apoptosis assay
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2022 ◽  
Vol 15 (1) ◽  
pp. 1-8
Yu-Zhen Li ◽  
Ya Shen ◽  
Lian-Di Gao ◽  
Xin-Xin Chen ◽  

AIM: To explore the effect of miR-184 and miR-205 on the proliferation and metastasis of conjunctival mucosa associated lymphoid tissue (MALT) lymphoma. METHODS: Tissue of tumor and adjacent normal control from 5 patients with conjunctival MALT was included. RPMI8226 cell line was selected to verify the effect of miRNAs in B cells. The function of microRNA on the RPMI8226 cell apoptosis, migration and invasion was evaluated by apoptosis assay and Transwell assay. The mRNA and protein expression were examined by quantitative RT-PCR and Western blotting. The effect of microRNA on regulation of downstream gene expression was evaluated by luciferase report assay. RESULTS: A decreased level of miR-184 and miR-205 was observed in MALT lymphoma tissue. Exogenous miR-184 and miR-205 analogues promoted apoptosis, and inhibited the survival, migration, and invasion of RPMI8226 cells. miR-184 and miR-205 inhibitor reversed the process. The RNA and protein level of RasL10B and TNFAIP8 were downregulated in MALT lymphoma tissue. The exogenous of miR-184 and miR-205 promoted the expression of RasL10B and TNFAIP8. Meanwhile, inhibition of miR-184 and miR-205 repressed the expression of target gene, RasL10B and TNFAIP8. CONCLUSION: miR-184 and miR-205 suppresses the tumorigenesis of conjunctival MALT lymphoma through regulating RasL10B and TNFAIP8.

2022 ◽  
Vol 20 (1) ◽  
Changming Tan ◽  
Jianming Li ◽  
Zhaoshun Yuan ◽  
Yongxin Mu

Abstract Background This study aimed to analyze the role of circular RNA ciRs-126 in hypoxia/reoxygenation cardiac injury (H/R). Methods Expression of ciRs-126 and miR-21 in plasma samples from patients with H/R and healthy controls was determined by RT-qPCR. Correlations were analyzed by linear regression. Overexpression of ciRs-126 and miR-21 was achieved in cardiomyocytes to explore their crosstalk. The roles of ciRs-126 and miR-21 in H/R-induced apoptosis of cardiomyocytes were analyzed using cell apoptosis assay. Results CiRs-126 was upregulated and miR-21 was downregulated in H/R patients. They were inversely correlated across plasma samples from H/R patients. In H/R cardiomyocytes, ciRs-126 was upregulated and miR-21 was downregulated. In cardiomyocytes, ciRs-126 overexpression decreased miR-21 level and reduced the inhibitory effects of miR-21 overexpression on H/R-induced cell apoptosis. Conclusions Circular RNA ciRs-126 may suppress miR-21 expression to promote H/R cardiac injury.

Miganoosh Simonian ◽  
Mozhan Haji Ghaffari ◽  
Ali Salimi ◽  
Ebrahim Mirzadegan ◽  
Niloufar Sadeghi ◽  

Background: Sortilin has an important role in various malignances and can be used as a promising target to eradicate cancer cells. Methods: In this study, the expression of sortilin in 4T1 and MDA-MB231 cell lines was evaluated by flow cytometry and immunocytochemistry. Apoptosis assay was also applied to evaluate apoptosis induction in 4T1 and MDA-MB231 cell lines. Results: Based on cell surface flow cytometry results, anti-sortilin (2D8-E3) mAb could recognize sortilin molecules in 79.2% and 90.3% of 4T1 and MDA-MB231 cell-lines, respectively. The immunocytochemistry staining results confirmed sortilin surface expression. Apoptosis assay indicated that anti-sortilin mAb could induce apoptosis in 4T1 and MDA-MB231 cell lines. Conclusion: Our study revealed the important role of surface sortilin in breast carcinoma cell survival and its possible application as a therapeutic agent in cancer targeted therapies.

2021 ◽  
Katarzyna Niesyto ◽  
Magdalena Skonieczna ◽  
Malgorzata Adamiec-Organisciok ◽  
Dorota Neugebauer

Abstract In vitro cytotoxicity evaluation of linear copolymer (LC) containing choline ionic liquid units and its conjugates with an antibacterial drug in anionic form, i.e. p-aminosalicylate (LC_PAS), clavulanate (LC_CLV), or piperacillin (LC_PIP) was carried out. These systems were tested against normal: human bronchial epithelial cells (BEAS-2B), and cancers: adenocarcinoma human alveolar basal epithelial cells (A549), and human non-small cell lung carcinoma cell line (H1299). Cells viability, after linear copolymer LC and their conjugates addition for 72 hours, was measured at concentrations range of 3.125‑100 μg/mL. The MTT test allowed the designation of IC50 index, which was higher for BEAS-2B, and significantly lower in the case of cancer cell lines. The cytometric analyzes, i.e. Annexin-V FITC apoptosis assay and cell cycle analysis as well as gene expression measurements for interleukins IL6 and IL8 were carried out, and showed pro-inflammatory activity of tested compounds towards cancer cells, while it was not observed against normal cell line. In summary, the anticancer property of linear copolymers with antibacterial drugs was shown.

Dongming Xu ◽  
Ling Lin ◽  
Zhen Chen

Objectives: In this study, we aimed to investigate the effects of LncRNA cardiac autophagy inhibitory factor (CAIF) and miR-20a on the apoptosis of synovial cells in rheumatoid arthritis (RA) and the regulatory mechanism. Patients and methods: Between May 2018 and March 2020, a total of 62 RA patients (24 males, 38 females; mean age: 55.2±4.9 years; range, 42 to 68 years) and 62 controls (24 males, 38 females; mean age: 55.3±4.8 years; range, 41 to 68 years) were included in this study. Plasma samples were collected from all participants. The expression levels of CAIF, mature miR-20a, and miR-20a precursor in these plasma samples were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Correlations were analyzed using linear regression analysis. Overexpression of CAIF was achieved in human fibroblast-like synoviocytes (HFLSs) and the expression levels of mature miR-20a and miR-20a precursor were determined using RT-qPCR. Cell apoptosis was analyzed by cell apoptosis assay. Results: The CAIF was downregulated in RA and positively correlated with the expression of mature miR-20a. In HFLSs, LPS treatment resulted in downregulation of both CAIF and miR-20a in a dose-dependent manner. In HFLSs, overexpression of CAIF did not affect the expression of miR-20a precursor, but upregulated the expression of mature miR-20a. Cell apoptosis analysis showed that overexpression of CAIF and miR-20a inhibited the apoptosis of HFLSs induced by LPS. The combination of overexpression of CAIF and miR-20a showed a stronger effect. Conclusion: The CAIF may suppress the apoptosis of HFLSs in RA by promoting the maturation of miR-20a.

2021 ◽  
Vol 10 (23) ◽  
pp. 5606
Michele Massimino ◽  
Paolo Vigneri ◽  
Stefania Stella ◽  
Elena Tirrò ◽  
Maria Stella Pennisi ◽  

Background: The management of Philadelphia Chromosome-positive (Ph+) hematological malignancies is strictly correlated to the use of BCR-ABL1 tyrosine kinase inhibitors (TKIs). However, these drugs do not induce leukemic stem cells death and their persistence may generate a disease relapse. Published reports indicated that Venetoclax, a selective BCL2 inhibitor, could be effective in Ph+ diseases, as BCL2 anti-apoptotic activity is modulated by BCR-ABL1 kinase. We, therefore, investigated if BCL2 inhibition, alone or combined with Nilotinib, a BCR-ABL1 inhibitor, affects the primitive and committed Ph+ cells survival. Methods: We used Ph+ cells isolated from leukemic patients at diagnosis. To estimate the therapeutic efficacy of BCL2 and BCR-ABL1 inhibition we employed long-term culture, proliferation and apoptosis assay. Immunoblot was used to evaluate the ability of treatment to interfere with the down-stream targets of BCR-ABL1. Results: Blocking BCL2, we observed reduced proliferation and clonogenic potential of CML CD34-positive cells and this cytotoxicity was improved by combination with BCR-ABL1 inhibitor. However, BCL2 inhibition, alone or in combination regiment with BCR-ABL1 inhibitor, did not reduce the self-renewal of primitive leukemic cells, while strongly induced cell death on primary Ph+ Acute Lymphoblastic Leukemia (ALL). Conclusion: Our results suggest that primitive CML leukemic cells are not dependent on BCL2 for their persistence and support that committed CML and Ph + ALL cells are dependent by BCL2 and BCR-ABL1 cooperation for their survival. The antileukemic activity of BCL2 and BCR-ABL1 dual targeting may be a useful therapeutic strategy for Ph+ ALL patients.

2021 ◽  
Xin He ◽  
Jie Sheng ◽  
Yingxi Chen ◽  
Xuan Long ◽  
Wei Yu ◽  

Abstract Purpose Temozolomide-based therapeutic resistance has become the crucial cause of chemotherapy failure in glioma treatment. Long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5) is reported to be downregulated in glioma and to inhibit tumor progression and metastasis. This study aimed to investigate function and potential regulatory mechanism of GAS5 in temozolomide (TMZ) chemoresistance to glioma.Methods qRT-PCR, western blotting and immunofluorescence were used to measure the levels of GAS5 and proteins. RNA-binding protein immunoprecipitation assay was used to analyze the interaction between GAS5 and PTBP1. Flow cytometry apoptosis assay, CCK-8 assay, colony formation assay and nude mice xenograft experiments were used to detect the effects of GAS5 on TMZ resistance in glioma.Results Downregulation of GAS5 might predict a poor prognosis in glioma patients. Overexpression of GAS5 improves the sensitivity to TMZ in glioma cells. Mechanistically, GAS5 could interact with polypyrimidine tract binding protein 1 (PTBP1) to downregulate its expression, thereby inactivating the Wnt/β-catenin pathway. Moreover, GAS5 could increase the anti-tumor effect of TMZ in vivo. Conclusion This study indicated that GAS5 contributed to TMZ chemoresistance of glioma through interacting with PTBP1, and then inhibiting Wnt/β-catenin pathway, which provides a novel approach to develop promising therapeutic strategy.

2021 ◽  
Catherine M. Worsley ◽  
Rob B. Veale ◽  
Elizabeth S. Mayne

Cell death is a key component of mammalian physiology, and can happen as a result of structural damage, or actively as a sequence of programmed cellular processes known as apoptosis. Pathogenic alterations in apoptosis occur in a number of diseases, including cancer, viral infections, autoimmune diseases, immunodeficiencies and degenerative conditions. Developing accurate and reproducible laboratory methods for inducing and detecting apoptosis is vital for research into these conditions. A number of methods are employed to detect cell death, including DNA fragmentation, the TUNEL assay, and electron microscopy although each has its limitations. Flow cytometry allows for the distinction between live, early apoptotic, late apoptotic and necrotic cells. In this protocol we successfully induce apoptosis using chemical treatment and treatment with low pH in solid tumour cell lines, and have optimized detection using the Annexin V apoptosis assay.

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Yiming Pan ◽  
Lingyan Chen ◽  
Ruibai Li ◽  
Yu Liu ◽  
Mengdie Nan ◽  

Tanshinone IIa (TanIIa), an ingredient of Radix Salviae Miltiorrhizae, has an anticancer effect on various solid tumors with high efficiency and low toxicity. Nonetheless, the underlying role of TanIIa in acute promyelocytic leukemia (APL) remains unclear. Here, we revealed that TanIIa drastically inhibited NB4 cell viability with an IC50 value of 31.25 μmol/L. Using flow cytometry apoptosis assay, we identified that TanIIa dose-dependently exacerbated NB4 cell apoptosis. Mechanistically, TanIIa upregulated apoptotic factor levels, namely, cleaved-caspase 9, cleaved-caspase 3, and cleaved-PARP-1. Moreover, we noticed that TanIIa dose-dependently suppressed the PI3K/Akt/mTOR axis. This axis not only functions as an essential antiapoptotic modulator but also serves as a suppressant regulator of autophagy. Correspondingly, we detected the levels of autophagic marker, namely, LC3B, which were increased after the TanIIa treatment. Furthermore, the autophagy inhibitor Baf-A1 could effectively reverse the TanIIa-induced apoptosis, manifesting that TanIIa eliminated NB4 cells in an autophagy-dependent manner. In conclusion, tanshinone IIa exerts anti-APL effects through triggering autophagy and apoptosis in NB4 cells.

2021 ◽  
Vol 11 ◽  
Jiancheng Lv ◽  
Zijian Zhou ◽  
Jingzi Wang ◽  
Xiao Yang ◽  
Hao Yu ◽  

IntroductionCircular RNAs (circRNAs) are non-coding RNAs that have the structure of a covalently closed loop. Increasing data have proven that circRNAs can influence the progression and chemotherapy sensitivity of tumors. Therefore, the underlying function and mechanisms of more circRNAs in progression and chemotherapy resistance are important.MethodsWe conducted RNA sequencing on five pairs of urothelial carcinoma samples and screened for circRNAs. CircFAM114A2 was found to be low expressed in urothelial carcinoma. The functions of circFAM114A2 in urothelial carcinoma were explored by cell cycle assay, IC50 determination assay, cell proliferation assay, apoptosis assay, and tumorigenesis assay.ResultsWe discovered that the levels of circFAM114A2 were decreased in urothelial carcinoma cell lines and tissues. According to follow-up data, urothelial carcinoma patients with higher circFAM114A2 expression had better survival. Importantly, the levels of circFAM114A2 were associated with the histological grade of urothelial carcinoma. CircFAM114A2 could inhibit cell proliferation and block more urothelial carcinoma cells in the G1 phase and then increase the sensitivity of urothelial carcinoma to cisplatin chemotherapy. Mechanistically, circFAM114A2 directly sponged miR-222-3p/miR-146a-5p and subsequently influenced the expressions of the downstream target genes P27/P21, which, in turn, inhibited the progression of urothelial carcinoma and increased the sensitivity of cancer cells to cisplatin chemotherapy.ConclusionCircFAM114A2 could inhibit progression and promote cisplatin sensitivity in urothelial carcinoma through novel circFAM114A2/miR-222-3p/P27 and circFAM114A2/miR-146a-5p/P21 pathways. CircFAM1142 has therefore great potential as a prognostic biomarker and therapeutic target for urothelial carcinoma.

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