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1.
Article in Chinese | WPRIM | ID: wpr-1038699

ABSTRACT

Objective@#To investigate the changes in the levels of interleukin-6 (IL-6) and let-7e in rats induced by coal mine dust, so as to provide the basis for the mechanism of coal worker's pneumoconiosis (CWP).@*Methods@#Sixty-four clean and healthy male Sprague-Dawley rats were randomly divided into the control group, coal dust group, mixed dust group (mixed coal and silica dust) and quartz group. The rats in the control group were exposed to 1 mL physiological saline by non-exposure tracheal perfusion, and the rats in the dust-exposed groups were exposed to 1 mL dust suspension. Rats were sacrificed by anesthesia after 1 month and 6 months, lung tissue was observed using hematoxylin-eosin staining, the pathological change in the lungs was scored using the Szapiel scoring system, the levels of IL-6 in the bronchoalveolar lavage fluid were detected using enzyme-linked immunosorbent assay, and the expression of let-7e was determined by quantitative real-time PCR.@*Results@#A month after exposure, a small amount of coal spots and inflammatory exudation were observed in the lung tissue of the coal dust group and the mixed dust group. The quartz group showed tissue structure destruction and mild fibrosis and thickening of alveolar septum. Six months after exposure, there were more coal spots and slightly thickened alveolar septum in the coal dust group, and hyperplasia of pulmonary interstitial fibers, destruction of alveolar structure and silica nodules were observed in the mixed dust group. In the quartz group, the alveolar structure was obviously destroyed, the interstitial fiber proliferation was significant and silica nodules were seen. Two-factor analysis of variance showed that the interaction between duration of exposure and dust type significantly influenced the pathological score of lung tissue, IL-6 levels, and let-7e expression levels (P<0.05). Under the same dust type, the pathological score of lung tissue and IL-6 levels were higher at 6 months after exposure than at 1 month, while the relative expression of let-7e was lower at 6 months after exposure than at 1 month (all P<0.05). Under the same duration of exposure, the pathological score of lung tissue and IL-6 levels were higher in the dust-exposed groups than in the control group, while the relative expression of let-7e was lower in the dust-exposed groups than in the control group (all P<0.05).@*Conclusions@#Coal dust can cause an increase in levels of IL-6 and a decrease in let-7e expression in rats. The type of dust and duration of exposure can interactively affect IL-6 and let-7e.

2.
Article in Chinese | WPRIM | ID: wpr-1036346

ABSTRACT

Background Coal workers' pneumoconiosis (CWP) is a serious occupational lung disease and one of the prescript occupational diseases in China. Epithelial-mesenchymal transition (EMT) is involved in the diffuse fibrosis of lung tissue of pneumoconiosis patients, and its mechanism may be related to the polarization of macrophages regulated by let-7c. Objective To investigate the effect of let-7c on the regulation of macrophage polarization in EMT in rats induced by coal dust exposure with different content of free SiO2. Methods SD rats were randomly divided into a control group, a 5% SiO2 group, a 30% SiO2 group, and a 99.9% SiO2 group, with 16 rats in each group. The rats in each group were tracheally titrated with 100 μL of 20 mg·mL−1 suspension (5% SiO2, 30% SiO2, and 99.9% SiO2) or normal saline, respectively. Alveolar lavage fluid was collected at the ends of the 1st month and the 3rd month. The relative expression levels of M1 or M2 markers, CD86 or CD206, in alveolar macrophages (AMs) were detected by immunofluorescence. The inflammation and fibrosis of lung tissue were observed by hematoxylin-eosin (HE) staining and Masson staining. The expression levels of transforming growth factor-β1 (TGF-β1), E-cadherin, and vimentin were detected by Western blotting. The expression levels of let-7c and c/EBP-δ genes were detected by real-time fluorescence quantitative PCR. Results The HE and Masson staining results showed that compared with the control group, the degree of pulmonary fibrosis in the 5% SiO2 group, the 30% SiO2 group, and the 99.9% SiO2 group gradually increased with the increase of dust exposure time. Compared with the control group, the expressions of CD86 and CD206 in the 5% SiO2 group, the 30% SiO2 group, and the 99.9% SiO2 group gradually increased at the end of the 1st month (F=330.904, 146.801, P<0.05), and the expression of CD86 in each group decreased gradually at the end of the 3rd month (F=331.781, P<0.05), but the expression of CD206 increased (F=1164.190, P<0.05). At the end of the 1st month, the expressions of TGF-β1 (F=8.847, P<0.05) and vimentin (F=13.275, P<0.05) gradually increased, and the expression of E-cadherin (F=6.253, P<0.05) gradually reduced in the 5% SiO2 group, the 30% SiO2 group, and the 99.9% SiO2 group. At the end of the 3rd month, the expressions of TGF-β1 (F=16.833, P<0.05) and vimentin (F=55.021, P<0.05) increased, and the expression of E-cadherin (F=12.790, P<0.05) gradually decreased in all groups. The PCR results showed that compared with the control group, the expression of let-7c mRNA in the 5% SiO2 group, the 30% SiO2 group, and the 99.9% SiO2 group increased at the ends of the 1st month and the 3rd month (F=11.251, 28.136, P<0.05). The expression of c/EBP-δ mRNA decreased in all groups at the ends of the 1st month and the 3rd month (F=49.204, 177.090, P<0.05). Conclusion In response to dust stimulation, let-7c promotes EMT by modulating macrophage polarization, which is involved in the formation of pulmonary fibrosis and thus influences the progression of CWP .

3.
Frontiers of Medicine ; (4): 143-155, 2023.
Article in English | WPRIM | ID: wpr-971621

ABSTRACT

Gliomas are the most common central nervous system tumours; they are highly aggressive and have a poor prognosis. RGS16 belongs to the regulator of G-protein signalling (RGS) protein family, which plays an important role in promoting various cancers, such as breast cancer, pancreatic cancer, and colorectal cancer. Moreover, previous studies confirmed that let-7c-5p, a well-known microRNA, can act as a tumour suppressor to regulate the progression of various tumours by inhibiting the expression of its target genes. However, whether RGS16 can promote the progression of glioma and whether it is regulated by miR let-7c-5p are still unknown. Here, we confirmed that RGS16 is upregulated in glioma tissues and that high expression of RGS16 is associated with poor survival. Ectopic deletion of RGS16 significantly suppressed glioma cell proliferation and migration both in vitro and in vivo. Moreover, RGS16 was validated as a direct target gene of miR let-7c-5p. The overexpression of miR let-7c-5p obviously downregulated the expression of RGS16, and knocking down miR let-7c-5p had the opposite effect. Thus, we suggest that the suppression of RGS16 by miR let-7c-5p can promote glioma progression and may serve as a potential prognostic biomarker and therapeutic target in glioma.


Subject(s)
Humans , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , MicroRNAs/metabolism , Glioma/genetics , Genes, Tumor Suppressor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Cell Line, Tumor
4.
Zhonghua zhong liu za zhi ; (12): 471-481, 2023.
Article in Chinese | WPRIM | ID: wpr-984746

ABSTRACT

Objective: To investigate the effects of lncRNA DRAIC on proliferation, apoptosis, migration and invasion of lung adenocarcinoma cells and its mechanism. Methods: Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of DRAIC in lung cancer tissues and corresponding adjacent normal tissues of 40 patients with lung adenocarcinoma who underwent surgery in Tangshan People's Hospital from 2019 to 2020. Lung adenocarcinoma cells A549 and H1299 were cultured in vitro and divided into si-NC group, si-DRAIC group, miR-NC group, let-7i-5p mimics group, si-DRAIC+ inhibitor-NC group, and si-DRAIC+ let-7i-5p inhibitor group. CCK-8 method and clone formation experiment were used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. Transwell array was used to detect the cell migration and invasion. Western blot was used to detect the protein expressions of Caspase-3, Caspase-9, Bcl-2 and Bax. The double luciferase reporter gene experiment was used to verify the regulatory relationship between DRAIC and let-7i-5p. Independent sample t test was used for comparison between two groups, one-way ANOVA was used for comparison between multiple groups, and Pearson correlation analysis was used for correlation analysis. Results: Compared with adjacent tissues, the expression level of DRAIC in lung adenocarcinoma tissues increased (P<0.05), but the expression level of let-7i-5p decreased (P<0.05). The expression levels of DRAIC and let-7i-5p in lung adenocarcinoma tissues were negatively correlated (r=-0.737, P<0.05). The absorbance value of A549 and H1299 cells in the si-DRAIC group at 48, 72 and 96 hours were lower than those in the si-NC group (P<0.05), the number of clones formed [(91.00±6.08 vs. 136.67±6.51); (50.67±1.53 vs. 76.67±4.51)], the number of migration [(606.67±31.34 vs. 960.00±33.06); (483.33±45.96 vs. 741.67±29.67)], the number of invasion [(185.00±8.19 vs. 447.33±22.05); (365.00±33.87 vs. 688.00±32.97)] were lower than those in the si-NC group (P<0.05). However, the apoptosis rates of cells [(13.43±2.79)% vs. (4.53±0.42)%; (23.77±1.04)% vs. (6.60±1.42)%] were higher than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC group were higher than those in si-NC group, and the protein expression of Bcl-2 was lower than that in si-NC group (P<0.05). DRAIC is located in the cytoplasm. DRAIC targeted and negatively regulated the expression of let-7i-5p. The absorbance values of A549 and H1299 cells in the let-7i-5p mimics group at 48, 72 and 96 hours were lower than those in the miR-NC group (P<0.05), the number of clones formed [(131.33±14.47 vs. 171.33±6.11); (59.33±4.93 vs. 80.33±7.09)], the number of migration [(137.67±3.06 vs. 579.33±82.03); (425.00±11.14 vs. 669.33±21.13)], the number of invasion [(54.00±4.36 vs. 112.67±11.59); (80.00±4.58 vs. 333.33±16.80)] were lower than those in the miR-NC group (P<0.05). However, the apoptosis rates of cells [(14.57±1.10)% vs. (6.97±1.11)%; (23.97±0.42)% vs. (7.07±1.21)%] were higher than those in the miR-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in let-7i-5p mimics group were higher than those in miR-NC group, and the protein expression of Bcl-2 was lower than that in miR-NC group (P<0.05). The absorbance values of A549 and H1299 cells in the si-DRAIC+ let-7i-5p inhibitor group at 48, 72 and 96 hours were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05), the number of clones formed [(82.00±5.29 vs. 59.00±5.57); (77.67±4.93 vs. 41.33±7.57)], the number of migration [(774.33±35.81 vs. 455.67±19.04); (569.67±18.72 vs. 433.67±16.77)], the number of invasion [(670.33±17.21 vs. 451.00±17.52); (263.67±3.06 vs. 182.33±11.93)] were higher than those in the si-DRAIC+ inhibitor-NC group (P<0.05). However, the apoptosis rates of cells [(7.73±0.45)% vs. (19.13±1.50)%; (8.00±0.53)% vs. (28.40±0.53)%] were lower than those in the si-NC group (P<0.05). The protein expressions of Caspase-3, Caspase-9 and Bax in si-DRAIC+ let-7i-5p inhibitor group were higher than those in si-DRAIC+ inhibitor-NC group, and the protein expression of Bcl-2 was lower than that in si-DRAIC+ inhibitor-NC group (P<0.05). Conclusion: DRAIC is highly expressed in lung adenocarcinoma, and DRAIC promotes the proliferation, migration and invasion of lung adenocarcinoma cells and inhibits apoptosis by targeting let-7i-5p.


Subject(s)
Humans , Adenocarcinoma/genetics , Apoptosis/genetics , bcl-2-Associated X Protein/metabolism , Caspase 3/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cell Proliferation/genetics , Lung/metabolism , MicroRNAs/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Long Noncoding/genetics
5.
Zhongnan Daxue xuebao. Yixue ban ; (12): 909-919, 2023.
Article in English | WPRIM | ID: wpr-982363

ABSTRACT

MicroRNAs (miRNAs) are endogenous non-coding single-stranded small RNAs that regulate gene expression by recognizing homologous sequences and interfering with transcriptional, translational or epigenetic processes. MiRNAs are involved in a variety of disease processes, and regulate the physiological and pathological status of diseases by modulating target cell activity, migration, invasion, apoptosis, autophagy and other processes. Among them, let-7i is highly expressed in various systems, which participates in the process of tumors, cardiovascular and cerebrovascular diseases, fibrotic diseases, inflammatory diseases, neurodegenerative diseases and other diseases, and plays a positive or negative regulatory role in these diseases through different signal pathways and key molecules. Moreover, it can be used as an early diagnosis and prognostic marker for a variety of diseases and become a potential therapeutic target. As a biomarker, let-7i is frequently tested in combination with other miRNAs to diagnose multiple diseases and evaluate the clinical treatment or prognosis.


Subject(s)
Biomarkers , Apoptosis , Autophagy , Epigenesis, Genetic , MicroRNAs/genetics
6.
Journal of Chinese Physician ; (12): 401-405,410, 2022.
Article in Chinese | WPRIM | ID: wpr-932078

ABSTRACT

Objective:To investigate the predictive value of serum vascular endothelial growth factor (VEGF), squamous cell carcinoma-associated antigen (SCCAg) and miRNA let-7a in lymph node metastasis and postoperative recurrence in patients with laryngeal cancer.Methods:A total of 82 patients with laryngeal cancer in the Second Central Hospital of Baoding from November 2017 to October 2019 were selected as the research subjects, including 18 cases of lymph node metastasis (metastasis group) and 64 cases of non metastasis (non metastasis group). The blood routine was tested before operation, and the baseline data, serum VEGF, SCCAg and miRNA let-7a levels were compared between the two groups. Logistic regression was used to analyze the related influencing factors of lymph node metastasis in patients with laryngeal cancer. The correlation between serum VEGF, SCCAg, miRNA let-7a levels and clinicopathological characteristics was analyzed. The receiver operating characteristic (ROC)curve was used to analyze the value of each index and the combined diagnosis of lymph node metastasis in patients with laryngeal cancer. After 1 year of follow-up, the serum VEGF, SCCAg and miRNA let-7a levels of patients with or without recurrent laryngeal cancer were compared. ROC curve was used to evaluate the value of VEGF, SCCAg, and miRNA let-7a in predicting the recurrence of laryngeal cancer.Results:There were statistically significant differences in tumor node metastasis (TNM) stage, degree of infiltration, degree of differentiation, serum VEGF, SCCAg, and miRNA let-7a levels between the metastasis group and non metastasis group (all P<0.05). Serum VEGF, SCCAg, miRNA let-7a levels in patients with laryngeal cancer were related to TNM stage, degree of infiltration and degree of differentiation (all P<0.05). The combined diagnosis of serum VEGF, SCCAg and miRNA let-7a levels in the diagnosis of lymph node metastasis in patients with laryngeal cancer showed that the diagnostic sensitivity and specificity were 88.89% and 70.31%, respectively. The serum VEGF and SCCAg levels of patients with recurrence after operation were higher than those without recurrence, and the level of miRNA let-7a was lower than those without recurrence (all P<0.05). The sensitivity and specificity of combined serum VEGF, SCCAg and miRNA LET-7a levels in predicting postoperative recurrence of laryngeal cancer were 72.97% and 91.11%, respectively. Conclusions:VEGF, SCCAg, miRNA let-7a in patients with laryngeal cancer have a certain correlation with clinicopathological characteristics, which can assist in the diagnosis of lymph node metastasis and help clinical prediction of postoperative recurrence in patients with laryngeal cancer, and provide a reference for the formulation of clinical treatment plans.

7.
Journal of Chinese Physician ; (12): 676-681, 2022.
Article in Chinese | WPRIM | ID: wpr-932119

ABSTRACT

Objective:To investigate the effect of irinotecan combined with XELOX regimen on serum CD cells, miR-34a and let-7i in patients with advanced gastric cancer.Methods:A total of 72 patients with advanced gastric cancer treated in the Affiliated Hospital of Hubei University of Arts And Science from January 2015 to January 2020 were prospectively selected, and they were divided into control group and observation group with 36 cases in each group by random number table method. The control group was treated with XELOX regimen, while the observation group was treated with irinotecan on the basis of the control group. The efficacy, serum immune level, serum miR-34a and let-7i contents, incidence of toxic and side effects and life cycle of the two groups were compared.Results:The objective response rate (ORR) of the observation group was 33.33% , which were significantly higher than that of the control group (13.89%, χ 2=3.770, P>0.05). There was no significant difference in serum immune level, miR-34a and let-7i between the two groups before treatment (all P>0.05). After treatment, the levels of CD3 + , CD4 + and CD8 + in the two groups were significantly increased (all P<0.05), and the improvement of CD3 + , CD4 + and CD8 + in observation group was significantly better than that in control group (all P<0.05). After treatment, the serum miR-34a level increased significantly and serum let-7i level decreased significantly in the two groups (all P<0.05), and the serum miR-34a level in the observation group was higher than that in the control group, and the serum let-7i level was significantly lower than that in the control group (all P<0.05). Among the grade Ⅰ-Ⅱ adverse reactions, nausea and vomiting and leukocyte decline were the most common; The incidence of grade Ⅲ-Ⅳ leukopenia in the control group and the observation group were 5.56%(2/36) and 5.56%(2/36), respectively; The incidence of grade Ⅲ-Ⅳ thrombocytopenia was 5.56%(2/36) and 2.78%(1/36), respectively; One patient in the control group had grade Ⅲ-Ⅳ abnormal liver function, and one patient in the observation group had grade Ⅲ-Ⅳ nausea and vomiting. All patients with adverse reactions were effectively relieved after symptomatic treatment, and there were no treatment-related deaths. The progression free survival (PFS) of the observation group was 24.90 months (95% CI: 0.469-1.955), and that of the control group was 21.85 months (95% CI: 0.512-2.131). The PFS of the observation group was significantly longer than that of the control group (log rank=1.357, P=0.007). Conclusions:Irinotecan combined with XELOX regimen in the treatment of advanced gastric cancer can effectively improve the level of serum miR-34a and immune function, reduce the content of let-7i, and has good safety.

8.
Clinics ; Clinics;77: 100051, 2022. graf
Article in English | LILACS-Express | LILACS | ID: biblio-1384603

ABSTRACT

Abstract Objectives Some previous studies indicated that the excessive proliferation and migration of Pulmonary Artery Smooth Muscle Cells (PASMCs) could be observed in pulmonary artery intima after Pulmonary Embolism (PE) occurred. In addition, recent studies identified some miRNAs that are differentially expressed in the blood of PE patients, which might be used as a diagnostic biomarker for PE, including let-7a-5p, let-7b-5p, and miR-150-5p. Hence, the authors sought to explore the effects of let-7b-5p in PASMC proliferation and migration and the corresponding regulatory mechanism. Methods Platelet-Derived Growth Factor (PDGF) was utilized to induce the hyper-proliferation model in PASMCs. The mRNA and protein expression levels were detected by RT-qPCR and western blot, respectively. The proliferation of PASMCs was evaluated by the detection of PCNA expression, as well as CCK-8 and Edu assays. Wound healing and Transwell assays were exploited to assess the migration ability of PASMCs. The targets of let-7b-5p were predicted based on two bioinformatics online tools. Dual-luciferase and Ago2 pull-down assays were applied to confirm the interaction between let-7b-5p and IGF1. Results 40 ng/mL PDGF was selected as the optimal concentration to induce PASMCs. let-7b-5p mimics suppressed the proliferation and migration of PDGF-induced PASMCs, while let-7b-5p inhibitor led to the opposite result. In further mechanism exploration, IGF1 was predicted and confirmed as the direct target gene of let-7b-5p. The promotion role of IGF1 overexpression on the proliferation and migration of PDGF-induced PASMCs was dramatically countered by let-7b-5p mimics. Conclusion let-7b-5p prohibits the proliferation and migration of PDGF-induced PASMCs by modulating IGF1.

9.
Acta Universitatis Medicinalis Anhui ; (6): 1964-1970,1978, 2022.
Article in Chinese | WPRIM | ID: wpr-1038768

ABSTRACT

Objective @#To screen differentially expressed miRNAs and explore its effect and mechanism on cell mi- gration in lung adenocarcinoma (LUAD) .@*Methods @#Differentially expressed miRNAs in LUAD tissues and normal lung tissues were screened by miRNA microarrays,and then bioinformatics analysis was used to predict their poten- tial biological functions and signaling pathways.The cancer genome atlas (TCGA) analysis and quantitative real- time PCR (qRT-PCR) verified the expression level of hsa-let-7e-5p in LUAD tissues and cell lines.The effect of hsa-let-7e-5p cell migration in LUAD was examined by would healing experiment.After screening the underlying target genes by bioinformatics analysis ,the targeting relationship between hsa-let-7e-5p and DTX2,NME6, C8orf58,GATM and DHX57 were verified by qRT-PCR. @*Results @#The miRNA microarray results showed that 347 miRNAs were down-regulated while 229 miRNAs were up-regulated in lung adenocarcinoma tissues.Compared with normal lung tissue and cells,the expression level of hsa-let-7e-5p was significantly down-regulated.Besides,over- expression of hsa-let-7e-5p inhibitedLUAD cell migration.@*Conclusion @#Non-coding RNA hsa-let-7e-5p is down- regulated in LUAD and inhibits the migration of lung adenocarcinoma cells.DTX2,NME6,C8orf58,GATM and DHX57 are the potential target genes of hsa-let-7e-5p.

10.
Ciênc. rural (Online) ; 52(10): e20210171, 2022. tab, graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1364719

ABSTRACT

LIN28 is a RNA-binding protein including two highly conserved homologous, LIN28A and LIN28B. Proto-oncogenes such as LIN28A and LIN28B are generally targeted by the let-7 miRNAs in different types of human cancers. Here, we determined the expression of LIN28A in canine mammary tumor samples and the LIN28/let-7 pathway in canine mammary cell lines. In those cell lines, we identified a functional LIN28/let-7 pathway which exhibited high expression of let-7 members and low expression of its targets, including LIN28A and LIN28B. However, the mammary carcinoma tissue samples showed a frequent expression of LIN28A being expressed mainly in the epithelial cells. No association was observed between LIN28A expression and histopathological classification and grade, TNM and survival time. Our results suggested a possible role of the LIN28A protein in the development of canine mammary carcinomas due to the high frequency observed in the tumor samples (28 of 32). The in vitro experiments suggested that the LIN28/let-7 pathway is active in the tumor cells evaluated. However, more studies are necessary to elucidate the exact role of LIN28/let-7 pathway in canine mammary carcinomas.


LIN28 é uma proteína de ligação ao RNA, com duas formas homólogas altamente conservadas, LIN28A e LIN28B. Os proto-oncogenes LIN28A e LIN28B são regulados pela família de miRNAs let-7 em diferentes tipos de cânceres em humanos. No presente trabalho, o objetivo foi determinar a expressão de LIN28A em amostras de tumor mamário de cadelas e a via LIN28/let-7 em linhagens celulares mamárias caninas. Nestas linhagens, através das técnicas de qPCR e RNAseq, foi identificado que a via LIN28/let-7 apresenta-se funcional, com alta expressão dos membros da família let-7 e baixa expressão de seus alvos, entre eles LIN28A e LIN28B. No entanto, as amostras de tecidos de carcinomas mamários caninos demonstraram expressão frequente de LIN28A, sendo observada principalmente em células epiteliais. Não foram observadas associações entre expressão de LIN28A com classificação e gradação histopatológicas, TNM e tempo de sobrevida. Nossos resultados sugerem uma possível relação da proteína LIN28A no desenvolvimento de carcinomas mamários caninos devido à alta frequência observada nas amostras tumorais (28 de 32). Os experimentos in vitro sugerem que a via LIN28/let-7 é ativa nas linhagens celulares caninas avaliadas. Entretanto, estudos funcionais ainda são necessários para elucidar a função exata da via LIN28/let-7 nos carcinomas mamários caninos.


Subject(s)
Animals , Female , Dogs , Mammary Neoplasms, Animal/genetics , RNA-Binding Proteins/analysis , MicroRNAs/analysis , Polymerase Chain Reaction
11.
Article in Chinese | WPRIM | ID: wpr-906208

ABSTRACT

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<sup>-1</sup>) and high-dose group (1.6 g·L<sup>-1</sup>). 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 D<sub>1</sub> 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(IC<sub>50</sub>)<sub> </sub>of CPU Ⅱ and 5-FU on hepatocellular carcinoma HepG2 cells was 1.649, 0.044 648 g·L<sup>-1 </sup>respectively. As compared with the control group, CPU Ⅱ high-and low-dose groups (1.6, 0.8 g·L<sup>-1</sup>) 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 D<sub>1</sub> in CPU Ⅱ high-and low-dose groups (1.6, 0.8 g·L<sup>-1</sup>) 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.

12.
Article in Chinese | WPRIM | ID: wpr-906692

ABSTRACT

@#[摘 要] 目的:探讨miRNA let-7i-5p在肾细胞癌(renal cell carcinoma,RCC)组织中的表达水平及其对RCC细胞769-P的增殖、迁移、侵袭和透明质酸结合蛋白4(hyaluronan-binding protein 4,HABP4)表达的影响。方法:利用TCGA RCC数据库及GEO数据库对let-7i-5p在RCC组织中的表达进行meta分析。体外常规培养人RCC细胞769-P并对其进行转染,根据转染物不同分为过表达组(转染let-7i-5p模拟物)、抑制组(转染let-7i-5p抑制物)和对照组(转染NC序列)。采用CCK-8、细胞划痕实验、Transwell实验及WB法检测let-7i-5p对769-P细胞增殖活力、划痕愈合率、穿膜侵袭细胞数及HABP4表达水平的影响。双荧光素酶报告基因实验验证let-7i-5p与HABP4 mRNA的靶向关系。结果:分析TCGA RCC数据库及5个GEO数据集(GSE23085、GSE47582、GSE95385、GSE16441和GSE71302)中数据结果显示,let-7i-5p在RCC组织中的表达水平显著高于正常肾组织(均P<0.05)。体外实验结果显示,与对照组相比,过表达组在24、48及72 h时细胞增殖活力显著升高,而抑制组显著降低(均P<0.01);过表达组的划痕愈合率[(37.276±2.058)% vs(15.663±2.949)%,P<0.01]和穿膜侵袭细胞数[(377.000±34.044) vs (255.667±25.384)个,P<0.01]均显著升高,而抑制组的划痕愈合率[(8.791±2.568)% vs(15.663±2.949)%,P<0.05]和穿膜侵袭细胞数[(170.333±14.978)vs(255.667±25.384)个,P<0.01]均显著降低。在野生型HABP4-3’ URT质粒组中,过表达let-7i-5p可显著抑制细胞的荧光素酶活性(P<0.01);而在突变型HABP4-3’ URT质粒组中,过表达let-7i-5p对细胞的荧光素酶活性无影响(NS,P>0.05)。WB检测结果显示,与对照组相比,过表达组的HABP4和E-cadherin的水平均降低(均P<0.01)、CDK2的水平升高(P<0.01),而抑制组则相反(均P<0.01)。结论:Let-7i-5p在RCC组织中呈高表达,其可能通过靶向HABP4基因来促进769-P细胞的增殖、迁移和侵袭。

13.
J Biosci ; 2020 Apr; : 1-10
Article | IMSEAR | ID: sea-214303

ABSTRACT

Transforming growth factor b2 (TGF-b2)/Smad signaling is widely accepted as a key inducer of proliferationand epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs), contributing to thedevelopment of posterior capsule opacification (PCO). Increasing evidence shows that microRNAs (miRNAs)play important roles in PCO pathogenesis. Herein, we aimed to explore the role and molecular mechanism oflet-7a-5p on TGF-b2-induced proliferation and EMT in LECs. qRT-PCR was performed to detect theexpression of let-7a-5p and Smad2 mRNA. Western blot was used to determine the Smad2 level and theinduction of EMT. The targeted correlation between let-7a-5p and Smad2 was confirmed using dual-luciferasereporter and RNA immunoprecipitation assays. CCK-8 assay was employed to determine cell proliferation, andtranswell assays were performed to assess cell migration and invasion. We found that TGF-b2 induced EMT ofLECs, and TGF-b2 upregulated Smad2 expression and reduced let-7a-5p expression in LECs. Smad2 was adirect target of let-7a-5p. Moreover, let-7a-5p upregulation repressed proliferation, migration, invasion andEMT in TGF-b2-induced LECs. But, Smad2 expression restoration abrogated the inhibitory effect of let-7a-5pupregulation. In conclusion, our data indicated that let-7a-5p upregulation repressed TGF-b2-induced proliferation, migration, invasion and EMT at least partly by targeting Smad2 in LECs, highlighting that let-7a-5pmight act as a promising therapeutic target to intervene to the progression of PCO.

14.
Yonsei med. j ; Yonsei med. j;: 210-217, 2020.
Article in English | WPRIM | ID: wpr-811475

ABSTRACT

PURPOSE: The goal of this study was to explore the effects of hsa-let-7g on cell proliferation and apoptosis, and elucidate its role in lung cancer development.MATERIALS AND METHODS: The expression levels of has-let-7g and HOXB1 in tissues and cells were measured by qRT-PCR. An inhibitor of hsa-let-7g or one targeting a control messenger RNA were transfected into A549 and H1944 lung cancer cells, and the effects of hsa-let-7g dysregulation on cell viability and apoptosis were analyzed using CCK-8 and apoptosis detection assays. HOXB1 was confirmed as the target gene of hsa-let-7g, based on luciferase reporter assay results. The relationship between hsa-let-7g and HOXB1 was confirmed by co-transfection of inhibitors of hsa-let-7g and HOXB1 followed by Western blot, CCK-8, and apoptosis detection assays.RESULTS: We observed high expression of hsa-let-7g in lung cancer tissues compared to the corresponding normal tissues, and generally higher expression of hsa-let-7g in patients with advanced tumor classification. The results of CCK-8 and apoptosis detection experiments showed that the inhibition of hsa-let-7g significantly inhibited proliferation of A549 and H1944 cells, but also promoted apoptosis. HOXB1 is a specific target of hsa-let-7g, and downregulation of HOXB1 in lung cancer cells reversed the suppressive effects caused by knocking down hsa-let-7g.CONCLUSION: These data collectively suggest that the expression of hsa-let-7g inhibits lung cancer cells apoptosis and promotes proliferation by down-regulating HOXB1. The results from this study demonstrate the potential of hsa-let-7g/HOXB1 axis as a therapeutic target for the treatment of lung cancer.


Subject(s)
Humans , Apoptosis , Blotting, Western , Cell Proliferation , Cell Survival , Classification , Down-Regulation , Luciferases , Lung Neoplasms , Lung , MicroRNAs , RNA, Messenger , Sincalide
15.
Article in Chinese | WPRIM | ID: wpr-841575

ABSTRACT

Objetive: To detect the expressions of IncRNA H19 (H19) and IL-6/STAT3 pathway in the ulcerative colitis-associated colorectal cancer (CAC) tissue of the mice, and to explore its possible mechanism Methods: A total of 22 C57BL/6 mice were randomly divided into control group (n=10) and model group (n= 12). The CAC models were induced by azomethane (AOM) combined with dextran sodium sulfate (DSS) in the mice in model group. The mice were sacrificed on the 120th day, the disease activity index (DAI) of the mice was evaluated, the tumor formation rate was evaluated, the colon length was measured, and the pathomorphology of colon tissue of the mice was observed by HE staining. The serum IL-6 level of the mice was detected by ELISA. The expression levels of H19, let-7a, IL-6, STAT3 and c-Myc mRNA in colon tissue of the mice were detected by qPCR method. The expression levels of p-STAT3 and c-Myc proteins in colon tissue of the mice were detected by Western blotting method. Results: Compared with control group, the tumor formation rate of the mice in model group was 100%, the colon length was significantly shortened (P<0. 01), the DAI score was increased (P< 0.01), the colon tissue showed the intraepithelial neoplasia by HE staining, the expression levels of H19, IL-6, STAT3 and c-myc mRNA in colon tissue were significantly increased (P<0. 01), the expression level of let-7a mRNA in colon tissue was significantly decreased (P<0. 01), the serum IL-6 level was increased (P<0. 01), and the expression levels of p-STAT3 and c-Myc proteins in colon tissue were increased (P<0. 01). Conclusion: The pathogenesis of CAC in the mice may be related to the up-regulation of c-Myc and H19 and down-regulation of let-7a, which are mediated by IL-6/STAT3 pathway.

16.
Article in Chinese | WPRIM | ID: wpr-847600

ABSTRACT

BACKGROUND: Cyclic RNA plasmacytoma variant translocation 1 (circPVT1) is involved in the senescence of fibroblasts, but the relationship of circPVT1 with nucleus pulposus senescence and its mechanism are still unclear. OBJECTIVE: To investigate the expression of circPVT1 in nucleus pulposus cell senescence and to explore its possible mechanism. METHODS: Human nucleus pulposus cells were cultured in vitro, and the senescence of nucleus pulposus cells was induced by ionizing radiation (5 Gy, 6 days). The expression of circPVT1 and let-7 mRNA was detected by real-time quantitative polymerase chain reaction (qRT-PCR). CircPVT1 siRNA and anti-let-7 were transfected into normal nucleus pulposus cells, which were divided into control group, si-NC+anti-NC group, si-circPVT1+anti-NC group, si-NC+anti-let-7 group, and si-circPVT1+anti-let-7 group. The expressions of circPVT1 and let-7 mRNA were detected by qRT-PCR. Cell counting kit-8 assay was used to detect the inhibition of cell proliferation. Plate cell clone formation assay was used to detect colony formation. Cell senescence was detected by SA-β-gal staining. The expressions of p21, p27, let-7 target high mobility group protein A2 (HMGA2) and KRAS were detected by western blot assay. Double luciferase activity assay was used to verify the relationship between let-7 and target regulation of HMGA2 and KRAS. RESULTS AND CONCLUSION: (1) Compared with normal nucleus pulposus cells, the expression of circPVT1 was decreased, while let-7 expression and the positive rate of SA-β-gal staining were increased in the irradiated cells (P < 0.05). (2) Compared with the control group and si-NC+anti-NC group, the si-circPVT1+anti-NC group appeared to have decreased expression of circPVT1 mRNA, HMGA2 and KRAS proteins and number of clones formed as well as increased let-7 mRNA expression, p21, p27 protein expression, cell inhibition rate and positive rate of SA-β-gal staining (P < 0.05). However, opposite changes were found in the si-NC+anti-let-7 group in relative to the control group (P < 0.05). (3) The expression of circPVT1 mRNA, clone formation, and expressions of HMGA2 and KRAS proteins in the si-circPVT1+anti-let-7 group were higher than those in the si-circPVT1+anti-NC group, and lower than those in the si-NC+anti-let-7 group. Let-7 mRNA expression, cell inhibition rate, positive rate of SA-β-gal staining, and expressions of p21 and p27 proteins in the si-circPVT1+anti-let-7 group were lower than those in the si-circPVT1+anti-NC group, and higher than those in the si-NC+anti-let-7 group (P < 0.05). Double luciferase activity assay showed that HMGA2 and KRAS were the targets of let-7. These findings indicate that inhibition of circPVT1 can inhibit the aging of nucleus pulposus cells. The mechanism may be through binding let-7 to inhibit the targeting of HMGA2 and KRAS proteins.

17.
Article in Chinese | WPRIM | ID: wpr-815604

ABSTRACT

@#Objective: To investigate the effect of panax japlcus var polysaccharide (PJPS) on the proliferation and apoptosis of gastric cancer MKN45 cells and its regulatory mechanism. Methods: Human gastric cancer cell lines (HGC27, MGC803, MKN45) and gastric mucosal epithelial cell line GES-1 were selected for this study. Let-7a mimics and let-7a inhibitor were transfected into MKN45 cells; Gastric cancer cell lines were treated with 100 μg/ml PJPS and MKN45 was selected as the subsequent experimental cell line. MKN45 cells were cultured with 0, 10, 50, 100 and 120 μg/ml PJPS, respectively. The proliferation and apoptosis rate of MKN45 cells were detected by CCK-8 and flow cytometry, respectively. Expressions of cell cycle dependent kinase 6 (CDK6) and apoptosis-related proteins in MKN45 cells were detected by Western blotting, and the expression level of miRNAs regulating the proliferation of gastric cancer cells was detectedbyReal-timequantitativePCR(qPCR).TheDualluciferasereportergeneassaywasusedtovalidatethetargeting relationship between let-7a and CDK6. Results: Compared with other gastric cancer cells, 100 μg/ml PJPS significantly inhibited the proliferation of MKN45 cells (P<0.01). At the same time, 100 μg/ml PJPS significantly up-regulated the expression of let-7a in MKN45 cells (P<0.01). The Dual luciferase reporter gene assay confirmed that CDK6 was the target gene of let-7a. Furthermore, PJPS inhibited the expression of CDK6 by up-regulating let-7a, thereby inhibiting the proliferation and inducing apoptosis of MKN45 cells (all P<0.01). Conclusion: PJPS inhibits proliferation and induces apoptosis of gastric cancer MKN45 cells by regulating the let-7a/ CDK6 axis.

18.
Article in Chinese | WPRIM | ID: wpr-880783

ABSTRACT

OBJECTIVE@#To investigate the expression of CDC25A in non- small cell lung cancer (NSCLC) tissues and explore its correlation with the clinicpathological features of the patients and the expressions of let-7a1 and let-7c.@*METHODS@#We collected surgical specimens of pathologically confirmed NSCLC tissues and paired adjacent lung tissues from 44 patients and tissues of benign lung lesions from 9 patients. The expressions of CDC25A protein and mRNA in the tissues were detected by immunohistochemistry and fluorescence quantitative RT-PCR, respectively; the expressions of let-7a1 and let-7c mRNA were detected using tail-adding fluorescence quantitative RT-PCR.@*RESULTS@#The positivity rate of CDC25A protein expression was significantly higher in NSCLC tissues than in the adjacent tissues and benign pulmonary lesions (@*CONCLUSIONS@#The expression level of CDC25A is significantly increased in NSCLC with a negative correlation with Let-7c expression, which identifies CDC25A as a possible downstream target gene of Let-7c.


Subject(s)
Humans , Carcinoma, Non-Small-Cell Lung/genetics , Gene Expression Regulation, Neoplastic , Lung , Lung Neoplasms/genetics , Lymphatic Metastasis , MicroRNAs , RNA, Messenger/genetics , cdc25 Phosphatases
19.
J Cancer Res Ther ; 2019 Oct; 15(5): 1321-1327
Article | IMSEAR | ID: sea-213530

ABSTRACT

Aim of the Study: The purpose of this study was to identify specific circulating microRNAs (miRNAs) and investigate expression level of their target genes for evaluation of pathogenesis of epithelial ovarian cancer (EOC). Materials and Methods: In this study, we have studied on EOC patients' serum and whole blood, healthy control (HC) serum, and whole blood samples. Sixteen serum samples were collected to compare miRNA expression analysis through microarray. According to microarray results, one of the dysregulated miRNA in serum, hsa-let-7d-3p, was validated by RT-qPCR for discriminate two groups. The hsa-let-7d-3p is one of the tumor suppressive let-7d family members. Let-7d is downregulated in numerous types of cancer, including ovarian cancer and directly targets various oncogenes. We analyzed the let-7d targets, which are High Mobility Group A2 (HMGA2) and (Kirsten Rat Sarcoma Viral Oncogene Homolog), as the oncogenes that are associated with EOC. The relation between target genes of hsa-let-7d-3p and EOC has been examined by Pathway Studio. Twenty serum and whole blood samples collected to analyze expression level of target genes were analyzed by real-time PCR. Results: 31 significantly dysregulated miRNAs were identified by microarray in serum. Hsa-let-7d-3p has been selected for the validation, according to P-value and dysregulated level. RT-qPCR results showed that hsa-let-7d-3p could discriminate EOC patients from HC (P = 0.0484, AUC = 0.7). Furthermore, we identified hsa-let-7d-3p's target genes (HMGA2, KRAS) by bioinformatic analysis. The expression level of genes could discriminate patients with EOC from HC, with a power area under the ROC curves (AUC) of 62 and 64.2, respectively. Conclusion: HMGA2 and KRAS could be translationally downregulated by the hsa-let-7d-3p, and the loss of hsa-let-7d-3p expression led to the progression of EOC related to the tumorigenesis, invasion, and metastasis

20.
Article in Chinese | WPRIM | ID: wpr-744537

ABSTRACT

Objective To explore the expression of miRLet-7 family members in breast cancer and its correlation with overall survivals (OS), and to find more effective molecular targets for breast cancer prevention and treatment.Methods Kaplan-Meier (KM) plotter online database was used to analyze the correlation among the expression of Let-7 family members (Let-7a, 7b, 7c, 7d, 7e, 7f, 7g, 7i, miR-98, miR-202) correlated with overall survival (OS) and the prognosis and clinical pathological parameters of breast cancer patients, and Hazard ratio (HR), 95%confidence interval (CI), and P value were determined.ResultsThe study showed that the high expression level of Let-7a, Let-7b, Let-7c, Let-7e, Let-7f, Let-7g, miR-98 and the low expression level of miR-202 was associated with better OS for breast cancer patients (P<0.05).We further assessed the prognostic value of Let-7 in different subtypes and clinical stage.The expression of Let-7a, Let-7b, Let-7f, Let-7g, miR-98, miR-202 was related to clinical stage (P<0.05).Let-7a, Let-7b, Let-7c, Let-7e, Let-7f, Let-7g, miR-98 and miR-202 was related to lymph node status (P<0.05).In triple-negative breast cancers (TNBC), with breast cancer subtype, the expression of Let-7b, Let-7c, Let-7g and miR-202 was significantly correlated to overall survival (P<0.05).Conclusion The Let-7 is significantly correlated with OS in breast cancer patients.The results suggested that members of the Let-7 have different values in predicting the prognosis of breast cancer.Among them, Let-7b, Let-7g and miR-202 are closely related with clinical stage and TNBC, and might promote development of Let-7 as targeted inhibitors for the treatment of breast cancer.

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