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Objective:To discuss the effect of CD40 ligand(CD40L)on the biological behavior of the human monocytic leukemia THP-1 cells through long non-coding RNA(lncRNA)linc00239,and to clarify its potential mechanism.Methods:The linc00239 over-expression vector(pcDNA-linc00239)and interference vector(sh-linc00239)were constructed and transfected into the THP-1 cells.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the transfection efficiency.The THP-1 cells were divided into control group,vector group,pcDNA-linc00239 group,sh-linc00239 group,vector+CD40L group,pcDNA-linc00239+CD40L group,and sh-linc00239+CD40L group.RT-qPCR method was used to detect the expression levels of linc00239 in the cells in various groups;CCK-8 assay was used to detect the proliferation activities of the cells in various groups;flow cytometry was used to detect the percentages of the cells at different cell cycles and the apoptotic rates of the cells in various groups;RT-qPCR and Western blotting methods were used to to detect the expression levels of B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax)mRNA and proteins in the cells in various groups;Western blotting method was used to detect the expression levels of protein kinase B(AKT)and phosphorylated AKT(p-AKT)proteins in the cells in various groups,and the ratio of p-AKT/AKT was calculated.Results:Compared with vector group,the proliferation activity of the cells and the percentage of the cells at G2 phase in pcDNA-linc00239 group were significantly increased(P<0.05 or P<0.01),the expression levels of linc00239,Bcl-2 mRNA and protein,and the ratio of p-AKT/AKT were significantly increased(P<0.05 or P<0.01),the percentage of the cells at G1 phase,apoptotic rate,and expression levels of Bax mRNA and protein in the cells were significantly decreased(P<0.05);compared with vector group,the proliferation activity of the cells and percentage of the cells at G2 phase,expression levels of linc00239,Bcl-2 mRNA and protein,and ratio of p-AKT/AKT in the cells in sh-linc00239 group and vector+CD40L group were significantly decreased(P<0.05 or P<0.01),while the percentage of the cells at G1 phase,apoptotic rate,and the expression levels of Bax mRNA and protein in the cells were significantly increased(P<0.05 or P<0.01);compared with pcDNA-linc00239 group,the proliferation activity of the cells and percentage of cells at G2 phase in pcDNA-linc00239+CD40L group were significantly decreased(P<0.05 or P<0.01),the expression levels of linc00239,Bcl-2 mRNA and protein,and ratio of p-AKT/AKT were significantly decreased(P<0.05 or P<0.01),while the percentage of cells at G1 phase,apoptotic rate,and the expression levels of Bax mRNA and protein were significantly increased(P<0.05 or P<0.01);compared with sh-linc00239 group,the proliferation activity of the cells and percentage of cells at G2 phase in sh-linc00239+CD40L group were significantly decreased(P<0.05 or P<0.01),the expression levels of linc00239,Bcl-2 mRNA and protein,and ratio of p-AKT/AKT were significantly decreased(P<0.05 or P<0.01),and the percentage of the cells at G1 phase,apoptotic rate,and expression levels of Bax mRNA and protein were significantly increased(P<0.05 or P<0.01).Conclusion:CD40L can inhibit the proliferation and cell cycle progression of the THP-1 cells through linc00239 and induce the apoptosis.
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Objective To investigate the expression of long non-coding RNA(lncRNA)LINC02695 in human retinal microvascular endothelial cells(HRMECs)in high glucose(HG)environment and its effect on the proliferation,migration and neovascularization of HRMECs.Methods HRMECs was divided into four groups:the normal glucose(NG)group(5.5 mmol/L),the HG group(30.0 mmol/L),the HG+LINC02695 silenced group(HG+si-LINC02695),and the HG+silenced control group(HG+si-NC).Real-time quantita-tive fluorescent PCR(qPCR)was used to detect the expression of LINC02695 and vascular endothelial growth factor(VEGF)mRNA in HRMECs of each group.The cell proliferation of each group was measured by Cell Counting Kit-8(CCK-8)method.The migration ability of cells in each group was detected by Transwell as-say.The tube forming ability of cells in each group was detected by tube forming experiment.Results The qPCR results showed that compared with the NG group,LINC02695 and VEGF were highly expressed in the HG group(P＜0.05).Compared with the HG group,VEGF mRNA expression level in the HG+si-LINC02695 group was significantly decreased(P＜0.05).The results of CCK-8 experiment showed that the proliferation ability of the HG group was significantly enhanced compared with the NG group(P＜0.05).Compared with the HG group,the cell proliferation ability of the HG+si-LINC02695 group was significantly decreased(P＜0.05).The results of Transwell experiment showed that the cell migration ability of the HG group was significantly increased compared with the NG group(P＜0.05).Compared with the HG group,the cell migration ability of the HG+si-LINC02695 group was significantly decreased(P＜0.05).The results of tube formation experiment showed that compared with the NG group,the tube formation ability of the HG group was significantly increased(P＜0.05).Compared with the HG group,canalization ability of cells in the HG+si-LINC02695 group was significantly decreased(P＜0.05).Conclusion LINC02695 may be involved in promoting the proliferation,migration and angiogenesis of HRMECs induced by HG.
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Objective To investigate the expression and clinical significance of serum long non-coding RNA small nucleolar RNA host gene 16(lncRNA SNHG16)and mothers against decapentaplegic homolog 4(SMAD4)in elderly patients with chronic obstructive pulmonary disease(COPD)and pulmonary infection(PI).Methods A total of 237 elderly COPD patients admitted to the hospital from January 2021 to January 2023 were enrolled in the study.Among them,117 patients with concomitant PI were classified as the concur-rent group,and 120 patients without concomitant PI were classified as the COPD group.Real-time fluores-cence quantitative polymerase chain reaction(qRT-PCR)was applied to detect the expression level of serum lncRNA SNHG16 in two groups.Enzyme linked immunosorbent assay(ELISA)was applied to detect the lev-el of SMAD4 in patients'serum.Simplified clinical pulmonary infection scale(sCPIS)was used to evaluate the degree of PI of patients in the concurrent group.Multivariate Logistic regression was applied to analyze the in-fluencing factors of PI in elderly COPD patients.Correlation between serum lncRNA SNHG16,SMAD4 levels and sCPIS in elderly COPD patients with PI was analyzed by using Spearman correlation analysis.Receiver op-erating characteristic(ROC)curve was applied to analyze the diagnostic value of serum lncRNA SNHG16 and SMAD4 levels in elderly COPD patients with PI.Results The serum relative expression level of lncRNA SNHG16 in the concurrent group was higher than that in the COPD group,but the serum SMAD4 level was lower than that in the COPD group(P<0.05).In addition,the proportions of patients with age≥70 years,smoking history,complicated with diabetes and COPD course≥5 years and the levels of tumor necrosis fac-tor-α(TNF-α),interferon-γ(INF-γ)in the concurrent group were higher than those in the COPD group,and FEV1/FVC and the level of interleukin-10(IL-10)in concurrent group were lower than those in COPD group(P<0.05).Multivariate Logistic analysis showed that age≥70 years old,complicated with diabetes,COPD course≥5 years,high levels of TNF-α,INF-γ and lncRNA SNHG16 were risk factors for elderly patients with COPD complicated with PI(P<0.05),but high FEV1/FVC and high levels of SMAD4 and IL-10 were protective factors(P<0.05).Spearman correlation analysis showed that serum relative expression level of ln-cRNA SNHG16 was positively correlated with sCPIS in COPD patients with PI(r=0.505,P<0.001),while SMAD4 level was negatively correlated with sCPIS(r=-0.550,P<0.001).The area under the curve(AUC)of the combined diagnosis of serum lncRNA SNHG16 and SMAD4 for PI in elderly COPD patients was higher than those of individual diagnosis(Z=2.416,P=0.016;Z=2.375,P=0.018).Conclusion The serum relative expression level of lncRNA SNHG16 increases and SMAD4 level decreases in elderly COPD pa-tients with PI,both are influencing factors for elderly COPD patients complicated with PI,and both are related to the degree of PI in patients,and both have diagnostic value for elderly COPD patients complicated with PI,and the diagnostic efficacy of combined detection is better.
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Objective To investigate the relationship between the expression of long non-coding RNA HOXA-AS2(lncRNA HOXA-AS2),long non-coding RNA FOXD2-AS1(lncRNA FOXD2-AS1),and long non-coding RNA CRNDE(lncRNA CRNDE)in endometrial carcinoma and the clinical pathological character-istics and prognosis of patients.Methods Collect samples of endometrial carcinoma cancer tissues and adja-cent tissues excised during surgery from 119 endometrial carcinoma patients admitted to a hospital from Octo-ber 2017 to February 2020.The relative expression levels of HOXA-AS2,FOXD2-AS1 and CRNDE in tissues were retrospectively analyzed,as well as their relationship with clinicopathological features and 3-year survival rate of patients.Results The relative expression levels of HOXA-AS2,FOXD2-AS1 and CRNDE in cancer tissues of endometrial carcinoma patients were higher than those in adjacent tissues,with statistical signifi-cance(P＜0.05).The relative expression levels of HOXA-AS2,FOXD2-AS1 and CRNDE in cancer tissues of endometrial carcinoma patients were positively correlated(rHOXA-As2 vs.FOXD2-AS1=0.384,P=0.001;rHoXA-AS2 vs.CRNDE=0.576,P＜0.001;rFoXD2-AS1 vs.CRNDE=0.326,P=0.003).In the HOXA-AS2,FOXD2-AS1 and CRNDE high expression group,the proportion of patients with international federation of gynecology and ob-stetrics(FIGO)stage Ⅲ+Ⅳ,lymph node metastasis,deep infiltration and low differentiation was higher than that in the low expression group,with statistical significance(P＜0.05).The 3-year survival rate of low HOXA-AS2 expression group in endometrial cancer patients(52/60,86.67％)was higher than that of high HOXA-AS2 expression group(40/59,67.79％),the difference was statistically significant(x2=6.039,P＜0.05).The 3-year survival rate of patients with endometrial cancer with low FOXD2-AS1 expression group(53/59,89.83％)was higher than that of patients with endometrial cancer with high FOXD2-AS1 expression group(39/60,65.00％),and the difference was statistically significant(x2=10.456,P＜0.05).The 3-year sur-vival rate of low CRNDE expression group in endometrial cancer patients(51/60,85.00％)was higher than that of high CRNDE expression group(41/59,69.49％),and the difference was statistically significant(x2=4.079,P＜0.05).HOXA-AS2,FOXD2-AS1,and CRNDE were risk factors for death in endometrial carcinoma patients(P＜0.05).Conclusion The expression of HOXA-AS2,FOXD2-AS1,and CRNDE in endometrial carcinoma cancer tissue is closely related to the clinical pathological characteristics and prognosis of patients.
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Objective To investigate the relationship between serum levels of long non-coding RNA(ln-cRNA)nuclear-enriched abundant transcript 1(NEAT1)and microRNA miR-23c in patients with diabetic ne-phropathy(DN).Methods A total of 136 DN patients admitted to the hospital from May 2019 to May 2020 were enrolled in the study as the DN group.Fifty-eight healthy people who underwent physical examination in the hospital during the same period were enrolled as the control group.Real-time fluorescence quantitative PCR(qPCR)was used to detect serum lncRNA NEAT1,miR-23c,kidney injury molecule-1(KIM-1),neutro-phil gelatinase-associated lipocalin(NGAL),tumor necrosis factor-α(TNF-α)mRNA and interleukin-6(IL-6)mRNA in the two groups.Pearson/Spearman correlation was used to analyze the correlation of serum ln-cRNA NEAT1 and miR-23c with KIM-1,NGAL,TNF-α,IL-6 mRNA levels and eGFR in DN patients.DN pa-tients were divided into different CKD stages,and the levels of serum lncRNA NEAT1,miR-23c,KIM-1,NGAL,TNF-α,and IL-6 mRNA in patients in different CKD stages were compared.Multivariate ordered Lo-gistic regression was used to analyze whether serum levels of lncRNA NEAT1 and miR-23c were influencing factors for the progression of DN.Results Compared with the control group,the serum levels of lncRNA NEAT1,KIM-1,NGAL,TNF-α and IL-6 mRNA in the DN group were increased,while miR-23c and esti-mated glomerular filtration rate(eGFR)were decreased,and the differences were all statistically significant(P＜0.05).The serum levels of lncRNA NEAT1,KIM-1,NGAL,TNF-α and IL-6 mRNA in DN patients in G1-G5 stages were increased in order,and the level of miR-23c was decreased in order(P＜0.05).Serum ln-cRNA NEAT1 in DN patients was positively correlated with KIM-1,NGAL,TNF-α and IL-6 mRNA levels(P＜0.05),and negatively correlated with miR-23c and eGFR(P＜0.05).The level of serum miR-23c was negatively correlated with the mRNA levels of KIM-1,NGAL,TNF-α and IL-6(P＜0.05),and positively cor-related with eGFR(P＜0.05).lncRNA NEAT1(OR=2.177,95％CI:2.113-2.441)was an independent risk factor for DN progression,while miR-23c(OR=0.595,95％CI:0.543-0.726)was an independent pro-tective factor(P＜0.05).Conclusion Elevated serum lncRNA NEAT1 levels and reduced miR-23c levels in DN patients are closely associated with the progression of DN disease.
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Objective:To investigate the expression of long non-coding RNA(lncRNA) ZFP36-AS1 in bladder cancer and the effect of ZFP36-AS1/miR-221 axis on the proliferation and immune escape of bladder cancer cells.Methods:The expression difference of ZFP36-AS1 in bladder cancer tissues was analyzed by cBioPortal database. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to analyze the expression difference of ZFP36-AS1 in bladder cancer cell lines (J82, RT-4, MGH-U3, 5637). MGH-U3 cells were randomly divided into negative control (NC) group and ZFP36-AS1 group, which were transfected with pcDNA3.1-NC plasmid and pcDNA3.1-ZFP36-AS1 plasmid, respectively. Colony formation assay and flow cytometry were used to analyze the proliferation activity and cell cycle of MGH-U3 cells, respectively. T lymphocytes were co-cultured with MGH-U3 cells in each group, and the levels of interleukin-10 (IL-10), γ-interferon (IFN-γ), and interleukin-4 (IL-4) in the supernatants of each group were detected by enzyme-linked immunosorbent assay (ELISA). The dual-luciferase reporter gene assay verified the targeting relationship between ZFP36-AS1 and miR-221. The effect of ZFP36-AS1 on the expression of miR-221 in MGH-U3 cells was detected by RT-qPCR. Western blotting was used to detect the effect of ZFP36-AS1/miR-221 axis on the protein expression of CDK3, Cyclin C, CDK5, Cyclin D1 and Cyclin D3 in MGH-U3 cells.Results:Compared with normal bladder tissue, ZFP36-AS1 was abnormally low-expressed in bladder cancer tissue ( P<0.01). Compared with SV-HUC-1 cells, ZFP36-AS1 was abnormally low-expressed in bladder cancer cell lines (J82, RT-4, MGH-U3, 5637) ( P<0.01), and the expression was lowest in MGH-U3 cells ( P<0.01). The number of MGH-U3 cell colonies formed in the NC group and the ZFP36-AS1 group were (220.80±34.65) and (77.84±19.11), respectively, and the number of MGH-U3 cell colonies formed in the ZFP36-AS1 group was significantly down-regulated, the difference was statistically significant ( P<0.01). The proportions of G 0/G 1 phase cells in NC group and ZFP36-AS1 group were (48.04±2.89)% and (72.89±3.46)%, respectively, and the proportion of S phase cells were (35.38±2.98)% and (20.62±2.56)%, respectively. The proportion of G 2/M stage cells was (16.59±1.46)% and (6.48±1.50)%, respectively. The proportion of cells in G 0/G 1 phase were up-regulated in ZFP36-AS1 group ( P<0.01), and the proportion of cells in S phase and G 2/M phase were both down-regulated ( P<0.01). Compared with the NC group, the levels of IL-4 and IFN-γ in the ZFP36-AS1 group were significantly up-regulated ( P<0.01), and the level of IL-10 was significantly down-regulated ( P<0.01). ZFP36-AS1 can target miR-221 ( P<0.01). The relative expression of miR-221 in the NC group and the ZFP36-AS1 group was 6.84±1.35 and 1.00±0.21, respectively. Compared with the NC group, overexpression of ZFP36-AS1 could significantly inhibit the expression of miR-221 ( P<0.01). Compared with the NC group, the expressions of CDK3, Cyclin C, CDK5, Cyclin D1, and Cyclin D3 in the ZFP36-AS1 group were significantly decreased. Conclusion:ZFP36-AS1 is abnormally low-expressed in bladder cancer, and it reduces the proliferation activity of bladder cancer cells and inhibits their immune escape by inhibiting the expression of miR-221.
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Objective To investigate the relationship between the expression of long non-coding RNA C-terminal binding protein 1 antisense RNA2(LncRNA CTBP1-AS2)and microRNA-140-5p(miR-140-5p)levels in nasopharyngeal carcinoma tissues and the radiotherapeutic effect and prognosis.Methods A total of 222 nasopharyngeal carcinoma patients diagnosed in Nantong Cancer Hospital from March 2018 to March 2020 were collected as the nasopharyngeal carcinoma group.The clinical data of these patients were recorded,the radiotherapeutic effect and prognosis were evaluated,and they were grouped into the survival group(n=194)and the death group(n=28).Meanwhile,another 219 patients with nasopharyngeal inflammation were collected as the control group.Correlation between LncRNA CTBP1-AS2 and miR-140-5p expression levels in nasopharyngeal carcinoma patients was calculated using Pearson correlation analysis.Kaplan-Meier survival curve was applied to analyze the relationship between the expression levels of LncRNA CTBP1-AS2 and miR-140-5p in nasopharyngeal carcinoma tissue and prognosis.Multivariate analysis was conducted on the prognosis of nasopharyngeal carcinoma patients using Cox proportional risk regression model.Results The expression level of LncRNA CTBP1-AS2 in the tissues of patients in nasopharyngeal carcinoma group(2.25±0.46)was higher than that in the control group(1.02±0.22),while the expression level of miR-140-5p(0.67±0.19)was lower than that in the control group(1.01±0.23),and the differences were statistically significant(t=35.742,16.934,all P<0.001).There was a negative correlation between LncRNA CTBP1-AS2 and miR-140-5p expression levels in nasopharyngeal carcinoma patients(r=-0.624,P<0.001).The total effective rate(74.11%)and 3-year survival rate(77.68%)of nasopharyngeal carcinoma patients with high expression of LncRNA CTBP1-AS2 after radiotherapy were lower than those with low expression(93.64%,97.27%),and the differences were statistically significant(χ2=15.578,19.331,all P<0.001).The total effective rate(93.58%)and 3-year survival rate(96.33%)of patients with high expression of miR-140-5p after radiotherapy were higher than those of patients with low expression(74.34%,78.76%),and the differences were statistically significant(χ2=15.119,15.538,all P<0.001).The magnetic resonance amide proton transfer(APT)value(2.10±0.26),the proportion of patients with radiotherapy failure(85.71%),high expression of LncRNA CTBP1-AS2(89.29%),and low expression of miR-140-5p(85.71%)in the death group were higher than those in the survival group(1.82±0.31,6.19%,44.85%,45.88%),and the differences were statistically significant(t/χ2=4.551,108.127,19.331,15.538,all P<0.001).The level of LncRNA CTBP1-AS2 was a risk factor for mortality within 3 years in nasopharyngeal carcinoma patients(HR=2.762,95%CI:1.510～5.051,P=0.001),while the level of miR-140-5p was a protective factor for mortality within 3 years in nasopharyngeal carcinoma patients(HR=0.817,95%CI:0.718～0.930,P=0.002).Conclusion LncRNA CTBP1-AS2 was highly expressed,while miR-140-5p was lowly expressed in nasopharyngeal carcinoma tissue,indicating the two may be closely related to the radiotherapeutic effect and prognosis.
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Objective To construct N6-methyladenosine related long non-coding RNA(LncRNA)pairing model for renal cell carcinoma based on bioinformatics analysis of the cancer ganome atlas(TCGA)database and to explore its prognosis value.Methods Transcriptome data of RNA-sep for renal cell carcinoma and its related clinical information were downloaded from the TCGA database.Perl software was used to organize and separate LncRNA and messenger RNA(mRNA)from the transcriptome data.A total of 564 tissues from renal cell carcinoma cases and 72 normal tissues were obtained,and thus 540 renal cancer patients were eventually included.Random data table method was used to divide 540 patients with renal cancer into a training group(n=275)and a validation group(n=265)by caret.M6A related LncRNA pairing models were established based on the single factor and multivariate COX regression analysis.The risk assessment equation was obtained using the LASSO regression algorithm.The risk scores were calculated based on this equation,and the optimal critical point of the median risk value was applied to divide all patients into high-risk and low-risk groups.Kaplan-Meier survival analysis was used to make a survival curve for the differences between high and low risk groups in the overall sample.The gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analyses were conducted using the Cluster Profiler software package.The relationship between N6-methyladenosine related LncRNA pairing model and immune cell infiltration was analyzed by R software.Results Kaplan-Meier survival analysis showed the total survival time of patients in the low-risk group was significantly higher than that of patients in the high-risk group of the training group(P<0.05).Compared with high risk group,the overall survival time of patients(G1～2,G3～4,Ⅰ～Ⅱ,or Ⅲ～Ⅳ,age≤65 years,or patients>65 years old)in low risk group was higher(P<0.05).Differential gene enrichment analysis was obtained for high and low risk groups,which mainly enriched with many differential genes such as muscle contraction,rhabdomytic cell differentiation,myofibril,receptor activation activity,and vascular smooth muscle contraction.The highest driver genes in high risk group and low risk group exhibited mutation frequency and mutation information,and their risk score was positively correlated with the degree of T cell and plasma cell infiltration(r=0.638,P=0.001).Conclusion Bioinformatics-based analysis of the N6-methyladenosine related LncRNA pairing models can be helpful to predict the prognosis of patients with renal cancer.It provides new ideas for the prognosis evaluation and optimal treatment strategy of renal cancer,and contributes to further analyzing the molecular mechanism of the occurrence and development of gastric cancer in the future.
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Objective To explore the impacts of long non-coding RNA(LncRNA)GNAS antisense RNA1(GNAS-AS1)on the proliferation and migration of gastric cancer(GC)cells by regulating the miR-449a/Notch1 axis.Method Tumor tissue and adjacent tissue samples were collected from 30 patients diagnosed with GC at Sichuan Provincial People's Hospital from September 2013 to September 2017;GC cells AGS were randomly divided into Control group,si-NC group,si-GNAS-AS1 group,si-GNAS-AS1+inhibitor NC group,and si-GNAS-AS1+miR-449a inhibitor group.Real-time fluorescence quantitative PCR method was applied to detect the expres-sion of GNAS-AS1,miR-449a,and Notch1 mRNA;MTT experiments and plate cloning experiments were applied to detect the proliferation;wound healing test was applied to detect cell migration;Transwell experiment was applied to detect cell invasion.Western Blot was applied to detect the expression of Notch1,E-cadherin,Vimentin,and N-cadherin proteins.Double Luciferase reporter gene experiment was applied to verify the relationship between GNAS-AS1 and miR-449a,between miR-449a and Notch1,respectively.Results Compared with adjacent tissues,the expression of GNAS-AS1 and Notch1 mRNA in tumor tissue was increased,the expression of miR-449a was reduced(P<0.05).Compared with the Control group and si-NC group,the expression of GNAS-AS1,OD490 value,number of clones formed,scratch healing rate,number of cell invasions,and the expression of Notch1,Vimentin,and N-cadherin proteins in AGS cells in the si-GNAS-AS1 group reduced,the expression of miR-449a and E-cadherin protein increased(P<0.05).Compared with the si-GNAS-AS1 group and the si-GNAS-AS1+inhibitor NC group,the OD490 value,scratch healing rate,number of cell invasions,Notch1,Vimentin,and N-cadherin expression in the si-GNAS-AS1+miR-449a inhibitor group increased,the expression of miR-449a and E-cadherin protein reduced(P<0.05).GNAS-AS1 targeted and negatively regulated miR-449a expression,while miR-449a targeted and negatively regulated Notch1 expression.Conclusion Silencing GNAS-AS1 may inhibit the expression of Notch1 protein by up-regulating miR-449a,thereby inhibiting the proliferation,migration,and invasion pro-cesses of GC cells.
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BACKGROUND:Umbilical cord mesenchymal stem cells(UMSCs)have been proven to have therapeutic effects on cartilage injury,and exosomes are the main carriers for UMSCs to exert therapeutic effects in vivo.Our research group previously found that lncRNA H19 is an important active molecule that mediates the activity of UMSCs-derived exosomes regulating chondrocytes.LncRNA H19 could adsorb miR-29b-3p to promote the proliferation and regeneration of chondrocytes,but its downstream mechanism is still unclear. OBJECTIVE:To reveal the specific mechanism of UMSCs in the treatment of cartilage injury from the perspective of exosomes and lncRNAs,so as to provide a new target for the treatment of cartilage injury. METHODS:UMSCs stably overexpressing lncRNA H19 were constructed.H19-Exos were extracted by ultra-centrifugation.The exosomes were identified by transmission electron microscopy,Nanosight,western blot assay and exosome uptake assay.The effect of miR-29b-3p overexpression and silencing on the TGF-β1/Smad3 pathway was detected by western blot assay,qPCR and dual luciferase reporter gene system.The biological effect of H19-Exos on cartilage regeneration was verified by the specific TGF-β1/Smad3 inhibitor in vitro and in vivo. RESULTS AND CONCLUSION:(1)H19-Exos showed a typical cup shape under an electron microscope,and the particle size was approximately 130 nm.H19-Exos expressed CD63,CD81 and TSG1010.(2)Overexpression of miR-29b-3p could down-regulate the mRNA and protein levels of TGF-β1 and Smad3,while silencing miR-29b-3p could up-regulate the mRNA and protein levels of TGF-β1/Smad3.(3)Dual-luciferase reporter gene system showed that miR-29b-3p had significant differences in the activities of downstream target genes TGF-β1 and Smad3.(4)The osteoarthritis models of rats were successfully established by injection of type II collagenase into the knee joint.H19-Exos significantly promoted cartilage regeneration.The specific TGF-β1/Smad3 inhibitor SB-431542 could block the biological effect of H19-Exos on cartilage regeneration in vitro and in vivo.(5)This study systematically demonstrated the promotion effect of UMSCs-derived exosomes highly expressing lncRNA H19 on cartilage regeneration,and the specific mechanism is that lncRNA H19 promotes cartilage regeneration by targeting miR-29b-3p/TGF-β1/Smad3 pathway.
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BACKGROUND:Studies have shown that long non-coding RNA small nucleolar RNA host gene 4(lncRNA SNHG4)is involved in the progress of many inflammatory diseases,but the effect of lncRNA SNHG4 on the osteogenic differentiation of human periodontal ligament stem cells during the treatment of periodontitis is still unclear. OBJECTIVE:To investigate the effect of lncRNA SNHG4 on the osteogenic differentiation of human periodontal ligament stem cells by regulating miR-152-3p. METHODS:Human periodontal ligament stem cells were isolated from periodontal membranes of premolars extracted for orthodontic purposes.After human periodontal ligament stem cells were induced to differentiate into osteoblasts for 0,7,and 14 days,the expression levels of RUNX family transcription factor 2,osteocalcin mRNA,lncRNA SNHG4 and miR-152-3p in human periodontal ligament stem cells were detected by qRT-PCR.The third-generation human periodontal ligament stem cells were divided into the NC group,pcDNA group,pcDNA-SNHG4 group,inhibitor NC group,miR-152-3p inhibitor group,pcDNA-SNHG4+mimic NC group,and pcDNA-SNHG4+miR-152-3p mimic group.The expression of lncRNA SNHG4 and miR-152-3p in human periodontal ligament stem cells was detected by qRT-PCR.The proliferation of human periodontal ligament stem cells was detected by CCK-8 assay.Alkaline phosphatase activity was detected by colorimetry.The formation of mineralized nodules was detected by alizarin red staining.Western blot assay was used to detect the expression of RUNX family transcription factor 2,osteocalcin and alkaline phosphatase proteins.A double luciferase reporter gene experiment was applied to verify the relationship between lncRNA SNHG4 and miR-152-3p. RESULTS AND CONCLUSION:(1)The expression of RUNX family transcription factor 2,osteocalcin mRNA and lncRNA SNHG4 in human periodontal ligament stem cells after 7 and 14 days of osteogenic induction was higher than that after 0 days of osteogenic induction,while the expression of miR-152-3p was lower(P<0.05).(2)Overexpression of lncRNA SNHG4 or inhibition of miR-152-3p was able to enhance the proliferation of human periodontal ligament stem cells,the alkaline phosphatase activity,mineralized nodule formation,the expression of RUNX family transcription factor 2,osteocalcin,and alkaline phosphatase proteins(P<0.05).miR-152-3p mimic attenuated the promoting effect of overexpression of lncRNA SNHG4 on osteogenic differentiation of human periodontal ligament stem cells.LncRNA SNHG4 had a targeting relationship with miR-152-3p.(3)These findings indicate that overexpression of lncRNA SNHG4 may promote the osteogenic differentiation of human periodontal ligament stem cells by inhibiting miR-152-3p.
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BACKGROUND:Wnt signaling pathway is overexpressed in degenerative intervertebral discs,and inhibition of its expression can delay the process of intervertebral disc degeneration.Therefore,Wnt signaling pathway is closely related to intervertebral disc degeneration. OBJECTIVE:To summarize the relationship between Wnt signaling pathway and intervertebral disc,especially the specific role and influence of Wnt signaling pathway in intervertebral disc degeneration. METHODS:The first author took"intervertebral disc,Wnt,cell proliferation,cell senescence,cell apoptosis,extracellular matrix"as the English search terms.PubMed,Web of science and OVID LWWSpringerlink were searched for articles published from 2000 to January 2023,and articles related to Wnt signaling pathway and disc degeneration were consulted.Totally 54 articles were reviewed by reading,collating and preserving. RESULTS AND CONCLUSION:(1)During intervertebral disc formation in embryos,Wnt signaling pathway is overexpressed,which is involved in intervertebral disc formation and promotes posterior extension of notochord.(2)During intervertebral disc degeneration,Wnt signaling pathway can inhibit cell proliferation by stagnating cell cycle,increase the expression of age-related proteins and promote cell aging by participating in oxidative stress,and participate in cell apoptosis by regulation of long non-coding RNA.(3)Wnt signaling pathway can also decrease extracellular matrix related protein synthesis,promote extracellular matrix degradation and accelerate intervertebral disc degeneration.(4)Wnt signaling pathway can promote cell regeneration by activating as early intervertebral disc formation signal and participate in inducing stem cells to differentiate into intervertebral disc cells to repair damaged intervertebral disc.For example,Wnt signaling pathway can induce stem cells from cartilage endplate cells to migrate and transform to intervertebral disc.
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BACKGROUND:In recent years,numerous studies have shown that long non-coding RNAs(lncRNAs)have an important role in the pathogenesis of periodontitis,including the immune response process and the biological activity and function of cells(periodontal stem cells and osteoblasts).Researchers attempt to regulate periodontal inflammation and periodontal regeneration by regulating lncRNA expression levels. OBJECTIVE:To review the progress of research on lncRNAs in periodontitis,thereby advancing the research of lncRNAs in periodontitis. METHODS:The first author searched PubMed,China National Knowledge Infrastructure(CNKI),Web of Science database,and WanFang Database for relevant literature published up to March 2023."Long non-coding RNA,lncRNA,periodontitis,periodontium,immunity,inflammation,periodontal ligament stem cells,osteoclasts,osteogenic differentiation,bone resorption,bone formation,recurrence,hypoxia,oxidative stress,static mechanical strain"were used as the Chinese search terms."lncRNA,periodontitis,periodontal,immunity,inflammation,periodontal membrane stem cells,osteoclasts,osteogenic differentiation,bone resorption,bone formation,recurrence,hypoxia,oxidative stress,static mechanical strain"were used as the English search terms.The title and abstract of each paper were read for initial screening,and 87 papers were finally selected for inductive analysis. RESULTS AND CONCLUSION:Periodontal pathogens stimulate the body,leading to immune imbalance that triggers inflammatory response and destroy periodontal tissue,and lncRNAs are involved in its regulatory mechanism.LncRNAs are involved in the pro-inflammatory regulation of periodontal ligament cells in an inflammatory environment,and their effects on osteoclast differentiation are regulated through ceRNA mechanism,which provides new clues for exploring the pathogenesis of periodontitis.For B cells and macrophages,lncRNAs can regulate the infiltration,cell activity and function of their subsets in periodontitis.LncRNAs participate in the immune response related to periodontitis mainly through two pattern recognition receptors,Toll-like receptors and NOD-like receptors,and nuclear factor-κB signaling pathway.To investigate whether lncRNAs can be used as a biomarker of periodontitis has great value in the diagnosis and prognosis of periodontitis.Animal experiments have demonstrated that the role of lncRNAs in periodontitis can be reversed by modulating the expression level of lncRNAs and thus lncRNAs act as an anti-inflammatory agent,which is of great value for the study of immunotherapy in periodontitis.The regulation of lncRNAs on periodontal ligament stem cells is mainly realized through endogenous competition mechanism and various signaling pathways,and its effects are influenced by various factors,such as inflammatory environment,mechanical strain,hypoxia and oxidative stress.Research on related mechanisms through these related factors provides new ideas for the treatment of periodontitis.
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BACKGROUND:Long non-coding RNAs(lncRNAs),as important regulators of the inflammatory response,are involved in the immune-inflammation-brain crosstalk mechanism after ischemic stroke and have the potential to become a therapeutic agent for neurological dysfunction after ischemic stroke. OBJECTIVE:To analyze and summarize the molecular mechanism of lncRNA acting on glial cells involved in the neuroimmuno-inflammatory cascade response after ischemic stroke and the associated signaling pathways,pointing out that lncRNAs have the potential to regulate inflammation after ischemic stroke. METHODS:PubMed was searched using the search terms of"ischemic stroke,long non-coding RNA,neuroinflammation,immune function,signal pathway,microglia,astrocytes,oligodendrocyte,mechanism,"and 63 relevant documents were finally included for review. RESULTS AND CONCLUSION:In the early stage of ischemic stroke,the death of nerve cells due to ischemia and hypoxia activates the innate immune response of the brain,promoting the secretion of inflammatory factors and inducing blood-brain barrier damage and a series of inflammatory cascades responses.As an important pathogenesis factor in ischemic stroke,the neuroimmuno-inflammatory cascade has been proved to seriously affect the prognosis of patients with ischemic stroke,and it needs to be suppressed promptly in the early stage.Neuroinflammation after ischemic stroke usually induces abnormal expression of a large number of lncRNAs that mediate a series of neuro-immune-inflammatory crosstalk mechanisms through regulating the polarization of microglia,astrocytes and oligodendrocytes to exert post-stroke neuroprotective effects.LncRNAs,as important regulatory factors of the inflammatory response,inhibit the neuroimmuno-inflammatory cascade response after ischemic stroke through regulating nuclear factor-κB,lncRNA-miRNA-mRNA axis,Rho-ROCK,MAPK,AKT,ERK and other signaling pathways to effectively improve neurological impairment after ischemic stroke.Most of experimental studies on the interaction between lncRNAs and ischemic stroke are based on a middle cerebral artery occlusion model or a cerebral ischemia-reperfusion injury model,but no clinical trials have been conducted.Therefore,it remains to be further explored about whether lncRNAs can be safely applied in clinical practice.At present,there are many therapeutic drugs for the treatment of ischemic stroke,but there are relatively few studies on the application of lncRNAs,exosomes and other transplantation technologies for the treatment of ischemic stroke using tissue engineering technology,which need to be further explored.lncRNA has become an important target for the treatment of ischemic stroke with its relative stability and high specificity.In future studies,more types of inflammatory lncRNAs that function under ischemic-hypoxia conditions should continue to be explored,in order to provide new research directions for the treatment of neuroinflammation after ischemic stroke.
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BACKGROUND:It has been shown that long non-coding RNA(lncRNA)plays an important role in the development and progression of cardiac diseases,and whether it is involved in daisy leaf gentianone antagonizing adriamycin-induced cardiac injury in mice has not been reported. OBJECTIVE:To screen differentially expressed lncRNAs in myocardial tissue of mice with adriamycin-induced myocardial injury antagonized with daisy leaf gentianone and conduct a bioinformatics analysis. METHODS:Forty-eight C57 mice were randomly divided into normal group,model group,daisy leaf gentianone group and positive drug group,with 12 mice in each group.The mice in the model group,daisy leaf gentianone group and positive drug group were injected with adriamycin intraperitoneally once every other day for 8 times in total.The daisy leaf gentianone group and positive drug group were given daisy leaf gentianone suspension and captopril solution by gavage based on adriamycin injection once a day for 21 continuous days.After medication,mice in each group underwent electrocardiogram examination and the myocardial tissue was taken for pathomorphological observation.At the same time,high-throughput sequencing analysis of mouse myocardial tissue was carried out,differentially expressed lncRNAs were screened,and target genes were predicted for differentially expressed lncRNAs.Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes signaling pathway analyses of target genes were performed. RESULTS AND CONCLUSION:The ST segment of the electrocardiogram of mice in the model group was significantly elevated.Compared with the model group,the degree of ST-segment elevation on the electrocardiogram was reduced in the daisy leaf gentianone group.Results from hematoxylin-eosin,Masson,and Sirius red staining indicated that in the model group,the myocardial cytoplasm was unevenly colored with varying shades of color,the integrity and continuity of myocardial fibers were poor,and a large number of collagen fibers were deposited.After treatment with gentianone daisy leaves,the abnormalities in myocardial tissue of mice were improved.The results of high-throughput sequencing analysis showed that compared with the model group,a total of 270 lncRNAs were identified in the myocardial tissue of mice in the daisy leaf gentianone group,including 165 up-regulated and 105 down-regulated lncRNAs.Combining the experimental results with related literature,three lncRNAs(NONMMUT149833.1,NONMMUT003237.2,and ENSMUST00000219015)and four related mRNAs(Alas2,Igf2,Acta1,and Cilp)were finally identified.The results of target gene prediction,gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes signaling pathway analyses showed that differentially expressed lncRNAs in myocardial tissue of mice with myocardial injury could regulate the expression of their target protein-coding genes through cis-and/or trans-regulation,and participate in regulating molecular functions and biological processes.To conclude,daisy leaf gentianone significantly improves cardiac function and partial lncRNA expression in mice with adriamycin-induced myocardial injury.
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BACKGROUND:The osteogenic differentiation of mesenchymal stem cells is regulated by a variety of molecules.Long non-coding RNA(lncRNA)has attracted much attention because they can participate in regulating a variety of biological processes,but the regulatory role of lncRNA on osteogenic differentiation of mesenchymal stem cells has not been fully explored. OBJECTIVE:To explore the effect of lncRNA Gm16104 on osteogenic differentiation of C3H10T1/2 mesenchymal stem cells. METHODS:The C3H10T1/2 mesenchymal stem cells were induced into osteogenic differentiation by bone morphogenetic protein-2.Alkaline phosphatase staining was performed to identify the osteogenic differentiation of the cells 5 days after osteogenic induction.Expression levels of alkaline phosphatase and lncRNA Gm16104 were detected by qRT-PCR after 0,1,3,and 5 days of osteogenic differentiation.After transfection of the overexpression plasmid of pcDNA-Gm16104,the osteogenic differentiation was identified by alkaline phosphatase staining and qRT-PCR 4 days after osteogenic induction.The expression levels of osteogenesis-related signalling pathway proteins were detected by western blot assay. RESULTS AND CONCLUSION:(1)After 5 days of osteogenic induction,alkaline phosphatase activity was significantly increased.(2)Compared with 0 days,expression levels of the osteogenic marker gene alkaline phosphatase increased and expression levels of Gm16104 decreased after 1,3,and 5 days of osteogenic induction.(3)Transfection of C3H10T1/2 cells with pcDNA-Gm16104 plasmid significantly increased the expression level of Gm16104.(4)Overexpression of Gm16104 inhibited alkaline phosphatase activity,the expression levels of the osteogenic marker gene alkaline phosphatase and the osteogenesis-related transcription factor Osterix.(5)Overexpression of Gm16104 inhibited phosphorylated protein expression of PI3K and Akt.(6)The above results suggest that overexpression of Gm16104 may inhibit osteogenic differentiation of C3H10T1/2 mesenchymal stem cells through the PI3K/Akt signaling pathway.
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BACKGROUND:In recent years,there have been many studies on the mechanism of exosomal non-coding RNA in gestational diabetes mellitus,but there is a lack of the latest systematic review of exosomes from different sources,especially placental sources. OBJECTIVE:To summarize the changes and potential roles of microRNA(miRNA),long non-coding RNA(lncRNA),circular RNA(circRNA),and exosomes in gestational diabetes mellitus to provide potential targets for early screening and treatment of clinical gestational diabetes mellitus. METHODS:A literature search was conducted on PubMed,Web of Science,China National Knowledge Infrastructure,WanFang Data,and VIP databases to retrieve relevant articles on non-coding RNA or exosomal non-coding RNA in relation to gestational diabetes mellitus.A total of 74 articles were included for review. RESULTS AND CONCLUSION:(1)Non-coding RNAs play important pathological and physiological roles in the lifecycle activities,and increasing evidences suggest that non-coding RNAs are involved in the occurrence and development of gestational diabetes mellitus by regulating various physiological functions.This provides a new direction for the research of gestational diabetes mellitus.(2)Exosomes are widely present in the human body.Various cells can secrete exosomes,such as red blood cells,epithelial cells,and placental cells.Non-coding RNAs found in exosomes from different sources have been demonstrated to play a role in the pathogenesis,diagnosis,and treatment of gestational diabetes mellitus.(3)MiRNA and gestational diabetes mellitus:The role of peripheral blood miRNA in gestational diabetes mellitus is mainly to affect the functions of trophoblast cells,pancreatic beta cells and blood glucose levels in gestational diabetes mellitus;placental miRNA can reflect the severity of gestational diabetes and impair the function of trophoblast cells.(4)LncRNA and gestational diabetes mellitus:Peripheral blood lncRNA can induce insulin resistance through the phosphatidylinositol 3-kinase/protein kinase B pathway and may provide new insights for the diagnosis and treatment of gestational diabetes mellitus;placental lncRNA can regulate proliferation and migration of placental trophoblast cells,promoting the occurrence and development of gestational diabetes mellitus.(5)CircRNA and gestational diabetes mellitus:Peripheral blood and placental circRNA can induce the occurrence and development of gestational diabetes mellitus by impairing the proliferation,migration and metabolism of placental trophoblast cells.(6)Non-coding RNA in exosomes and gestational diabetes mellitus:Peripheral blood non-coding RNA in exosomes can affect gestational diabetes mellitus blood glucose levels and glucose homeostasis,and participate in the occurrence and development of gestational diabetes mellitus by influencing placental function.(7)Non-coding RNA has the potential to serve as biomarkers for early diagnosis of gestational diabetes mellitus.Additionally,engineered exosomes can better achieve targeted therapy for gestational diabetes mellitus.These latest findings provide a reference for both basic research and clinical translation of gestational diabetes mellitus.(8)In the future,improvements in the extraction and purification methods of peripheral blood exosomes should be improved,and factors such as race,diet and physical activity should be excluded to improve the reproducibility of results.Further prospective clinical studies are required to explore the clinical application of circulating non-coding RNA and exosomes in the prediction and diagnosis of gestational diabetes mellitus.
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Objective To investigate the expression of LncRNA HOXA-AS2 in gastric cancer tissues and its effect on the malig-nant biology of gastric cancer.Methods The expression levels of lncRNA HOXA-AS2 in gastric cancer tissues and gastric cancer cell lines were detected by qPCR;the effect of lncRNA HOXA-AS2 on the prognosis of gastric cancer patients was analyzed by Kaplan-Meier Plotter,an online website for bioinformatics analysis;the correlation between the expression levels of lncRNA HOXA-AS2 and the clinical and pathological characteristics of gastric cancer patients;cell lines interfering with the expression of lncRNA HOXA-AS2 were constructed,and the effects of down-regulation of lncRNA HOXA-AS2 on the proliferation ability,migration ability and invasion ability of gastric cancer cells were analyzed using CCK8,clone formation assay,scratch assay and Transwell assay.Results The expression lev-el of lncRNA HOXA-AS2 was significantly upregulated in gastric cancer tissues compared with paraneoplastic tissues;the expression lev-el of lncRNA HOXA-AS2 was significantly higher in gastric cancer cell lines compared with human normal gastric mucosal cells GES(P＜0.05);survival analysis showed that high expression of lncRNA HOXA-AS2 was associated with poor prognosis of gastric cancer patients;lncRNA HOXA-AS2 expression level correlated with gastric cancer stage,lymph node metastasis and differentiation(P＜0.05);the expression level of lncRNA HOXA-AS2 was significantly decreased in gastric cancer cells transfected with SiRNA(P＜0.05),and their cell proliferation,migration,and invasion ability were also significantly decreased(P＜0.05).Conclusion lncRNA HOXA-AS2 plays an oncogene role in gastric cancer and is associated with prognosis.Down-regulation of lncRNA HOXA-AS2 ex-pression can inhibit the proliferation,migration,and invasion ability of gastric cancer cells.
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Objective To investigate the action mechanism of long non-coding RNA(lncRNA)-N1LR on blood-brain barrier(BBB)after cerebral ischemia-reperfusion(I/R)injury.Methods Primary rat brain microvascular endothelial cells(BMECs)were cultured and treated with OGD/R to simulate cerebral I/R injury.The experiment was divided into normal control group,ln-cRNA-N1LR OGD group,overexpression group(lncRNA-N1LR overexpression after OGD treat-ment)and silence group(lncRNA-N1LR silence after OGD treatment).The mRNA levels of ln-cRNA-N1LR,claudin-5 and occludin in each group were detected by RT-qPCR.The BBB permea-bility was detected by FITC-dextran infiltration assay.The expression of claudin-5 and occludin were detected by Western blotting.Results The mRNA levels of lncRNA-N1LR,occludin and claudin-5 were significantly decreased(0.31±0.01 vs 1.00±0.10,0.42±0.03 vs 1.01±0.13,0.38±0.03 vs 1.00±0.15,P＜0.05),and the BBB permeability was significantly increased(58.79± 3.04 vs 8.87±0.63,P＜0.05)in the OGD group than the control group.The lncRNA-N1LR over-expression group increased the mRNA expression of lncRNA-N1LR,occludin and claudin-5(0.67±0.07 vs 0.31±0.01,0.92±0.02 vs 0.42±0.03,0.70±0.08 vs 0.38±0.03,P＜0.05),and decreased the BBB permeability(41.57±2.43 vs 58.79±3.04,P＜0.05)than the OGD group.lncRNA-N1LR silence resulted in lower mRNA levels of lncRNA-N1LR,occludin and claudin-5(0.21±0.02 vs 0.31±0.01,0.31±0.03 vs 0.42±0.03,0.22±0.02 vs 0.38±0.03,P＜0.05),and enhanced BBB permeability(72.34±1.43 vs 58.79±3.04,P＜0.05)when compared with the OGD group.Conclusion Up-regulation of lncRNA-N1LR may play a neuroprotective role by reducing BBB permeability.
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@#Objective A competing endogenous RNA (ceRNA) regulatory network associated with long non-coding RNA (lncRNA) specific for lung adenocarcinoma (LUAD) was constructed based on bioinformatics methods, and the functional mechanism of actinfilament-associated protein 1-antisense RNA1 (AFAP1-AS1) in LUAD was analyzed, in order to provide a new direction for the study of LUAD therapeutic targets. Methods The gene chip of LUAD was downloaded from the Gene Expression Omnibus (GEO), and lncRNA and mRNA with differential expression between LUAD and normal tissues were screened using GEO2R online software, and their target genes were predicted by online databases to construct ceRNA networks and perform enrichment analysis. In cell experiments, AFAP1-AS1 was genetically knocked down and siRNA was constructed and transfected into LUAD cells A549 by cell transfection. CCK8, transwell, scratch assay and flow cytometry were used to detect the ability of cells to proliferate, invade, migrate and apoptosis. Results A total of 6 differentially expressed lncRNA and 494 differentially expressed mRNA were identified in the microarray of LUAD. The ceRNA network involved a total of 6 lncRNA, 22 miRNA, and 55 mRNA. Enrichment analysis revealed that mRNA was associated with cancer-related pathways. In cell assays, knockdown of AFAP1-AS1 inhibited cell proliferation, invasion, and migration, and AFAP1-AS1 promoted apoptosis. Conclusion In this study, we construct a lncRNA-mediated ceRNA network, which may help to further investigate the mechanism of action of LUAD. In addition, through cellular experiments, AFAP1-AS1 is found to have potential as a therapeutic target for LUAD.