Significance of leukocyte-specific transcript 1 levels in nasal mucosal tissue to predict recurrence of nasal polyps

Abstract Objective: Chronic Rhinosinusitis with Polyps (CRSwNP) is characterized by high heterogeneity and postoperative recurrence rate. This study aims to explore the clinical significance of tissue Leukocyte-Specific Transcript 1 (LST1) in predicting CRSwNP recurrence. Methods: We enrolled 62 CRSwNP patients including 30 primary CRSwNP and 32 recurrent CRSwNP patients, and 40 Healthy Controls (HC). Tissue samples were collected. Tissue LST1 expression was assessed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Western Blotting (WB) and Immunofluorescence (IF) staining. The predictive values of LST1 expression for CRSwNP postoperative recurrence were assessed through the Receiver Operating Characteristic (ROC) curves. Results: The tissue levels of LST1 were significantly increased in the CRSwNP group than the HC group, especially in the recurrent group, and the elevated LST1 mRNA levels were positively correlated with the peripheral eosinophil percentages, tissue eosinophil counts and percentages. IF staining results showed that the LST1 protein levels were higher in CRSwNP patients, especially in the recurrent patients than in the HC group. ROC curves highlighted that tissue LST1 levels were associated with recurrent CRSwNP and exhibited a higher predictive ability for postoperative CRSwNP recurrence. Conclusion: This was the first report suggesting that LST1 expression was upregulated and associated with mucosal eosinophil infiltration and CRSwNP recurrence. Tissue LST1 could be a promising biomarker for predicting postoperative recurrence in CRwNP patients.

Effect of LncRNA LUCAT1 targeting miR-502-5p on the biological behavior of human retinoblastoma cells / 中华实验眼科杂志

Objective:To investigate the effects of long noncoding RNA (LncRNA) lung cancer associated transcript 1 (LUCAT1) targeting microRNA (miR)-502-5p on the proliferation, migration and invasion of human retinoblastoma (RB) cells.Methods:RB tissue samples were collected from 27 RB patients who underwent eyeball enucleation in Henan Eye Hospital from May 2019 to January 2021.Another 27 normal retinal tissue specimens were collected from 12 patients with eyeball rupture, 7 with eyeball atrophy, and 8 with eyeball penetrating injury combined with pigment film incarceration who underwent the eyeball enucleation in Henan Eye Hospital during the same period.The expressions of LncRNA LUCAT1 and miR-502-5p in RB tissues, cell lines (Y-79, WERI-Rb-1, HXo-RB44) and human retinal epithelial cells (ARPE-19) were detected by real-time quantitative PCR.Y-79 RB cell was divided into control group, small interfering RNA (si)-LncRNA LUCAT1 group, si-control (con) group, pcDNA group, pcDNA-LncRNA LUCAT1 group, miR-con group, miR-502-5p group, si-LncRNA LUCAT1+ anti-miR-con group and si-LncRNA LUCAT1+ anti-miR-502-5p group, and cells in different groups were transfected with corresponding reagents.The expressions of MMP2 and MMP9 proteins were detected by Western blot.Cell proliferation activity was assayed by cell counting kit 8.Cell proliferation capability was detected by colony formation assay.Cell migration and invasion ability were determined by Transwell assay.The targeting regulation of LncRNA LUCAT1 against miR-502-5p was confirmed by dual luciferase reporter assay and real-time quantitative PCR.The study protocol was approved by the Ethics Committee of Henan Eye Hospital (No.HNEECKY-2021[32]). Written informed consent was obtained from guardians of subjects.Results:LncRNA LUCAT1 expression level in RB tissue was 2.73±0.34, which was significantly higher than 1.00±0.15 in normal retinal tissue ( t=24.190, P<0.001). The miR-502-5p expression level in RB tissues was 0.42±0.06, which was significantly lower than 1.00±0.13 in normal retinal tissue ( t=21.049, P<0.001). LncRNA LUCAT1 expression level was significantly higher and the miR-502-5p expression level was significantly lower in human RB cell lines Y-79, WERI-Rb-1 and HXO-RB44 than those in ARPE-19 cells, with statistically significant differences (all at P<0.05). The LncRNA LUCAT1 expression, the relative expressions of MMP2 and MMP9 proteins, the absorbance ( A) value, and the number of proliferated, migrating and invading Y-79 cells in si-LncRNA LUCAT1 group were significantly reduced in comparison with control group, and the differences were statistically significant (all at P<0.05). The miR-502-5p expression level was higher, and the relative expression levels of MMP2 and MMP9, A value, as well as the number of proliferated, migrating and invading Y-79 cells were lower in miR-502-5p group than in miR-con group, showing statistically significant differences ( t=20.274, 14.884, 14.181, 12.692, 17.749, 20.889, 21.913; all at P<0.001). The miR-502-5p expression level was lower and the relative expression levels of MMP2 and MMP9, A value as well as the number of proliferated, migrating and invading Y-79 cells were higher in si-LncRNA LUCAT1+ anti-miR-502-5p group than in si-LncRNA LUCAT1+ anti-miR-con group, showing statistically significant differences ( t=14.097, 15.839, 15.757, 11.860, 16.235, 16.565, 16.487; all at P<0.001). When co-transfected with LncRNA LUCAT1-wild type, the relative luciferase activity of miR-502-5p group was lower than that of miR-con group, and the difference was statistically significant ( t=16.379, P<0.001). The LncRNA LUCAT1 expression level was higher and the miR-502-5p expression level was lower in pcDNA-LncRNA LUCAT1 group than in pcDNA group, and the differences were statistically significant (both at P<0.05). The LncRNA LUCAT1 expression level was lower and the miR-502-5p expression level was higher in si-LncRNA LUCAT1 group than in si-con group, and the differences were statistically significant (both at P<0.05). Conclusions:Inhibition of LncRNA LUCAT1 can attenuate the proliferation, migration and invasion ability of human RB cells by the targeting up-regulation of miR-502-5p.

Study on RHD and RHCE gene mRNA based on three generation sequencing technology / 中国输血杂志

【Objective】 To establish RH gene mRNA sequencing method based on nanopores sequencing and to explore the RHD and RHCE mRNA transcripts in D positive and Del individuals. 【Methods】 From March 2021 to May 2022, 5 RhD positive samples and 5 Del samples screened out by hospitals in Chengdu were sent to our laboratory for futher examination. The erythrocytes and buff coat were isolated, then DNA and RNA were extracted.All 10 samples were genotyped by PCR-SSP. After the mRNA was reversely transcribed into cDNA, the full-length mRNA of RHD and RHCE genes were simultaneously amplified by a pair of primers. Sanger sequencing and third-generation sequencing technology based on Nanopore were used to sequence the amplified products, and the types and expressions of different splices of RHD and RHCE gene mRNA transcripts were analyzed. 【Results】 The method established in this study can simultaneously amplify the full length transcripts of RHD and RHCE. Ten different RHD gene mRNA transcripts and nine RHCE gene mRNA transcripts were detected in 10 samples. RHD full-length transcript (RHD-201) can be detected in RhD Del type, but the expression amount was significantly lower than that in RhD positive samples. The expression amount of transcript RHD-207 (Del789) in Del samples was significantly higher than that in RhD positive samples. The transcript RHD-208 (Del8910+ 213) was only detected in RhD Del type individuals, and no significant difference was found between other RHD transcripts and all RHCE transcripts in the two phenotypes. 【Conclusion】 In this study, an analytical method for sequencing full-length transcript isomers of RHD and RHCE mRNA via the third generation was successfully established, and complex alternative splicing patterns were found in RHD and RHCE genes, providing a new method for the study of alternative splicing of blood group gene variants mRNA.

Influence of GLCCI1 gene rs37973 polymorphism on the efficacy of ICSs in the treatment of asthma-chronic obstructive pulmonary disease overlap / 中国药房

OBJECTIVE To investigate the association between the functional GLCCI1 gene rs37973 polymorphism and inhaled corticosteroids （ICSs） response in patients with asthma-chronic obstructive pulmonary disease overlap （ACO）. METHODS Totally 173 newly diagnosed ACO patients were recruited from Shanghai Pudong New Area People’s Hospital during April 1st， 2019 to December 31st， 2020. All patients were treated with Salmeterol fluticasone inhalation powder， twice a day， for 24 weeks. The genotype of rs37973 locus was determined， and lung function indicators [forced expiratory volume in one second （FEV1）， FEV1/forced vital capacity （FVC）， the percentage of FEV1 to expected value （FEV1%pred）]， and lung function improvement （ΔFEV1 and ΔFEV1%pred） were all detected. RESULTS Totally 111 patients completed the whole 24-week follow-up and lung function detection. Among them， there were 42 cases of AA genotype， 52 cases of AG genotype， and 17 cases of GG genotype. After 12， 24 weeks of treatment， lung function indexes of patients were significantly better than baseline lung function indexes before treatment （P＜0.05）. After 24 weeks of treatment， ACO patients with AA and AG genotypes showed significantly better lung function improvement than GG genotype， and ΔFEV1%pred of AA genotype was significantly better than AG genotype （P＜ 0.05）. After 12， 24 weeks of treatment， the improvement of lung function in patients with a smoking history ≤20 pack year was significantly better than those with a smoking history ＞20 pack year， and among patients with a smoking history ≤20 pack year， only AA genotype had significantly better FEV1%pred than AG genotype （P＜0.05）. After 12 weeks of treatment， among patients with a smoking history ＞20 pack year， the improvement of lung function in AA genotype and AG genotype was significantly better than GG genotype， and the FEV1%pred in AA genotype was significantly better than AG genotype （P＜0.05）. After 24 weeks of treatment， the improvement of lung function of AA genotype and AG genotype was significantly better than GG genotype （P＜0.05）. CONCLUSIONS GG genotype of GLCCI1 gene rs37973 locus is associated with the poor treatment response to ICSs in patients with ACO， especially in patients with smoking history ＞20 pack year.

Effect of negative regulation of nucleophosmin 1 by long non-coding RNA growth arrest specific transcript 5 on cisplatin resistance of gastric cancer cells / 中国生物制品学杂志

@#Objective To investigate the effects of long non-coding RNA(LncRNA) growth arrest specific transcript 5(GAS5) negatively regulating nucleophosmin 1(NPM1) on cisplatin(DDP) resistance of gastric cancer cells.Methods The normal human gastric mucosa cell line GES-1 and human gastric cancer cell lines BG3-823,MGC-803 and AGS were selected as the research objects,of which the level of LncRNA GAS5 in each cell was measured by qRT-PCR.The drug resistance of AGS cells to DDP(AGS/DDP) was induced,and the experiment was divided into control group,empty plasmid group(BC group),GAS5 nonsense interference group(pLJM-GAS5 NC group) and GAS5 overexpression group(pLJM-GAS5 group).MTT method was used to determine the effect of DDP on the proliferation of AGS and AGS/DDP cells;and the levels of NPM1,multidrug resistance 1(MDR1),excision repair cross complementation group 1(ERCC1),multidrug resistance-associated protein 1(MRP1) and N-cadherin in AGS and AGS/DDP cells were measured by Western blot.Results Compared with the normal gastric mucosa GES-1 cells,the level of LncRNA GAS5 in BG3-823 and AGS cells decreased significantly,and among them,the level of LncRNA GAS5 in AGS cells was the lowest,so AGS cells were used for the follow-up experiments.Compared with the control group,the level of LncRNA GAS5 in AGS cells of BC group and pLJM-GAS5 NC group decreased significantly,while the levels of NPM1,MDRl,ERCC1,MRP1 and N-cadherin increased significantly;compared with BC group and pLJM-GAS5 NC group,the level of LncRNA GAS5 in AGS/DDP cells of pLJM-GAS5 group increased significantly,while the levels of NPM1,MDR1,ERCC1,MRP1 and N-cadherin decreased significantly;after treatment with DDP of the same concentration(except 0 μmol/L),compared with the control group,the inhibition rate of AGS/DDP cell proliferation in BC group and pLJM-GAS5 NC group decreased significantly;compared with BC group and pLJM-GAS5 NC group,the inhibition rate of AGS/DDP cell proliferation in pLJM-GAS5group was significantly higher.The semi inhibitory concentration(IC_(50)) of DDP on AGS/DDP cells in pLJM-GAS5 group for 48 h was(65.38±5.04) μmol/L,which was significantly lower than(120.74±4.17) μmol/L and(120.24±4.29) μmol/L in BC group and pLJM-GAS5 NC group.Conclusion Up-regulating the level of LncRNA GAS5 in AGS/DDP cells can reverse the drug resistance of AGS/DDP cells,which may be related to the down-regulation of NPM1expression

Construction of eukaryotic expression plasmid of human promyelocytic leukemia gene of six transcripts and subcellular location analysis of its recombinant proteins / 中国生物制品学杂志

@#Objective To construct eukaryotic expression plasmids of human promyelocytic leukaemia(hPML) gene of six transcripts and analyze the subcellular location of the recombinant proteins.Methods Primers were designed according to the hPML gene sequences registered in GenBank databases.Six transcripts of hPML gene fragments(hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ,Ⅵ and Ⅶ) were amplified by RT-PCR,which were linked to the eukaryotic expression vector pCAGGS respectively.The obtained eukaryotic expression plasmids of six transcripts of hPML gene were transfected into 293T cells respectively and detected for their protein expression by Western blot,while transfected into Vero cells and detected for their subcellular location by indirect immunofluorescence assay(IFA).Results The target gene fragments of the six eukaryotic expression plasmids were consistent with the hPML gene sequences registered in GenBank.All the six recombinant proteins showed specific binding with Myc antibody,among which the recombinant protein hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ and Ⅵ were located in the nucleus and cytoplasm,while the recombinant protein hPML Ⅶ was mainly located in the cytoplasm,rarely in the nucleus.Conclusion The eukaryotic expression plasmids of six transcripts of hPML gene all can be expressed correctly in mammalian cells,and the expressed recombinant proteins were located in nucleus and cytoplasm simultaneously or mainly in cytoplasm.This study provides an experimental basis for subsequent study on the antiviral and other biological functions of recombinant protein hPML.

Progress of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 in hematological malignancies / 白血病·淋巴瘤

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in the development of various tumors. Recent studies have shown that MALAT1 is highly expressed in hematological tumors and can participate in development, progression and prognosis of hematological tumors at transcriptional and post-transcriptional levels as a competitive endogenous RNA. Therefore, MALAT1 might be a novel marker as a valuable basis for the diagnosis, treatment and prognosis evaluation of hematological tumors.

Effects of arsenic and its metabolites on expressions of BCL-2α and BCL-2β transcripts / 环境与职业医学

Background Arsenic is a toxicant that can affect the expressions of the cellular anti-apoptotic gene BCL-2 and its protein, but the effects of arsenic on BCL-2α and BCL-2\begin{document}$\beta $\end{document} transcripts have not been reported. Objective To investigate the potential effects of arsenic and its metabolites, methylarsonic acid (MMA) and dimethylarsonic acid (DMA), on BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T (total of α and \begin{document}$\beta $\end{document} transcripts) in human bronchial epithelial cells (16HBE) and human lung adenocarcinoma cells (A549). Methods 16HBE cells and A549 cells were randomly divided into three categories of exposure after in vitro culture: single-selected arsenic compound exposure groups with isoconcentration (16HBE cells were treated with 4.5 μmol·L−1 of MMA, DMA, and sodium arsenite, respectively, while A549 cells were treated with 60 μmol·L−1 of MMA, DMA, and sodium arsenite, respectively), sodium arsenite exposure groups with different concentrations (16HBE cells were treated with 1.5, 3.0, and 4.5 μmol·L−1 of sodium arsenite respectively, while A549 cells were treated with 20, 40, and 60 μmol·L−1 of sodium arsenite respectively), and combined exposure groups (i.e. MMA+sodium arsenite, and DMA+sodium arsenite; the exposure concentrations of 16HBE cells were both 1.5 μmol·L−1 and both 4.5 μmol·L−1 respectively, and those of A549 cells were both 20 μmol·L−1 and both 60 μmol·L−1 respectively). Meanwhile, a blank control group was also set up in each exposure category. After 48 h of continuous exposure, the relative expressions of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T in both cells were detected by real-time PCR. Results Regarding the single-selected arsenic compound exposure, in 16HBE cells, the expression levels of BCL-2α and BCL-2T under 4.5 μmol·L−1 MMA treatment were lower than those in their control groups (q=3.27, 2.93, both P<0.05), and the expression levels of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T under 4.5 μmol·L−1 sodium arsenite were lower than those in their respective control groups (q=11.06, 3.65, 10.70, all P<0.05). In A549 cells, the expression level of BCL-2T treated with 60 μmol·L−1 DMA was lower than that in the control group (q=3.12, P<0.05), and the expression levels of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T treated with 60 μmol·L−1 sodium arsenite were lower than those in their respective control groups (q=7.59, 7.27, 8.06, all P<0.05). Regarding the sodium arsenite exposure: 16HBE cells treated with 1.5 μmol·L−1 sodium arsenite had a lower expression level of BCL-2α and a higher expression level of BCL-2\begin{document}$\beta $\end{document} than those in their respective control groups (q=6.06, 11.92, both P<0.05); the expression level of BCL-2α under 3.0 μmol·L−1 sodium arsenite was lower than that in the control group (q=12.72, P<0.05); and under 4.5 μmol·L−1 sodium arsenite treatment, the expression levels of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T were lower than those in their respective control groups (q=15.72, 6.79, 6.62, all P<0.05). The expression levels of BCL-2α gradually decreased with increasing concentrations of sodium arsenite (Fα trend=144.80, P<0.001), while BCL-2\begin{document}$\beta $\end{document} and BCL-2T decreased in a dose-dependent manner in the range of 1.5-4.5 μmol·L−1 (F\begin{document}${}_{\beta } $\end{document} trend=135.40, FT trend=38.24, both P<0.001). In A549 cells, the expression levels of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T under each concentration of sodium arsenite treatments were lower than those in their respective control groups (all P<0.05); the results of further trend tests showed that their expression levels gradually decreased with increasing concentrations of sodium arsenite (Fα trend =31.97, F\begin{document}${}_{\beta} $\end{document} trend=549.50, FT trend=252.40, all P<0.001). Regarding the combined exposure, under MMA+sodium arsenite treatment at both 60 μmol·L−1, the expression levels of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T in A549 cells were higher than those in their respective control groups (q=6.37, 14.91, 5.33, all P<0.05); under DMA+sodium arsenite treatment at both 60 μmol·L−1, their expression levels in A549 cells were also higher than those in their respective control group (q=8.60, 17.29, 6.91, all P<0.05). Conclusion Exposure to a high concentration (16HBE: 4.5 μmol·L−1, A549: 60 μmol·L−1) of a single arsenic metabolite has no effect on BCL-2 transcripts in 16HBE cells and A549 cells. Exposure to a low concentration (1.5 μmol·L−1) of sodium arsenite alone would decrease the expression level of BCL-2α and increase the expression level of BCL-2\begin{document}$\beta $\end{document} in 16HBE cells, and exposure to all designed concentrations of sodium arsenite alone would decrease the expressions of all transcripts in A549 cells. The combined exposure to high concentrations (both 60 μmol·L−1) of MMA plus sodium arsenite or high concentrations (both 60 μmol·L−1) of DMA plus sodium arsenite would increase the expressions of BCL-2α, BCL-2\begin{document}$\beta $\end{document}, and BCL-2T in A549 cells, which are different from the effects presented by single exposure.

Research progress of the metastasis-associated lung adenocarcinoma transcript 1 in eye diseases / 国际眼科杂志(Guoji Yanke Zazhi)

@#Metastasis-associated lung adenocarcinoma transcript 1(MALAT1)is one of the first identified LncRNA associated with human diseases. Unlike most members of the LncRNA family, MALAT1 is found in almost all human tissues and expressed at a relatively high level. At present, MALAT1 is known to play a vital role in the pathophysiological process of many diseases such as tumors, cardiovascular diseases, and nervous system diseases. In recent years, studies have found that MALAT1 may be involved in many ocular diseases(such as diabetic retinopathy, cataracts, glaucoma, retinoblastoma, neonatal retinopathy, <i>etc</i>.)play an important role in the pathological development process, and it is expected to become an effective target for the diagnosis and treatment of eye diseases. This article summarizes the research progress of eye diseases in which MALAT1 has participated in recent years.

Progress of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 in breast cancer / 肿瘤研究与临床

The occurrence and development of breast cancer is a complicated process, in which many kinds of bioactive substances are involved. As a long non-coding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) not only plays a role in multiple sites of breast cancer, but also plays an important role in the treatment and prognosis evaluation of breast cancer patients. This article reviews the research progress of MALAT1 in breast cancer.

Effects of berberine on necroptosis of non-alcoholic fatty liver disease in mice: Relationship with AMPK/STAT6 pathway / 中华内分泌代谢杂志

Objective:To evaluate the effects of berberine on necroptosis of non-alcoholic fatty liver disease in mice and its relationship with adenosine monophosphate-activated protein kinase(AMPK)/ signal transducer and activator of transcription 6(STAT6) pathway.Methods:Twenty-five 8-week-old male C57BL/6N mice were divided into control group, steatotic liver group, berberine treatment group(200 mg·kg -1·d -1), AMPK inhibitor Compound C treatment group(0.2 mg·kg -1·d -1), and STAT6 inhibitor AS1517499 treatment group(10 mg·kg -1·d -1). After 12 weeks of intervention, the mice and liver tissue were weighed, and serum aspartate aminotransferase(AST), alanine aminotransferase(ALT), triglyceride, tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) as well as liver malondialdehyde and superoxide dismutase were measured; liver tissue HE, Masson, and oil red O staining were performed. Western blotting was used to detect the expressions of necroptosis related proteins[receptor interaction protein kinase 3(RIPK3), phosphorylated(p-) mixed lineage kinase domain-like(MLKL)], AMPK, p-AMPK, and p-STAT6. Results:Compared with control group, the steatotic liver group had higher quality of liver and liver index, and higher levels of serum AST, ALT, triglyceride, TNF-α, IL-1β, and oxidative stress( P<0.05); Liver tissue was full of cavity changes and inflammatory cell infiltration, widely distributed red lipid droplets and obvious blue fiber dyeing; The expressions of RIPK3 and p-MLKL were up-regulated ( P<0.05), but the levels of p-AMPK and p-STAT6 were relatively reduced ( P<0.05). Compared with the steatotic liver group, berberine intervention decreased liver quality and liver index, improved liver function, reduced blood lipid levels, pro-inflammatory factor expression and oxidative stress level, and significantly alleviated the degree of liver steatosis and fibrosis, the levels of RIPK3 and p-MLKL ( P<0.05), while the expressions of p-AMPK and p-STAT6 were increased significantly ( P<0.05). As compared with the berberine treatment, AMPK and STAT6 inhibitor treatment could offset the protective effect of berberine on steatotic liver, moreover, the expressions of RIPK3 and p-MLKL were increased ( P<0.05). There was no statistical difference in AMPK total protein content among the five groups ( P>0.05). Conclusion:Berberine can activate AMPK/STAT6 pathway to inhibit the necroptosis of hepatocyte, thus plays a protective role on non-alcoholic fatty liver disease in mice.

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.

Inhibitory effect of siRNA-KCNQ1OT1 on human lens epithelial cell apoptosis by targeting miR-199a-5p / 中华实验眼科杂志

Objective:To explore the inhibitory effect of long non-coding RNA (lncRNA) KCNQ1 overlapping transcript 1 (KCNQ1OT1) by targeting microRNA-199a-5p (miR-199a-5p) on the apoptosis of human lens epithelial cells (LECs).Methods:The anterior lens capsule tissue of 23 age-related cataract patients who underwent cataract surgery in Xinxiang First People's Hospital from December 2018 to August 2019 was collected.At the same time, anterior lens capsules from 20 healthy donor were collected.The expressions of KCNQ1OT1 and miR-199a-5p in the tissues were detected by real-time fluorescence PCR.Human LECs SRA01/04 cultured in vitro were divided into blank control group, model control group, small interfering RNA-negative control (siR-NC) group, siR-KCNQ1OT1 group, miR-NC group, miR-199a-5p group, siR-KCNQ1OT1+ anti-miR-NC group and siR-KCNQ1OT1+ anti-miR-199a-5p group.No intervention was administered to blank control group.Cells in model control group were cultured with 100 μmol/L H 2O 2 for 24 hours to establish oxidative stress injured model, and cells in the other six groups were transfected with corresponding transfection reagents for 6 hours by liposome method according to grouping, and then treated with 100 μmol/L H 2O 2 for 24 hours.The expressions of KCNQ1OT1 and miR-199a-5p in lens anterior capsule tissue and LECs cells were determined by real-time fluorescent quantitative PCR.Cell viability was detected with thiazolyl blue (MTT). Cell apoptosis was analyzed by flow cytometry.The expressions of B-cell lymphoma/leukemia-2 (bcl-2) and bcl-2 related X protein (Bax) proteins were assayed by Western blot.The superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured by enzyme-linked immunosorbent assay (ELISA). The targeting relationship between KCNQ1OT1 and miR-199a-5p was verified by dual luciferase reporter experiment.The study protocol was approved by an Ethics Committee of Xinxiang First People's Hospital (No.2019-001). Written informed consent was obtained from relatives of patient. Results:The relative expression of KCNQ1OT1 in the anterior capsule of patients with age-related cataract was 2.41±0.42, which was significantly higher than 0.97±0.19 of normal people, and the relative expression of miR-199a-5p in the capsule of patients with age-related cataract was 0.36±0.12, which was lower than 1.04±0.15 of normal people, and the differences were statistically significant ( t=14.112, 16.507; both at P<0.001). Compared with blank control group, the relative expressions of KCNQ1OT1 and bax protein, cell apoptosis rate and MDA content were significantly increased, and the relative expressions of miR-199a-5p and bcl-2 protein, cell viability and SOD activity were significantly reduced in model control group, showing statistically significant differences (all at P<0.001). Compared with siR-NC group, the relative expressions of KCNQ1OT1 and bax protein, cell apoptosis rate and MDA content in cells of siR-KCNQ1OT1 group were decreased, while the relative expression of bcl-2 protein, cell survival rate and SOD activity were increased, and the differences were statistically significant (all at P<0.05). Compared with miR-NC group, the KCNQ1OT1-wild type (WT) luciferase activity in miR-199a-5p group was significantly decreased, with a statistically significant difference ( t=21.131, P<0.001). The relative expression levels of miR-199a-5p and bcl-2 proteins, cell survival rate and SOD activity were significantly increased, and the relative expression of bax protein, cell apoptosis rate and MDA content were significantly decreased in miR-199a-5p group than those in miR-NC group, and the differences were statistically significant (all at P<0.05). The relative expression levels of miR-199a-5p and bcl-2 proteins, cell survival rate and SOD activity were significantly lower, and the cell apoptosis rate, relative expression of bax protein and MDA content were significantly higher in siR-KCNQ1OT1+ anti-miR-199a-5p group than those in siR-KCNQ1OT1+ anti-miR-NC group, and the differences were statistically significant (all at P<0.05). Conclusions:The inhibition of KCNQ1OT1 can promote the cell viability of human LECs, inhibit H 2O 2-induced cell apoptosis and oxidative stress, and the mechanism may be related to the up-regulation of miR-199a-5p.

Expression and significance of long non-coding RNA metastasis associated lung adenocarcinoma transcript 1 in bronchopulmonary dysplasia of neonatal rats induced by hyperoxia / 中国小儿急救医学

Objective:To investigate the expression of long non-coding RNA metastasis associated lung adenocarcinoma transcript 1(lncRNA MALAT1) in bronchopulmonary dysplasia (BPD) of neonatal rats induced by hyperoxia and its effect on alveolar type 2 epithelial cells (AEC Ⅱ).Methods:The lung injury model of neonatal SD rats induced by hyperoxia(model group, n=50, inhaled oxygen concentration of 80%-85%) and the control group(inhaled air, n=50) were prepared.Lung tissue samples were taken and retained on days 1, 3, 7, 14 and 21, and the physiological and pathological changes of lung tissue were detected by paraffin-embedded sections and hematoxylin-eosin staining; The dynamic expression of lncRNA MALAT1 in lung tissue was detected by real-time fluorescent quantitative polymerase chain reaction; The dynamic expression of surfactant protein C(SPC) in lung tissue and AECⅡ was detected by Western blot.AECⅡ was extracted from lung tissue of normal newborn rats, and lncRNA MALAT1 was knocked down by siRNA.The cells were collected and Western blot as well as immunofluorescence were used to detect the changes of SPC. Results:The lung tissue of model group gradually became thickened with alveolar compartments, and the alveolar cavity was enlarged with the disappearance of alveolar spine and other pathological structural changes.Compared with the control group, there was no difference in the expression of lncRNA MALAT1 and SPC in the lung tissue from model group on days 1, 3( P>0.05), but the expression of lncRNA MALAT1 and SPC significantly increased on days 7, 14 and 21( P<0.05). When lncRNA MALAT1 was inhibited, SPC expression showed a decrease trend. Conclusion:Hyperoxia can lead to the stagnation of lung development in neonatal rats, and the structure and function of alveolar disorders are impaired.The expression of lncRNA MALAT1 is involved in the process of hyperoxia-induced BPD in neonatal rats.The increase of lncRNA MALAT1 may promote the proliferation of AECⅡ.

Long non-coding RNA KCNQ1OT1 promotes cell viability and migration as well as inhibiting degradation of CHON-001 cells by regulating miR-126-5p/TRPS1 axis

Abstract Background: Osteoarthritis (OA) is defined as a degenerative disease. Pivotal roles of long non-coding RNA (lncRNAs) in OA are widely elucidated. Herein, we intend to explore the function and molecular mechanism of lncRNA KCNQ1OT1 in CHON-001 cells. Methods: Relative expression of KCNQ1OT1, miR-126-5p and TRPS1 was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was examined by MTT assay. The migratory ability of chondrocytes was assessed by transwell assay. Western blot was used to determine relative protein expression of collagen II, MMP13 and TRPS1. Dual-luciferase reporter (DLR) assay was applied to test the target of lncRNA KCNQ1OT1 or miR-126-5p. Results: Relative expression of KCNQ1OT1 and TRPS1 was reduced, whereas miR-126-5p was augmented in cartilage tissues of post-traumatic OA patients compared to those of subjects without post-traumatic OA. Increased KCNQ1OT1 or decreased miR-126-5p enhanced cell viability and migration, and repressed extracellular matrix (ECM) degradation in CHON-001 cells. MiR-126-5p was the downstream target of KCNQ1OT1, and it could directly target TRPS1. There was an inverse correlation between KCNQ1OT1 and miR-126-5p or between miR-126-5p and TRPS1. Meantime, there was a positive correlation between KCNQ1OT1 and TRPS1. The promoting impacts of KCNQ1OT1 on cell viability and migration as well as the suppressive impact of KCNQ1OT1 on ECM degradation were partially abolished by miR-126-5p overexpression or TRPS1 knockdown in CHON-001 cells. Conclusions: Overexpression of KCNQ1OT1 attenuates the development of OA by sponging miR-126-5p to target TRPS1. Our findings may provide a possible therapeutic strategy for human OA in clinic.

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.

Hypoxia preconditioning promotes bone marrow mesenchymal stem cells survival and vascularization through the activation of hif-1α/malat1/vegfa pathway / 中国组织工程研究

BACKGROUND: Previous study demonstrated that hypoxia preconditioning promoted mesenchymal stem cells survival and their therapeutic efficacy, and this effect was mediated by hypoxia induced factor-1α (HIF-1α). However, specific downstream mechanism remained unclear. OBJECTIVE: To observe the influence of hypoxia preconditioning on the survival and vascularization potential of bone marrow mesenchymal stem cells in vitro and explore the regulatory mechanism of HIF-1α/MALAT1/VEGFA pathway. METHODS: Bone marrow mesenchymal stem cells were obtained and cultured in vitro. Cells were divided into hypoxia (1% O2) and normoxia control groups (20% O2), and cultured for 24 hours. Cells proliferation, apoptosis and vascularization were evaluated. The expression of HIF-1α, MALAT1, and VEGFA was detected. HIF-1α and MALAT1 were inhibited by their siRNAs separately. HIF-1α siRNA scramble and MALAT1 siRNA scramble were used as negative controls before hypoxia preconditioning. Alterations of the molecules were examined and compared in different groups. RESULTS AND CONCLUSION: (1) Compared with the normoxia control group, cell viability was significantly enhanced; and cell apoptosis percentage was significantly declined in the hypoxia group; vascular lumen like structure was also increased significantly in the hypoxia group (P < 0.01); expression of HIF-1α, MALAT1, and VEGFA was significantly increased in the hypoxia group (P < 0.01). (2) After the inhibition of HIF-1α and hypoxia preconditioning, both MALAT1 and VEGFA expression levels were significantly reduced (P < 0.01). The expression of VEGFA was also significantly suppressed after the blockage of MALAT1 (P < 0.01). (3) This study suggested that hypoxia preconditioning effectively promoted bone marrow mesenchymal stem cell survival and vascularization through the activation of HIF-1α/MALAT1/VEGFA pathway.

Role of long non-coding RNA MALAT1 in the development and progression of hepatocellular carcinoma / 临床肝胆病杂志

Hepatocellular carcinoma (HCC) has the features of high incidence rate, low survival rate, poor treatment outcome, and complex pathogenesis. In recent years, many studies have shown that long non-coding RNA (lncRNA) MALAT1 is upregulated in HCC and can promote the proliferation, invasion, and metastasis of HCC cells, and it can also guide the diagnosis, prognostic evaluation, and treatment of HCC in clinical practice. This article reviews the current status of research on lncRNA MALAT1 in HCC and discusses its expression pattern, mechanism of action, and clinical significance in predicting and monitoring the progression of HCC, so as to gain a deep understanding of the role of lncRNA MALAT1 in the progression of HCC. It is pointed out that lncRNA MALAT1 is expected to become a potential biomarker for the diagnosis and prognostic evaluation of HCC and may be used as a therapeutic target in clinical practice.

Long non-coding RNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 regulates the proliferation and osteogenic differentiation of human periodontal ligament stem cells by targeting miR-24-3p / 华西口腔医学杂志

OBJECTIVES@#This study aims to explore the effect and molecular mechanism of long non-coding RNA (lncRNA) potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) on proliferation and osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs).@*METHODS@#The hPDLSCs of normal periodontal tissues were isolated and cultured. The mineralized solution induced the osteoblast differentiation of hPDLSCs. The down-regulation of lncRNA KCNQ1OT1, the overexpression of anti-miR-24-3p on the proliferation and the levels of osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) of hPDLSCs were investigated. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the levels of lncRNA KCNQ1OT1, miR-24-3p, OCN, OPN, and ALP. Methyl thiazolyl tetrazolium (MTT) method was used to detect cell viability and activity. Cell proliferation was evaluated by MTT. Western blot was used to detect protein expression. The targeted relationship between lncRNA KCNQ1OT1 and miR-24-3p was detected by double-luciferase experiment.@*RESULTS@#The expression level of lncRNA KCNQ1OT1 increased, and that of miR-24-3p decreased during the osteogenesis of hPDLSCs (@*CONCLUSIONS@#Down-regulation of lncRNA KCNQ1OT1 inhibited the proliferation and osteogenic differentiation of hPDLSCs by targeting the up-regulated expression of miR-24-3p.

Effect of Compound Phylanthus urinaria Ⅱ in Regulating Expression of lncRNA CCAT1 in Hepatocellular Carcinoma HepG2 Cells / 中国实验方剂学杂志

Objective:To explore the anti-hepatoma effect of compound <italic>Phylanthus urinaria</italic> Ⅱ （ CPU Ⅱ） by inhibiting the expression of the long non-coding RNA （lncRNA） colon cancer associated transcript-1 （CCAT1） and restoring the expression of microRNA let-7a. Method:Real-time fluorescence quantitative polymerase chain reaction （PCR） was used to detect the expression of lncRNA CCAT1 in normal liver cells （LO2 cells） and hepatocellular carcinoma HepG2 cells， and the differences in expression between these two types of cells were compared. The methylthiazolyl tetrazolium（MTT） assay was used to detect the proliferation of HepG2 cells after treatment with different concentrations of CPU Ⅱ and 5-fluorouracil（5-FU） for 24， 48 and 72 h. Hepatocellular carcinoma HepG2 cells were cultured <italic>in vitro </italic>and set into three gropes： cell control group， CPU Ⅱ low-dose group （0.8 g·L^-1） and high-dose group （1.6 g·L^-1）. Real-time PCR was used to detect the mRNA expression of lncRNA CCAT1， microRNA let-7a and its target genes high mobility group protein A2（HMGA2）， and N-RAS in each grope. Western blot was used to detect the protein expression of HMGA2， and Cyclin D₁ in each grope. Result:As compared with LO2 cells， expression of lncRNA CCAT1 in HepG2 cells was significantly up-regulated （<italic>P</italic><0.05）. Results of MTT assay showed that the 50% inhibiting concentration（IC₅₀）of CPU Ⅱ and 5-FU on hepatocellular carcinoma HepG2 cells was 1.649， 0.044 648 g·L^-1respectively. As compared with the control group， CPU Ⅱ high-and low-dose groups （1.6， 0.8 g·L^-1） significantly inhibited the proliferation of HepG2 cells （<italic>P</italic><0.05）， and the effect was most remarkable in CPU Ⅱ high-dose group （<italic>P</italic><0.05）. The results of Real-time PCR showed that as compared with control group， the expression of lncRNA CCAT1 mRNA was significantly inhibited in CPU Ⅱ high-and low-dose groups （<italic>P</italic><0.05）， and the expression of microRNA let-7a mRNA was obviously up-regulated in high-dose group （<italic>P</italic><0.05）， but the expression of HMGA2 mRNA in CPU Ⅱ high-and low-dose groups as well as the expression of N-RAS mRNA in CPU Ⅱ low-dose group were down-regulated （<italic>P</italic><0.05）. Western blot results showed that as compared with the cell control group， the protein expression of HMGA2 and Cyclin D₁ in CPU Ⅱ high-and low-dose groups （1.6， 0.8 g·L^-1） was significantly down-regulated （<italic>P</italic><0.05）. Conclusion:CPU Ⅱ can inhibit the expression of lncRNA CCAT1， recover the expression of microRNA let-7a， and suppress the mRNA and protein expression of related downstream target genes in hepatoma cells line HepG2， thereby inhibiting the proliferation of hepatocellular carcinoma cells and exerting anti-hepatocellular carcinoma effect.