonsense control sequence+si-TRIM52-AS1, miR-28-5p inhibitor+si-TRIM52-AS1; 6 h after transfection, H/R-induced cell damage models were prepared. RT-qPCR, CCK-8, and flow cytometry were used to detect the miR-28-5p expression, proliferation and apoptosis of neurons in the two groups, respectively; ELISA and Western blotting were used to detect the LDH, MDA and SOD levels and protein expressions of Cyclin D1, Bcl-2 and Bax. Results:(1) As compared with those in the control group, the TRIM52-AS1 expression statistically increased, but the miR-28-5p expression significantly decreased in the model group ( P<0.05). (2) As compared with the control group, the model group and nonsense sequence transfection group had significantly increased LDH content in the culture supernatant, statistically decreased MDA and SOD content in the cells, significantly decreased A value and protein expressions of Cyclin D1 and Bcl-2, but significantly increased apoptosis rate and Bax protein expression ( P<0.05). As compared with the model group and nonsense sequence transfection group, si-TRIM52-AS1 transfection group had significantly decreased si-TRIM52-AS1 expression, statistically decreased LDH content in the culture supernatant and MDA content in the cells, significantly increased SOD content in the cells, significantly increased A value and protein expression of Cyclin D1 and Bcl-2, significantly decreased apoptosis rate and Bax protein expression ( P<0.05). (3) As compared with the nonsense sequence transfection group, the miR-28-5p transfection group had significantly increased miR-28-5p expression, significantly decreased LDH content in the culture supernatant and MDA content in the cells, and significantly increased SOD content in the cells, significantly increased A value and protein expressions of Cyclin D1 and Bcl-2, but significantly decreased apoptotic rate and Bax protein expression ( P<0.05). (4) The luciferase activity of the wild-type TRIM52-AS1+miR-28-5p mimic transfection group was significantly lower than that of the wild-type TRIM52-AS1+miR-28-5p nonsense control sequence transfection group (0.43±0.04 vs. 1.00±0.09, P<0.05). (5) As compared with the nonsense sequence transfection group, the miR-28-5p inhibitor transfection group had significantly decreased miR-28-5p expression, significantly increased LDH content in the culture supernatant and MDA content in the cells, significantly decreased SOD content in the cells, significantly decreased A value and protein expressions of Cyclin D1 and Bcl-2, but significantly increased apoptosis rate and Bax protein expression ( P<0.05). As compared with the nonsense sequence+si-TRIM52-AS1 transfection group, the miR-28-5p inhibitor+si-TRIM52-AS1 transfection group had significantly decreased miR-28-5p expression, significantly increased LDH content in the culture supernatant and MDA content in the cells, significantly decreased SOD content in the cells, significantly decreased A value and protein expressions of Cyclin D1 and Bcl-2, but significantly increased apoptosis rate and Bax protein expression ( P<0.05). Conclusion:TRIM52-AS1 down-regulation may inhibit H/R-induced oxidative stress and apoptosis of rat brain cortical neurons by negatively regulating the miR-28-5p expression, which reduces neuronal cell damage.

The Long Non-coding RNA ILF3-AS1 Inhibited Proliferation of Cervical Cancer Cells Through the MiR-130a-3p/PTEN Axis / 中国生物化学与分子生物学报

ILF3 antisense RNA 1 (ILF3-AS1), the antisense RNA of interleukin enhancer binding factor 3 (ILF3), is a lncRNA located on chromosome 19p13. 2. ILF3-AS1 played a key role in the occurrence and development of a variety of tumors, but its role in cervical cancer had not been explored yet. Therefore, we first used TCGA and GTEx database to conduct bioinformatics analysis. The results suggested that ILF3-AS1 was down-regulated in cervical cancer tissues (P < 0. 001) and was associated with a good prognosis (P = 0. 045). The qRT-PCR experiment showed that expression of ILF3-AS1 in cervical cancer tissues and SiHa, HeLa, CaSki cervical cancer cell lines was lower than that in control groups. Subsequently, overexpressing of ILF3-AS1 can significantly inhibit the cancer cell viability and stimulate apoptosis (P<0. 001). Analysis using the Star Base v3. 0 database suggested that ILF3-AS1 can target miR-130a-3p; while miR-130a-3p may target PTEN. The qRT-PCR test showed that the expression of miR-130a-3p in cervical cancer was significantly higher than that in normal cervical tissues (P < 0. 01). The results of the luciferase reporter assay showed that ILF3-AS1 can specifically bind to miR-130a-3p (P<0. 01). After overexpression of ILF3-AS1 in HeLa cells, the expression of miR-130a-3p was significantly down-regulated (P < 0. 01). Co-transfection with pcDNA3. 1-ILF3-AS1 and miR-130a-3p mimics, the inhibitory effect of LF3-AS1 on cell proliferation can partially be reversed (P<0. 001). After HeLa cells overexpressed ILF3-AS1, the expression of phosphatase and tensin homolog deleted on chromosome ten (PTEN) mRNA (P < 0. 001) and proteins (P < 0. 001) significantly increased; when miR-130a-3p mimics was simultaneously used in HeLa cell, the increased expression of PTEN mRNA (P <0. 001) and proteins (P < 0. 001) was notably inhibited. In summary, ILF3-AS1 inhibited the proliferation of cervical cancer cells by sponging miR-130a-3p to regulate the expression of PTEN.

Post-transcriptional gene silencing: Basic concepts and applications

Post-transcriptional gene silencing (PTGS)-mediated gene silencing exploits the cellular mechanism whereintranscripts having sequence similarity to the double-stranded RNA (dsRNA) molecules present in the cell will besubjected to degradation. PTGS is closely related to natural processes such as RNA-mediated virus resistance andcross-protection in plants. Gene silencing and the cellular machinery for affecting this phenomenon might haveevolved as a natural protective measure against viral infection in plants. In PTGS, small interfering RNA (siRNA)molecules of 21–23 nucleotides length act as homology guides for triggering the systemic degradation of transcriptshomologous to the siRNA molecules. PTGS phenomenon, first discovered in transgenic petunia plants harbouringchalcone synthase gene and termed co-suppression, has been subsequently exploited to target specific gene transcripts for degradation leading to manifestation of desirable traits in crop plants. Targeted gene silencing has beenachieved either through the introduction of DNA constructs encoding dsRNA or antisense RNA or by deploying cosuppression constructs producing siRNAs against the transcript of interest. Understanding the mechanism of genesilencing has led to the development of several alternative strategies for inducing gene silencing in a precise andcontrolled way. This has paved the way for using PTGS as one ofthe chief functional genomicstools in plants and hashelped in unraveling the mechanism of many cellular processes and identifying the focal points in pathways, besides,opening new vistas in genetic engineering of plants for human benefits. PTGS has shown great potential in silencingthe deleterious genes efficiently so that value-added plant products could be obtained. Thus, PTGS has ushered in anew era in the genetic manipulation of plants for both applied and basic studies. In this review, we have outlined thebasics of RNAi-mediated gene silencing and summarized the work carried out at our institute using this approach, ascase studies. In particular, adopting RNAi-mediated gene silencing (a) as a method to restore fertility in transgenicmale sterile lines developed based on orfH522 gene from sunflower PET1-CMS source, (b) as a tool to suppress theproduction of toxic proteins, ricin and RCA, in castor, and (c) as an approach to induce bud necrosis virus resistancein sunflower has been discussed. Examples from other plant systems also have been mentioned to exemplify theconcept and utility of gene silencing in crop plants.

Prognostic value of long non-coding RNA TP73-AS1 expression in different types of cancer: a systematic review and meta-analysis

Background: TP73 antisense RNA 1 (TP73-AS1), a newly discovered long non-coding RNA (lncRNA), has been reported to be upregulated in various kinds of tumors, and shows a variable influence on living quality and prognosis of patients. Thus, we conducted a meta-analysis to evaluate the overall prognostic value of the lncRNA TP73-AS1 in cancer patients. Results: A systematic literature retrieval was carried out using the PubMed, Cochrane Library, EMBASE, and Web of Science databases. We calculated the pooled hazard ratio (HR) and odds ratio (OR) with 95% confidence intervals (CIs) to evaluate the association of TP73-AS1 expression with prognostic and clinicopathological parameters. A total of 15 studies including 1057 cancer patients were finally selected for the meta-analysis. The results demonstrated that high TP73-AS1 expression was significantly associated with shorter overall survival (OS) (HR = 1.97, 95% CI: 1.68­2.31, P b 0.001). According to a fixed-effects or random-effects model, elevated TP73-AS1 expression markedly predicted advanced clinical stage (OR = 3.30, 95% CI: 2.35­4.64, P b 0.001), larger tumor size (OR = 2.37, 95% CI: 1.75­3.22, P b 0.001), earlier lymph node metastasis (OR = 3.28, 95% CI: 1.59­6.76, P = 0.001), and distant metastasis (OR = 4.94, 95% CI: 2.61­9.37, P b 0.001). Conclusions: High lncRNA TP73-AS1 expression appears to be predictive of a worse OS and clinicopathologic features for patients with various types of malignant tumors. These results provide a basis for utilizing TP73- AS1 expression as an unfavorable indicator to predict survival outcomes.

Expression and clinical significance of lncRNATTN-AS1 in lung adenocarcinoma / 中国肿瘤生物治疗杂志

@#[Abstract] Objective：To study the expression of long non-coding RNA(lncRNA) titin antisense RNA1 (TTN-AS1) in lung adenocarcinoma (LUAD) tissues, and explore its relationship with clinicopathologic characteristics and prognosis of LUAD patients. Methods: The TTN-AS1 expression in LUAD data set was analyzed using TCGAdatabase. 52 pairs of tumor tissues and matched para-carcinoma tissues from LUAD patients, who underwent surgical resection and were later pathologically conformed in Fourth Hospital of Hebei Medical University between Jan. 2014 and Jan. 2015, were used in this study. qPCR was performed to detect TTN-AS1 expression in the specimens. Then, the correlations between TTN-AS1 expression and clinicopathologic characteristics were analyzed. Survival analysis was used to determine the significance of TTN-AS1 expression for predicting the prognosis of LUAD patients. Results: TCGAdatabase analysis and qPCR results showed that TTN-AS1 expression in LUAD tissues was significantly higher than that in normal lung and para-carcinoma tissues (both P<0.01). TTN-AS1 expression in LUAD tissues was significantly correlated with the TNM stage and lymph node metastasis (P<0.05), but not correlated with gender, age, tumor invasion range (P>0.05). Kaplan-Meier univariate analysis result demonstrated that the patients with high TTN-AS1 expression had shorter post-operative disease-free survival (DFS) and overall survival (OS) than those patients with low TTN-AS1 expression (all P<0.01). Cox proportional hazard regression model result demonstrated that wider tumor invasion range, positive lymph node metastasis and high TTN-AS1 expression were significantly correlated with shorter postoperative DFS and OS (P<0.05). Conclusion: TTN-AS1 was highly expressed in LUAD tissues, and closely correlated with TNM stage and lymph node metastasis of LUAD patients (all P<0.05). High expression of TTN-AS1 is significantly correlated with shorter DFS and OS, indicating that TTN-AS1 may be a biomarker for predicting poor prognosis of LUAD patients.

Expression of HOXA terminal transcript antisense RNA in hepatocellular carcinoma tissues and its effect on proliferation, invasion and migration of hepatocellular carcinoma HepG2 cells / 肿瘤研究与临床

Objective@#To investigate the expression of HOXA terminal transcript antisense RNA (HOTTIP) in hepatocellular carcinoma (HCC) tissues and to explore its effect on proliferation, invasion and migration in HepG2 cells.@*Methods@#A total of 60 cases with HCC tissues undergoing excision surgery and 60 cases of corresponding paracancerous tissues from January 2012 to June 2018 in Dandong First Hospital of Liaoning Province were collected. The expressions of HOTTIP in HCC tissues and paracancerous tissues were detected by using real-time quantitative polymerase chain reaction (RT-qPCR), and the relationship between the expression and clinicopathological features was analyzed. HepG2 cell line with high expression of HOTTIP constructed by cell transfer technique was treated as the experimental group, and the empty plasmid pcDNA3.1-NC was treated as the control group. The effect of HOTTIP on the proliferation of HepG2 cells was detected by using CCK-8 method, and the effect of HOTTIP on invasion and migration of HepG2 cells was detected by using Transwell assay.@*Results@#The expression of HOTTIP mRNA in HCC tissues was higher than that in paracancerous tissues, and there was no statistically significant difference (1.9±0.6 vs. 0.9±0.7, t = 6.069, P < 0.01). The whole HCC cases were divided into the high expression group (30 cases) and the low expression group (30 cases) according to the median value (1.92) of the expression of HOTTIP mRNA. The expression of HOTTIP was related with TNM stage, differentiation degree and lymph node metastasis (χ2 values were 10.800, 8.076, 5.711, all P < 0.05), but not with age, gender, tumor diameter, number of tumors, hepatitis and alpha fetoprotein (AFP) levels (all P > 0.05). The results of RT-qPCR showed that the expression of HOTTIP mRNA in HepG2 cells was increased after transfection of overexpressed HOTTIP and the differences was statistically significant compared with the control group (63±6 vs. 13±9, t = 9.129, P < 0.01). The results of CCK-8 method showed that the proliferation activity of cells was enhanced after the overexpression of HOTTIP in HepG2 cells (24 h, 36 h, 72 h at 490 nm absorbance was 1.497 ± 0.017 vs. 0.826±0.006, 2.002±0.025 vs. 1.211±0.020, 3.257±0.042 vs. 1.772±0.021), and the differences were statistically significant (t values were 5.321, 7.349, 8.793, all P < 0.01). In Transwell invasion assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (101±9 vs. 41±11), and the difference was statistically significant (t = 6.839, P < 0.01). In Transwell migration assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (112±9 vs. 53±11), and the difference was statistically significant (t = 7.105, P < 0.01).@*Conclusion@#The expression of HOTTIP in HCC tissues is up-regulated, and the overexpression of HOTTIP can promote the proliferation, invasion and migration of HCC cells.

Expression of HOXA terminal transcript antisense RNA in hepatocellular carcinoma tissues and its effect on proliferation, invasion and migration of hepatocellular carcinoma HepG2 cells / 肿瘤研究与临床

Objective To investigate the expression of HOXA terminal transcript antisense RNA (HOTTIP) in hepatocellular carcinoma (HCC) tissues and to explore its effect on proliferation, invasion and migration in HepG2 cells. Methods A total of 60 cases with HCC tissues undergoing excision surgery and 60 cases of corresponding paracancerous tissues from January 2012 to June 2018 in Dandong First Hospital of Liaoning Province were collected. The expressions of HOTTIP in HCC tissues and paracancerous tissues were detected by using real-time quantitative polymerase chain reaction (RT-qPCR), and the relationship between the expression and clinicopathological features was analyzed. HepG2 cell line with high expression of HOTTIP constructed by cell transfer technique was treated as the experimental group, and the empty plasmid pcDNA3.1-NC was treated as the control group. The effect of HOTTIP on the proliferation of HepG2 cells was detected by using CCK-8 method, and the effect of HOTTIP on invasion and migration of HepG2 cells was detected by using Transwell assay. Results The expression of HOTTIP mRNA in HCC tissues was higher than that in paracancerous tissues, and there was no statistically significant difference (1.9±0.6 vs. 0.9±0.7, t=6.069, P<0.01). The whole HCC cases were divided into the high expression group (30 cases) and the low expression group (30 cases) according to the median value (1.92) of the expression of HOTTIP mRNA. The expression of HOTTIP was related with TNM stage, differentiation degree and lymph node metastasis (χ2 values were 10.800, 8.076, 5.711, all P<0.05), but not with age, gender, tumor diameter, number of tumors, hepatitis and alpha fetoprotein (AFP) levels (all P>0.05). The results of RT-qPCR showed that the expression of HOTTIP mRNA in HepG2 cells was increased after transfection of overexpressed HOTTIP and the differences was statistically significant compared with the control group (63±6 vs. 13±9, t=9.129, P<0.01). The results of CCK-8 method showed that the proliferation activity of cells was enhanced after the overexpression of HOTTIP in HepG2 cells (24 h, 36 h, 72 h at 490 nm absorbance was 1.497 ± 0.017 vs. 0.826 ±0.006, 2.002 ±0.025 vs. 1.211 ±0.020, 3.257 ±0.042 vs. 1.772 ±0.021), and the differences were statistically significant (t values were 5.321, 7.349, 8.793, all P < 0.01). In Transwell invasion assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (101 ±9 vs. 41 ±11), and the difference was statistically significant (t= 6.839, P< 0.01). In Transwell migration assay, the number of cells transferred into the basement membrane in the experimental group was more than that in the control group (112 ±9 vs. 53 ±11), and the difference was statistically significant (t=7.105, P<0.01). Conclusion The expression of HOTTIP in HCC tissues is up-regulated, and the overexpression of HOTTIP can promote the proliferation, invasion and migration of HCC cells.

Expression and functions of long non-coding RNA actin filament-associated protein 1-antisense RNA1 in oral squamous cell carcinoma / 华西口腔医学杂志

OBJECTIVE@#To analyze the expression and clinical significance of long non-coding RNA (lncRNA) actin filament-associated protein 1-antisense RNA1 (AFAP1-AS1) in oral squamous cell carcinoma (OSCC) and its effect on the biobehavior of OSCC cells.@*METHODS@#Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of lncRNA AFAP1-AS1 in the tumor tissue and matching adjacent normal tissue of OSCC patients (n=55), SCC25 cells, and normal oral keratinocyte lines (NOK) cells. The correlation between AFAP1-AS1 expression and the clinicopathological characteristics of OSCC patients was analyzed. The relationship between AFAP1-AS1 and prognosis was also studied with the Kaplan-Meier method. AFAP1-AS1 siRNA was transfected into the SCC25 cells. Cell counting kit-8 (CCK-8) and Trans-well were used to detect cell proliferation, invasion, and migration. The expression of the invasion-associated protein, AFAP1, and Rho GTPase family members, was detected by Western blot. In addition, the immunofluorescence of the cytoskeletal actin filament was observed.@*RESULTS@#The expression of AFAP1-AS1 was higher in the OSCC tissues than in the NOK cells, and the relative expression of AFAP1-AS1 was higher in the SCC25 cells than in the NOK cells (P<0.001). AFAP1-AS1 expression was associated with the degree of diffe-rentiation, TNM stage, lymphatic metastasis, and poor prognosis of OSCC (P<0.05). Patients with a high expression of AFAP1-AS1 had lower survival rates than those with a low expression of AFAP1-AS1 (P<0.05). After transfected by AFAP1-AS1 siRNA, the expression of AFAP1-AS1 was downregulated. The inhibition of AFAP1-AS1 expression consequently suppressed the proliferation, invasion, and migration of SCC25. Moreover, AFAP1-AS1 siRNA upregulated the expression levels of AFAP1, RhoA, Rac2, Rab10, RhoGDI, and Pfn1 but downregulated the expression of RhoC. Immunofluorescence showed that AFAP1-AS1 also reduced the formation of stress filaments in the cytoskeleton and affected the integrity of the actin fila-ment.@*CONCLUSIONS@#The expression of AFAP1-AS1 was high in the OSCC tissues and SCC25 cells and is associated with the development and prognosis of OSCC. The knockdown of AFAP1-AS1 might inhibit the proliferation and invasion of OSCC cells by regulating the integrity of the actin filament.

Progress of long non-coding RNA in colorectal cancer / 肿瘤研究与临床

Colorectal cancer is one of the most familiar malignant neoplasms,but the pathogenesis of colorectal cancer is not clear now.Recently the studies show that the long non-coding RNA(lncRNA)plays a regulatory role in various systemic tumors,including colorectal cancer.LncRNA is a non-coding RNA over 200 nucleotides.Five types of lncRNA including H19,colorectal cancer-associated transcript 1(CCAT1),HOX transcriptional antisense RNA(HOTAIR),lung adenocarcinoma-associated transcription factor 1(MALAT1),maternal imprinted expression gene 3(MEG3)are closely related to colorectal cancer.They are well correlated with the occurrence,development,clinical staging,overall survival,and prognosis of colorectal cancer.Therefore,lncRNA is expected to be a new marker for the diagnosis and evaluation of colorectal cancer in clinical work.

Antisense RNA: the new favorite in genetic research / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Antisense RNA molecule represents a unique type of DNA transcript that comprises 19-23 nucleotides and is complementary to mRNA. Antisense RNAs play the crucial role in regulating gene expression at multiple levels, such as at replication, transcription, and translation. In addition, artificial antisense RNAs can effectively regulate the expression of related genes in host cells. With the development of antisense RNA, investigating the functions of antisense RNAs has emerged as a hot research field. This review summarizes our current understanding of antisense RNAs, particularly of the formation of antisense RNAs and their mechanism of regulating the expression of their target genes. In addition, we detail the effects and applications of antisense RNAs in antivirus and anticancer treatments and in regulating the expression of related genes in plants and microorganisms. This review is intended to highlight the key role of antisense RNA in genetic research and guide new investigators to the study of antisense RNAs.

Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli

Background: Escherichia coli has been widely used as a host to clone and express heterologous genes. However, there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic and applied research on extremely toxic proteins. Results: In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA, ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli JM109(DE3). Conclusion: pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli.