Expression relationship and significance of NEAT1 and miR-27a-3p in serum and cerebrospinal fluid of patients with Alzheimer disease / 北京大学学报(医学版)

Objective:To explore the expression relationship and significance of long chain non-coding RNA nuclear-enriched abundant transcript 1(LncRNA NEAT1)and miR-27a-3p in serum and cerebro-spinal fluid of patients with Alzheimer disease(AD).Methods:Sixty-six AD patients received by the department of neurology of our hospital from October 2019 to September 2021 were gathered,according to the clinical dementia rating scale score,they were grouped into mild group(≤ 1 point,n=41)and moderate-to-severe group(＞1 point,n=25).Another 66 cases of serum and cerebrospinal fluid sam-ples from outpatient physical examination personnel were regarded as the control group.The general infor-mation on all subjects was recorded and cognition was assessed;real-time quantitative PCR was performed to measure the expression levels of miR-27a-3p and NEAT1 in serum and cerebrospinal fluid;enzyme-linked immunosorbent assay was performed to measure the protein levels of β-amyloid precursor protein cleaving enzyme 1(BACE1),β-amyloid(Aβ)40 and Aβ42 in cerebrospinal fluid;Spearman's method was performed to analyze the correlation of serum miR-27a-3p and NEAT1 levels with mini-mental state examination(MMSE)and montreal cognitive assessment(MoCA)scores;Pearson method was per-formed to analyze the correlation between serum miR-27a-3p and NEAT1 levels and Aβ deposition standard uptake value ratio(SUVR)and cerebrospinal fluid miR-27a-3p,NEAT1,BACE1,Aβ42 and Aβ40 levels.Results:The MMSE score[21(17,25),9(7,11)vs.27(21,34)],MoCA score[17(12,21),10(7,13)vs.27(21,31)],serum miR-27a-3p level(0.55±0.13,0.46±0.06 vs.0.97± 0.22),cerebrospinal fluid miR-27a-3p(0.48±0.10,0.35±0.10 vs.1.03±0.31),Aβ42 levels[(303.55±36.77)ng/L,(231.45±34.14)ng/L vs.(499.99±53.63)ng/L]and Aβ42/Aβ40 ra-tio(0.030±0.008,0.022±0.007 vs.0.048±0.010)of AD patients in mild group and moderate-to-severe group were all lower than those in the control group,and the moderate-to-severe group were lower than the mild group(all P＜0.05);the serum NEAT1 level(2.31±0.64,3.13±0.76 vs.1.05± 0.20),SUVR(1.50±0.29,1.76±0.52 vs.0.74±0.15),and cerebrospinal fluid NEAT1(3.51± 1.24,4.30±1.65 vs.1.01±0.23)and B ACE 1 levels[(55.78±5.98)μg/L,(72.32±16.08)μg/L vs.(21.39±3.73)μg/L]were higher than those in the control group,and the moderate-to-se-vere group were higher than the mild group(all P＜0.05).Serum NEAT1 level in AD patients was posi-tively correlated with SUVR,cerebrospinal fluid NEAT1 and BACE1(r=0.350,0.606,0.341,P＜0.05),and negatively correlated with MMSE score and MoCA score(r=-0.473,-0.482,all P＜0.05);serum miR-27a-3p level was positively correlated with cerebrospinal fluid miR-27a-3p level,MMSE score and MoCA score(r=0.695,0.424,0.412,all P＜0.05),and negatively correlated with SUVR and cerebrospinal fluid BACE1 level(r=-0.521,-0.447,all P＜0.05).Conclusion:The expression trends of NEAT1 and miR-27a-3p in the serum and cerebrospinal fluid of AD patients are con-sistent,the level of NEAT1 is increased,and the level of miR-27a-3p is decreased.The levels of the two are negatively correlated,which is related to the degree of Aβ deposition in the brain of AD patients and is involved in the progression of AD.

Effect of lncRNA NEAT1 on radiosensitivity in breast cancer cells by regulating miR-149-5p/GPT2 axis / 中华放射肿瘤学杂志

Objective:To evaluate the effect of long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) on the radiosensitivity in breast cancer cells by regulating the miR-149-5p/ glutamic pyruvic transaminase 2 (GPT2) axis.Methods:Real-time reverse transcription PCR (RT-qPCR) was used to detect NEAT1, miR-149-5p and glutamic pyruvic transaminase 2 (GPT2) mRNA levels in human breast cells MCF-10A, and human breast cancer cells MCF-7, MDA-MB-231 and MDA-MB-468, respectively. MCF-7 cells were divided into 0, 2, 4, 6 and 8 Gy irradiation groups. MCF-7 cells were divided into NEAT1 knockdown (si-NEAT1) group and control (si-NC) group, NEAT1 knockdown +miR-149-5p knockdown (si-NEAT1+anti-miR-149-5p) group and control (si-NEAT1+anti-miR-NC) group, NEAT1 knockdown + GPT2 overexpression (si-NEAT1+GPT2) group and control (si-NEAT1+NC) group. On the basis of the above grouping, irradiate each group of cells with 4 Gy radiation for 2 h, denoted as IR+si-NEAT1, IR+si-NC, IR+si-NEAT1+anti-miR-149-5p, IR+si-NEAT1+anti-miR-NC, IR+si-NEAT1+GPT2, IR+si-NEAT1+NC groups. Subsequently, MCF-7 cells were irradiated at a dose of 4 Gy and divided into the IR+si-NEAT1, IR+si-NC, IR+si-NEAT1+anti-miR-149-5p, IR+si-NEAT1+anti-miR-NC, IR+si-NEAT1+GPT2 and IR+si-NEAT1+NC groups. RT-qPCR was used to detect NEAT1, miR-149-5p, GPT2 mRNA levels in cells. Colony formation assay was used to detect cell radiosensitivity. CCK-8 assay was adopted to detect cell proliferation ability. The binding sites of NEAT1 and miR-149-5p were predicted by StarBase database. The binding sites of miR-149-5p and GPT2 were predicted by Targetscan database, and validated by dual luciferase assay. Single factor ANOVA was used for inter-group comparisons. LSD- t test was used for pairwise comparison. Results:Compared with MCF-10A cells, NEAT1 and GPT2 mRNA levels in cell lines were up-regulated, whereas miR-149-5p level was down-regulated (all P<0.05). Compared with the 0 Gy dose group, NEAT1 and GPT2 mRNA levels were down-regulated, while miR-149-5p levels were up-regulated in the 2, 4, 6, and 8 Gy dose groups (all P<0.05). Knockdown of NEAT1 expression or radiation alone could enhance cell radiosensitivity, and reduce cell proliferation ability (all P<0.05). Simultaneous radiation treatment with knockdown of NEAT1 expression could strengthen the above effects upon cells (all P<0.05). Knockdown of miR-149-5p expression or overexpression of GPT2 could partially reverse the aforementioned effects of knockdown of NEAT1 expression (all P<0.05). Conclusion:Knockdown of NEAT1 expression enhances breast cancer cell radiosensitivity, and reduces cell proliferation ability by regulating the miR-149-5p/GPT2 signal axis.

Effects of lncRNA NEAT1 silencing on microglial polarization via IL-10/STAT3 signaling pathway on cerebral ischemia reperfusion injury in rats / 中风与神经疾病杂志

@#Objective To investigate the effect of long noncoding RNA nuclear enriched abundant transcript 1 (lncRNA NEAT1) silencing on cerebral ischemia reperfusion injury (CIRI) in rats by regulating microglia polarization through interleukin (IL)-10/signal transducer and activator of transcription 3 (STAT3) signaling pathway.Methods SD rats were randomly divided into sham operation group (Sham group),CIRI group,CIRI+si-NC group,CIRI+si-NEAT1 group,each group of 24 rats.Except for the Sham group,the rats in the other groups were used to construct the CIRI model by thread occlusion method.After modeling,rats in each group were given corresponding treatments for 7 days.Neurological deficits of rats were scored; brain histopathological changes were observed; the percentage of cerebral infarction volume,the number of M1 and M2 polarization marker positive (Iba1+CD86+,Iba1+CD206+) cells in the positive cells of ionic calcic junction protein molecule 1 (Iba1+),IL-1β,tumor necrosis factor-α (TNF-α),IL-4,IL-10,lncRNA NEAT1,inducible nitric oxide synthase (iNOS),arginase-1 (Arg-1),IL-10 mRNA,IL-10,STAT3,phosphorylated STAT3 (p-STAT3) protein levels in rat brain tissue were detected.Results Compared with Sham group,the CIRI group showed severe pathological damage of brain tissue in rats,neurological deficit score,percentage of cerebral infarction volume,number of Iba1+CD86+,Iba1+CD206+ positive cells,CD86/CD206 ratio,IL-1β,TNF-α,IL-4,IL-10 levels,lncRNA NEAT1,iNOS,Arg-1,IL-10 mRNA levels,IL-10 and p-STAT3/STAT3 protein levels in brain tissue were significantly increased (P<0.05);compared with CIRI group and CIRI+si-NC group,CIRI+si-NEAT1 group had less pathological damage of brain tissue in rats,neurological deficit score,percentage of cerebral infarction volume,number of Iba1+CD86+ positive cells,CD86/CD206 ratio,IL-1β and TNF-α levels,lncRNA NEAT1,iNOS mRNA levels in brain tissue were significantly decreased (P<0.05),the number of Iba1+CD206+cells,IL-4,IL-10 levels,Arg-1,IL-10 mRNA,IL-10 and p-STAT3/STAT3 protein levels were significantly increased (P<0.05). Conclusion Silencing lncRNA NEAT1 may regulate microglial polarization by activating IL-10/STAT3 signaling pathway,thereby ameliorating brain tissue injury in CIRI rats.

LncRNA MALAT1 promotes hepatocellular carcinoma proliferation and invasion by regulating NEAT1 related exosomes secretion / 中华肝胆外科杂志

Objective:To investigate the correlations between expression of long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), nuclear-enriched abundant transcript 1 (NEAT1) and their functions on exosome secretion, proliferation and invasion in hepatocellular carcinoma (HCC).Methods:We used small interfering RNA of MALAT1 (si-MALAT1) to knockdown MALAT1 in HuH-7. At the meanwhile, cells which were transfected with si-NC were used as the negative control group. Expression of NEAT1, cell proliferation and invasion function were detected these two groups. HuH-7 cells were transfected with lentivirus NEAT1 over expressing vector (lv-NEAT1) or negative control (lv-control). Expression of exosomes secretion related genes were analyzed between lv-NEAT1 and lv-control groups. Cells of lv-NEAT1 were knockdown MALAT1 expression using si-MALAT1, which could be si-MALAT1+ lv-NEAT1 group. exosomes secretion was detected in si-NC, si-MALAT1 and si-MALAT1+ lv-NEAT1 group. We treated cells (si-MALAT1 group) with exosomes from cells with lv-NEAT1 or lv-control to divide cells as si-MALAT1+ exosomes of lv-NEAT1 cells and si-MALAT1+ exosomes of lv-control groups. Cell proliferation and invasion of cells were detected in two groups.Results:Low expression of NEAT1 were found in MALAT1 knockdown cells compared with si-NC group [(0.72±0.02) vs. (0.98±0.01), P<0.05]. Cells with MALAT1 knockdown shown diminished proliferation [(0.66±0.03) vs. (0.98±0.04), P<0.05)] and invasion [(88.33±7.26) vs. (147.70±13.62), P<0.05)]. Compared with si-NC group, CD9 and CD63 expression were decreased in exosomes of si-MALAT1 group. Compared with si-MALAT1 group, CD9 and CD63 expression was increased in exosomes of si-MALAT1+ lv-NEAT1 group. Compared with si-MALAT1+ exosomes of lv-control group, proliferation [(0.97±0.03) vs. (0.74±0.05), P<0.05)] and invasion [ (132.70±7.36) vs. (98.33±6.01), P<0.05) ] were increased in si-MALAT1+ exosomes of lv-NEAT1 group. Exosomes related genes expression including HSPA8 (5.53±0.31), SLC3A2 (0.32±0.07) and SLC7A5 (0.77±0.45) were changed in lv-NEAT1 group compared with lv-control group [(0.98±0.15), P<0.05]. Conclusion:MALAT1 induced exosomes secretion by NEAT1 and exosomes related genes regulation. This regulation might be related with increased proliferation and invasion function in HCC cells with MALAT1 and NEAT1 abnormal expression.

Effect of long non-coding RNA nuclear-enriched abundant transcript on hypoxia-reoxygenation rat astrocyte injury by targeting microRNA-761 / 解剖学报

Objective To investigate the effect of long non-coding RNA (IncRNA) nuclear-enriched abundant transcript 1 (NEATl) on hypoxia-reoxygenation (H/R) glial astrocyte injury, and to explore whether the mechanism was related to the regulation of micro RNA (miR)-761. Methods Rat cortical astrocytes were cultured to construct a H/R injury model. Astrocytes were divided into control group, model group, model+ small interfering RNA negative control (si-NC) group, model+ si-NEATl group, model+ miR-NC group, model + miR-761 group, model + si-NEATl + anti-miR-NC group, model+si-NEATl+anti-miR-761 group. Expression of NEATl and miR-761 were detected by Real-time PCR. The experiment was repeated 3 times. The content of malonaldefryde (MDA), and the activity of superoxide dismutase (SOD) and catalase (CAT) were detected by kits. Dual luciferase reporter experiment and Real-time PCR were used to analyze the targeting relationship between NEATl and miR-761. The experiment was repeated 3 times. Results Compared with the control group, the cell apoptosis rate and MDA content of the model group increased significantly, SOD and CAT activities decreased significantly, NEATl expression increased significantly, and miR-761 expression decreased significantly (P< 0. 05). Compared with the model+si-NC group, the apoptosis rate and MDA content of the model+si-NEATl group reduced significantly, and SOD and CAT activities increased significantly (P < 0 . 0 5) . Compared with the model + miR-NC group, the apoptosis rate and MDA content of the model + miR-761 group reduced significantly, and SOD and CAT activities increased significantly (P < 0 . 0 5) . MiR-761 was the target gene of N E A T l, and NEATl negatively regulated miR-761 expression. Compared with the model+si-NEATl+anti-miR-NC group, the apoptosis rate and MDA content of the model+siNEAT1+anti-miR-761 group increased significantly, and SOD and CAT activities decreased significantly (P < 0 . 0 5) . Conclusion Interference with NEATl expression can protect astrocytes from H / R injury by up-regulating miR-761.

Neuroprotective effect of long noncoding RNA-nuclear-enriched abundant transcript 1 on traumatic brain injury in mice and its possible mechanism / 中华创伤杂志

Objective:To explore the impact of the expression of long noncoding RNA-nuclear-enriched abundant transcript 1 (NEAT1) on neurological function and neuronal apoptosis after traumatic brain injury (TBI) in mice and the possible mechanism.Methods:According to the random number table, 90 C57BL/6J mice were divided into sham group, blank control group, empty virus group 1, empty virus group 2, NEAT1 over-expression group and NEAT1 knockdown group, with 15 mice per group. The traumatic brain injury (TBI) was simulated by controlled cortical injury (CCI) model, and NEAT1 was regulated by intracerebroventricular injection with recombinant adenovirus. The neurological severity score (NSS) and Morris water maze test were used to evaluate the neurological function in blank control group, NEAT1 over-expression group and NEAT1 knockdown group within 1 week and 14-21 days after injury. The Western blot was used to observe the expressions of P53-induced protein with a death domain 1 (Pidd1), caspase-2, caspase-9 and caspase-3 in blank control group at 6 hour and 1, 3, 7 days after injury. The TUNEL method and immunofluorescence were used to observe the neurological apoptosis and expression of Pidd1 in blank control group, NEAT1 over-expression group and NEAT1 knockdown group at 3 days after injury. The Western blot analysis was used to detect protein expressions of Pidd1, caspase-2, cytochrome c (Cyt c) and caspase-3 in sham group, blank control group, empty virus groups, NEAT1 over-expression group and NEAT1 knockdown group at 3 days after injury.Results:The NSS was significantly reduced in NEAT1 over-expression group [(3.5±0.7)points], and was significantly increased in NEAT1 knockdown group [(5.0±1.5)points]at day 1 after injury, when compared with blank control group [(4.9±1.0)points]( P<0.01). The Morris water maze test showed that the time to find platform was decreased in NEAT1 over-expression group[(10.9±2.8)seconds], and was prolonged in NEAT1 knockdown group [(30.7±6.2)seconds] at day 19 after injury ( P<0.05 or 0.01), when compared with blank control group [(20.1±5.6)seconds]. The Western blot analysis showed that the expressions of Pidd1, caspase-2, caspase-9 and caspase-3 had significant increase at day 3 after injury ( P<0.01). The TUNEL test showed that the apoptosis rate of neurons was significantly decreased in NEAT1 over-expression group [(18.0±2.7)%], and the apoptosis rate was significantly increased in NEAT1 knockdown group [(63.0±8.6)%] at day 3 after injury ( P<0.01). Immunofluorescence showed that the expression of Pidd1 protein in cytoplasm of the neurons was decreased in NEAT1 over-expression group [(22.7±2.2)%]( P<0.01), and was increased in NEAT1 knockdown group [(72.7±7.0)%]( P<0.01) at day 3 after injury, when compared with blank control group. The Western blot analysis showed that the expressions of Pidd1, capsase-2, Cyt c and caspase-3 were significantly reduced in NEAT1 over-expression group (0.5±0.0, 0.3±0.0, 0.5±0.0, 0.4±0.0) at day 3 after injury, when compared with blank control group. However, the results were opposite in NEAT1 knockdown group. Conclusion:After TBI, the NEAT1 can reduce the activation of caspase-3 through the Pidd1-caspase-2-Cyt c pathway after TBI, regulate neuronal apoptosis, and ultimately play a protective role in neurological function.

lncRNA NEAT1 promotes cell proliferation of lung adenocarcinoma PC-9 cells through inhibiting DNAdamage / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effects of long non-coding RNA nuclear enriched abundant transcript 1 (lncRNA NEAT1) on the proliferation of lung adenocarcinoma PC-9 cells and to explore its mechanism. Methods: qPCR was used to detect the expression level of lncRNA NEAT1 in human lung adenocarcinoma PC-9 cells and human embryonic lung diploid 2BS cells. The sequence of small interfering RNA(siRNA) targeting lncRNANEAT1 gene was designed and synthesized, and then transfected into PC-9 cells by liposome method. The expression level of NEAT1 in PC-9 cells before and after transfection was detected by qPCR. MTT and flow cytometry were used to detect the effect of lncRNANEAT1 knockdown on proliferation and cell cycle distribution of PC-9 cells, respectively. WB assay was used to detect the expressions of DNA damage-related proteins, namely, double-stranded DNA breaks (DSBs) biomarker γ-H2AX and ataxia-telangiectasia mutated (ATM), before and after transfection. Results: Compared with 2BS cells, lncRNA NEAT1 was highly expressed in PC-9 cells (P<0.05). The PC-9 cells with lncRNA NEAT1 knock-down were successfully established. After being transfected with siRNA for 12 h, the proliferation of PC-9 cells in siNEAT1 group and siNEAT2 group significantly decreased as compared with the blank control group and the empty transfection group (P<0.05). In the interference groups, cell cycle was arrested in G1 phase ([88.97±2.64]%, [88.15±1.48]% vs [84.5±1.72]%, P<0.05) and G2/M phase ([8.35±2.02]%, [8.11± 1.36]% vs [4.28±1.28]%, P<0.05). The expression levels of DNA damage-related proteinsATM and γ-H2AX in the interference groups were significantly increased (all P<0.05). Conclusion: lncRNA NEAT1 is highly expressed in lung adenocarcinoma PC-9 cells. lncRNA NEAT1 inhibits DNA damage and causes cell cycle at G1/M phase switch, and thus promotes the proliferation of lung adenocarcinoma cells.

Biological Function and Mechanism of Long Noncoding RNAs Nuclear-Enriched Abundant Transcript 1 in Development of Cervical Cancer / 中华医学杂志(英文版)

Background@#Accumulating documents have demonstrated that long noncoding RNAs (lncRNAs) play critical roles in tumorigenesis. As an lncRNA, nuclear-enriched abundant transcript 1 (NEAT1) has been identified to be involved in the progression of many types of cancers. However, the biological function of NEAT1 in cervical cancer is not fully investigated. The aim of this study was to disclose the specific biological function of lncRNA NEAT1 in cervical cancer progression.@*Methods@#Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to identify the expression of lncRNA NEAT1 in the cervical cancer tissues and cell lines. All cervical cancer samples used in this study were collected from the Affiliated Suzhou Hospital of Nanjing Medical University between September 2012 and September 2017. The correlation between NEAT1 expression and the overall survival rate of cervical cancer patients was analyzed by Kaplan-Meier analysis. The effects of NEAT1 knockdown or overexpression on cell proliferation were tested by performing MTT assays and colony formation assays. Transwell assays were conducted to detect the migratory ability of cervical cancer cells, in which NEAT1 was silenced or overexpressed. Western blotting was utilized to validate whether NEAT1 promotes cervical cancer progression through activating PI3K-Akt signaling pathway.@*Results@#High expression of NEAT1 predicted poor prognosis of cervical cancer patients (χ = 0.735, P = 0.005). Knockdown of NEAT1 decreased the number of colonies in CaSki cell from 136.667 ± 13.503 to 71.667 ± 7.506 (t = -18.76, P = 0.003) and decreased the number of colonies in HeLa cell from 128.667 ± 13.317 to 65.667 ± 7.024 (t = -5.54, P = 0.031). However, overexpression of NEAT1 increased the number of colonies in SiHa cell from 84.667 ± 12.014 to 150.667 ± 18.037 (t = 7.27, P = 0.018). Knockdown of NEAT1 decreased the migratory number of CaSki cell from 100.333 ± 9.866 to 58.333 ± 5.859 (t = -8.08, P = 0.015) and reduced the migratory number in HeLa cell from 123.667 ± 12.097 to 67.667 ± 7.095 (t = -6.03, P = 0.026). Overexpression of NEAT1 increased the migratory number of SiHa cell from 127.333 ± 16.042 to 231.333 ± 31.786 (t = 4.92, P = 0.039).@*Conclusion@#NEAT1 may exert oncogenic function in cervical cancer and serve as a novel therapeutic target for cervical cancer.

MicroRNA-449a inhibits cell growth in lung cancer and regulates long noncoding RNA nuclear enriched abundant transcript 1.

OBJECTIVE: Lung cancer has become the primary cause of cancer‑related death now. New therapies targeting the molecular regulatory machinery were required imperatively. MicroRNAs and long noncoding RNAs can respectively or cooperatively function as oncogenes or tumor suppressor genes in human cancers. The present study identified that miR‑449a was down‑regulated in tissue of human lung cancer. In this study, we aimed to investigate the function of miR‑449a in NL9980 and L9981 lung carcinoma cells lines and the relationship with lncRNA nuclear enriched abundant transcript 1 (NEAT1). MATERIALS AND METHODS: miR‑449a was profiled in several lung carcinoma cell lines by quantitative reverse transcription‑polymerase chain reaction RT‑PCR. We analyzed the effects of miR‑449a overexpression on proliferation, apoptosis and cell cycle in L9981 cells. The regulatory relationship between miR‑449a and NEAT1 was predicted in silico and further studied by miR‑449a inhibitor and mimics assay. RESULTS: miR‑449a was expressed in four cell lines, which we selected, however miR‑449a was in high level in NL9980 and in low level in L9981 (P < 0.05). When the miR‑449a was the overexpression in L9981 cells, the cell growth was suppressed, and the apoptosis cells were promoted compared with the control group (P < 0.05). The G1/G0 became longer and S, G2/M became shorter (P < 0.05) by miR‑449a overexpression. Further study of the interaction between miR‑449a and NEAT1 show that NEAT1 was up‑regulated when cells were transfected with miR‑449a inhibitor, and NEAT1 was down‑regulated when cells transfected with miR‑449a mimics. CONCLUSIONS: Our data indicate that miR‑449a may function as a suppressor of lung cancer, and affects the expression of NEAT1 in lung cancer cells.