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.

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.

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.

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 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.

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.

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.

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.

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.

Expression of vascular endothelial growth factor and endothelial growth factor receptor related Lnc RNA HOTAIR in hepatocellular carcinoma and their clinical significance / 中国综合临床

Objective To study the mechanism of the signaling pathway-related LncHOTAIR on the invasion and metastasis of hepatocellular carcinoma(HCC) and provide experimental basis for the treatment of liver cancer with target gene level.Methods Thirty specimens of liver cancer and tissue adjacent to carcinoma on liver and gallbladder surgery surgical resection were collected in the People′s Liberation Army General Hospital from September 2012 to June 2013.Expression of LncHOTAIR,vascular endothelial growth factor(VEGF) and endothelial growth factor receptor(VEGFR) in Hepatocellular Carcinoma and Paracancerous Tissues were detected by Real-time Quantitative Real-time PCR,the relationship between the pathological features and the pathological features of the patients with HCC was analyzed.Results The expression of LncRNA HOTAIR,VEGF and VEGFR were higher in HCC tissues than that in adjacent tissues,which was closely related to tumor size(P=0.512,0.003,0.008),TNM staging(P=0.094,0.001,0.014),portal vein thrombosis(P=0.065,0.046,0.031),invasion and metastasis (P=0.002,0.046,0.031) and 2-year recurrence((P=0.001,0.003,0.021).Conclusion Lnc HOTAIR-related VEGF signal pathways are closely related to the development of liver cancer,si-HOTAIR is expected to become a new focus in the clinical treatment of liver cancer in the future.

Regulatory effect of LncRNA HOTTIP in digestive system neoplasms / 国际外科学杂志

As the crucial member of noncoding RNA family,there is increasing evidence that long noncoding RNA has participated in numerous physiological and pathological processes of organisms.HOXA distal transcript antisense RNA is a long noncoding transcript located at the 5'tip of HOXA.According to research reports,the up-regulation of HOTTIP is closely associated with occurrence and progression of multiple digestive system cancers and promotes the carcinogenesis.The regulatory effect of long noncoding RNA HOXA distal transcript antisense in digestive system neoplasms will be summarized in this article.

Regulatory effect of LncRNA HOTTIP in digestive system neoplasms / 国际外科学杂志

As the crucial member of noncoding RNA family,there is increasing evidence that long noncoding RNA has participated in numerous physiological and pathological processes of organisms.HOXA distal transcript antisense RNA is a long noncoding transcript located at the 5'tip of HOXA.According to research reports,the up-regulation of HOTTIP is closely associated with occurrence and progression of multiple digestive system cancers and promotes the carcinogenesis.The regulatory effect of long noncoding RNA HOXA distal transcript antisense in digestive system neoplasms will be summarized in this article.

Long noncoding RNA HOTAIR promotes liver cancer HepG2 cell migra-tion and invasion by regulating PIK3R3 / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the effect of HOX transcript antisense RNA ( HOTAIR) on the migration and invasion abilities of liver carcinoma HepG 2 cells.METHODS:The expression of phosphoinositide-3-kinase regulatory sub-unit 3 (PIK3R3) in the liver cancer and normal liver tissues was detected by immunohistochemistry .The efficiency of gene silencing of HOTAIR or PIK3R3 by LV3-shHOTAIR or LV3-shPIK3R3 was determined by qPCR and Western blot .The mi-gration and invasion abilities of HepG 2 cells after silencing of HOTAIR and PIK3R3 were measured by wound healing assay and Transwell Matrigel invasion assay .The expression of miR-214 after silencing of HOTAIR and PIK3R3 was analyzed by qPCR.The expression of HOTAIR and PIK3R3 in the HepG2 cells was also evaluated by qPCR after transfected with miR-214 mimics or miR-214 inhibitor .Dual-luciferase reporter assay system was used to determine the regulatory effect of miR-214 on HOTAIR and PIK3R3 expression.RESULTS:PIK3R3 expression increased significantly in the liver cancer tissues compared with normal liver tissues .The abilities of invasion and metastasis of hepatocellular carcinoma were reduced after silencing of HOTAIR and PIK3R3.miR-214 expression was increased when silencing of HOTAIR and PIK3R3 was per-formed.HOTAIR and PIK3R3 expression was reduced after transfection with miR-214 mimics.HOTAIR and PIK3R3 ex-pression was increased after transfection with miR-214 inhibitor.The results of dual-luciferase reporter assay test showed that miR-214 directly regulated HOTAIR and PIK3R3 transcription activity .CONCLUSION: HOTAIR regulates the ex-pression of PIK3R3 through miR-214, thus promoting the migration and invasion abilities in the liver cancer cells .

Construction of recombinant vectors carrying antisense RNA to dualˉtarget BAGˉ1 and Bclˉ2 and its effect on proliferation of SGCˉ7901 cell / 国际检验医学杂志

Objective To construct the recombinant co-expression vector carrying antisense RNA to dual-target BAG-1 and Bcl-2 genes and recombinant single expression vector carrying antisense RNA to target BAG-1 and Bcl-2 gene respectively,then to pre-liminarily investigate their effect on the proliferation of gastric cancer cell SGC-7901 in order to lay the foundation for further study the effect of this recombinant vector on the tumor cells.Methods RT-PCR was used to amplify the full length of BAG-1 and Bcl-2 cDNA from total RNA of gastric cancer cell line SGC-7901.The BAG-1 cDNA fragment and the Bcl-2 cDNA fragment were insert-ed into pMD18-T simple vector respectively.The pMD18-T-BAG-1 was digested with BamH Ⅰ and Cla Ⅰ and the pMD18-T-Bcl-2 was digested with EcoR Ⅰ and Nhe Ⅰ.Then the BAG-1 cDNA fragment and the Bcl-2 cDNA fragment were inserted into the mcs1 and mcs2 of the eukaryotic co-expression vector pVITRO2 in the antisense orientation respectively.The construction of the single expression vector pVITRO2-AsBAG-1 and pVITRO2-AsBcl-2 was confirmed by restriction endonuclease treatment and se-quence identification.Then the Bcl-2 cDNA fragment was inserted into the mcs2 of the recombinant vector pVITRO2-AsBAG-1 in the antisense orientation to construct the co-expression vector pVITRO2-AsBAG-1-Bcl-2,and the recombinant vector was also iden-tified by restriction endonucleases digestion and sequence identification.Then the recombinant vector was transfected into SGC-7901 cell respectively.The proliferation of the cell was determined by the MTT assay.The level change of BAG-1 and Bcl-2 mRNA in SGC-7901 cell was detected by the semi quantitative RT-PCR and the change situation of cell cycle was detected by the flow cytom-etry(FCM).Results The restriction endonucleases digestion and sequencing identification indicated that the eukaryotic co-expres-sion vector pVITRO2-AsBAG-1-Bcl-2 and the single gene expression vector pVITRO2-AsBAG-1 and pVITRO2-AsBcl-2 were con-structed successfully.The MTT assay method demonstrated that compared with the control group,the recombinant vector could in-hibit the proliferation of the cells in time dependent manner,and the inhibiting effect was most notably in the 72 h transfection group(P 0. 05);the FCM detection results showed that the apoptosis rate of the recombinant vector groups was significantly higher than that of the control group and the pVITRO2 group with statistical difference(P <0.01),and the co-expression vector group was more nota-bly than single expression vector groups(P <0.01).In addition,the effect of the co-expression recombinant vector group was more significant than that of the single expression co-expression vector(P <0.01 ),the apoptosis rate was increased from 0.57% to 15.75%.Conclusion The co-expression recombinant vector pVITRO2-AsBAG-1-Bcl-2 and single expression vector pVITRO2-As-BAG-1 and pVITRO2-AsBcl-2 are successfully constructed and they can inhibit the proliferation of SGC-7901 cell and induce cell apoptosis,moreover the pVITRO2-AsBAG-1-Bcl-2 vector is most notably.

Expression of long non-coding RNA AFAP1-AS1 in human digestive system cancer and its clinical significance / 肿瘤

Objective: To investigate the expressions of actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1), a long non-coding RNA (IncRNA) in four common human digestive system cancers including esophageal cancer, gastric cancer, liver cancer and colorectal cancer, and their clinical significance. Methods: The expression of AFAP1-AS1 was preliminarily detected in several digestive system tumor tissues and their corresponding adjacent normal tissues from 82 cases by multi-tumor tissue microarrays. These 82 patients included 11 with esophageal cancer, 11 with gastric cancer, 26 with liver cancer, and 34 with colorectal cancer. The expression of AFAP1-AS1 which had significant difference in liver tumor tissues was further tested by in situ hybridization (additional 70 cases) and real-time fluorescence quantitative PCR (additional 30 cases). The relationship between the expression of AFAP1-AS1 and the clinicopathological features was analyzed. The role of AFAP1-AS1 in tumor lymph node metastasis was assessed. Results: The expression of AFAP1-AS1 in liver cancer was significantly lower than that in its corresponding adjacent normal liver tissue (P 0.05). Further test also revealed that the expression of AFAP1-AS1 was significantly down-regulated in liver cancer, and this effect was associated with clinical stage and lymph node metastasis (P < 0.05). The sensitivity, specificity, coincidence rate, positive predictive value and negative predictive value of AFAP1-AS1 serving as a molecular marker of metastasis were 68.75%, 65.00%, 65.63%, 28.21% and 91.23%, respectively. Conclusion: The expression of AFAP1-AS1 may play a role in the pathogenesis and progression of liver cancer and esophageal cancer, but this effect is different between these two cancer types. It is suggested that AFAP1-AS1 may become a noval molecular marker for clinical diagnosis of liver cancer. Copyright© 2014 by TUMOR.