scholarly journals Effect of CXCR4 on Apoptosis in Osteosarcoma Cells via the PI3K/Akt/NF-κβ Signaling Pathway

2018 ◽  
Vol 46 (6) ◽  
pp. 2250-2260 ◽  
Author(s):  
Chunming Jiang ◽  
Shenglin Ma ◽  
Runlei Hu ◽  
Xuepeng Wang ◽  
Maoqiang Li ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Luciferase Reporter ◽  
Osteosarcoma Cells ◽  
Down Regulation ◽  
Expression Levels ◽  
Apoptotic Protein ◽  
Effective Manner ◽  
Human Osteosarcoma Cells

Background/Aims: Osteosarcoma, the most common primary bone malignancy, arises from primitive transformed cells of mesenchymal origin with the worldwide increasing morbidity and mortality. Previous studies found apoptosis of osteosarcoma cells was essential for an effective manner to improve the progress of osteosarcoma, and CXCR4 has been demonstrated to be relevant with various tumor progress and metastasis. Methods: The proliferation of cells transfected with CXCR4 shRNA and control shRNA were measured by BrdU assay. Apoptosis was detected by flow cytometry. Apoptotic protein expression levels were detected by Western blot. Caspase activity was detected by Colorimetric Assay Kits using microplate reader. Activation of NF-κβ signaling after CXCR4 down-regulation in osteosarcoma cells was examined by constructing NF-κβ promoter luciferase reporter plasmid. The expression and activation of NF-κβ Signaling relevant protein were analyzed to investigate the relationship between Akt and NF-κβ signaling after the down-regulation of CXCR4 in osteosarcoma cells. Results: Down-regulation of CXCR4 significantly reduced the cell proliferation, while remarkably increased the cell apoptosis and apoptotic protein expression levels in osteosarcoma cells. Furthermore, down-regulation of CXCR4 induced cell apoptosis was caspase dependent in osteosarcoma cells. This study also showed CXCR4 down-regulation induced apoptosis through inhibiting PI3K/Akt/NF-κβ signaling pathway. In addition, endoplasmic reticulum stress (ERS) activation was involved in cell apoptosis induced down-regulation of CXCR4. Knockdown of partial ERS relevant proteins followed down-regulation of CXCR4 significantly inhibited cell apoptosis and the apoptotic protein expression levels. Conclusions: Taken together, the results demonstrated that down-regulation of CXCR4 could induce apoptosis of human osteosarcoma cells through inhibiting PI3K/Akt/NF-κβ signaling pathway, indicating that CXCR4 could be vital for the clinical therapy of osteosarcoma.

scholarly journals The Effect and Mechanism of KLF7 in the TLR4/NF-κB/IL-6 Inflammatory Signal Pathway of Adipocytes

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Meixiu Zhang ◽  
Cuizhe Wang ◽  
Jinxiu Wu ◽  
Xiaodan Ha ◽  
Yuchun Deng ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Transcriptional Activation ◽  
Inflammatory Cytokine ◽  
Luciferase Reporter ◽  
Signal Pathways ◽  
High Concentration ◽  
Inflammatory Signaling ◽  
Expression Levels ◽  
Mrna And Protein Expression

Objective. To investigate the role and possible molecular mechanism of Krüppel-like factor 7 (KLF7) in the TLR4/NF-κB/IL-6 inflammatory signaling pathway activated by free fatty acids (FFA). Methods. The mRNA and protein expression levels of KLF7 and the factors of TLR4/NF-κB/IL-6 inflammatory signal pathways were detected by qRT-PCR and Western blotting after cell culture with different concentrations of palmitic acid (PA). The expression of KLF7 or TLR4 in adipocytes was upregulated or downregulated; after that, the mRNA and protein expression levels of these key factors were detected. KLF7 expression was downregulated while PA stimulated adipocytes, and then the mRNA and protein expressions of KLF7/p65 and downstream inflammatory cytokine IL-6 were detected. The luciferase reporter assay was used to determine whether KLF7 had a transcriptional activation effect on IL-6. Results. (1) High concentration of PA can promote the expression of TLR4, KLF7, and IL-6 in adipocytes. (2) TLR4 positively regulates KLF7 expression in adipocytes. (3) KLF7 positively regulates IL-6 expression in adipocytes. (4) PA promotes IL-6 expression via KLF7 in adipocytes. (5) KLF7 has a transcriptional activation on IL-6. Conclusion. PA promotes the expression of the inflammatory cytokine IL-6 by activating the TLR4/KLF7/NF-κB inflammatory signaling pathway. In addition, KLF7 may directly bind to the IL-6 promoter region and thus activate IL-6.

scholarly journals MiR-103a-3p antagonism attenuates pneumonia via inactivating the PI3K/AKT/NF-κB signaling pathway by targeting PTEN

2020 ◽  
Author(s):  
Lin Xu ◽  
Qingying Song ◽  
Zhanghong Ouyang ◽  
Xiangyan Zhang ◽  
Cheng Zhang
Keyword(s):  
Cell Viability ◽  
Signaling Pathway ◽  
Cell Apoptosis ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Expression Levels ◽  
Tensin Homolog ◽  
Factor Α

Abstract Pneumonia accounts for approximately 15% mortalities in adolescents worldwide. MicroRNAs (miRNAs) regulate numerous diseases including pneumonia. miRNA and mRNA expression levels were detected by real time polymerase chain reaction (RT-qPCR). Protein expression levels were determined by enzyme-linked immunosorbent assay (ELISA) and western blot. The interaction between phosphatase and tensin homolog on chromosome ten (PTEN) and miR-103a-3p was explored by dual luciferase reporter assay. Cell viability and cell apoptosis were detected by cell Counting Kit-8 (CCK-8) and flow cytometry. Herein, we discovered that PTEN was decreased and miR-103a-3p was overexpressed in Ana-1 cells of in vitro pneumonia model. miR-103a-3p downregulated the expression levels of PTEN. AntagomiR-103a-3p reversed the increased cell apoptosis and decreased cell viability and inflammatory cytokine expression levels (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6) induced by LPS in Ana-1 cells by PTEN. AntagomiR-103a-3p inhibited the activation of PTEN/PI3K/AKT/NF-κB signaling pathway induced by LPS in Ana-1 cells. Taken together, our findings exhibited that miR-103a-3p attenuated LPS induced pneumonia by blocking the activation of PTEN/PI3K/AKT/NF-κB signaling pathway and the following cell apoptosis as well as release of proinflammatory cytokines, suggesting that miR-103a-3p might serve as a novel therapeutic target for the treatment of pneumonia.

scholarly journals microRNA-27a-3p down-regulation inhibits malignant biological behaviors of ovarian cancer by targeting BTG1

Open Medicine ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. 577-585 ◽  
Author(s):  
Enfang Li ◽  
Ke Han ◽  
Xuan Zhou
Keyword(s):  
Ovarian Cancer ◽  
Protein Expression ◽  
Cell Apoptosis ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Luciferase Reporter Gene ◽  
Down Regulation ◽  
Over Expression ◽  
Bax Protein ◽  
Gene Assay

AbstractOvarian cancer is the most deadly malignant tumor. MicroRNA-27a-3p (miR-27a-3p) was a tumor oncogene in various cancers. However, the role and mechanism of miR-27a-3p in ovarian cancer are still unknown. In this study, we found that miR-27a-3p over-expression could significantly promote the viability of SK-OV-3 cells, enhance cell migration and invasion, and reduce cell apoptosis. Besides, results from western blot assay showed that miR-27a-3p over-expression could increase Bcl-2 protein expression and decrease Bax protein expression. Furthermore, TargetScan and the dual luciferase reporter gene assay revealed that BTG anti-proliferation factor 1 (BTG1) was a direct target of miR-27a-3p. In addition, we found that miR-27a-3p down-regulation suppressed SK-OV-3 cell viability, migration and invasion, and promoted cell apoptosis. All the effects of miR-27a-3p down-regulation on SK-OV-3 cells were reversed by BTG1-siRNA. Therefore, miR-27a-3p/BTG1 axis may be a new potential target for the treatment of ovarian cancer.

scholarly journals Decitabine shows anti-acute myeloid leukemia potential via regulating the miR-212-5p/CCNT2 axis

2020 ◽  
Vol 15 (1) ◽  
pp. 1013-1023
Author(s):  
Lina Xing ◽  
Jinhai Ren ◽  
Xiaonan Guo ◽  
Shukai Qiao ◽  
Tian Tian
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Viability ◽  
Cell Apoptosis ◽  
Myeloid Leukemia ◽  
Luciferase Reporter ◽  
Expression Levels ◽  
Proliferation And Apoptosis ◽  
Polymerase Chain ◽  
The Relationship ◽  
Acute Myeloid

AbstractPrevious research has revealed the involvement of microRNA-212-5p (miR-212-5p) and cyclin T2 (CCNT2) in acute myeloid leukemia (AML). However, whether the miR-212-5p/CCNT2 axis is required for the function of decitabine in AML has not been well elucidated. Quantitative reverse transcription-polymerase chain reaction was used to examine enrichment of miR-212-5p. The relationship between CCNT2 and miR-212-5p was verified by the luciferase reporter assay. Cell apoptosis was evaluated by flow cytometry and western blot. CCK-8 assay was performed to determine cell viability. Decitabine significantly repressed cell viability, while promoted cell apoptosis. Meanwhile, the expression levels of cyclinD1, CDK4, and Bcl-2 were suppressed in cells with decitabine exposure, but Bax and caspase-3 expression levels were upregulated. Besides, miR-212-5p upregulation had the similar function with decitabine in AML cell proliferation and apoptosis. Subsequently, restoration of CCNT2 attenuated miR-212-5p overexpression-induced effects in Kasumi-1 and SKNO-1 cells. In addition, miR-212-5p depletion reversed decitabine-induced CCNT2 downregulation. The miR-212-5p/CCNT2 axis had an implication in the anti-leukemic effect of decitabine in AML.

scholarly journals miR-636 inhibits EMT, cell proliferation and cell cycle of ovarian cancer by directly targeting transcription factor Gli2 involved in Hedgehog pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jiong Ma ◽  
Chunxia Zhou ◽  
Xuejun Chen
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Luciferase Reporter ◽  
Cell Proliferation And Migration ◽  
Hh Signaling ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background Hedgehog (Hh) signaling pathway, which is essential for cell proliferation and differentiation, is noted to be aberrantly activated in tumor from increasing studies in recent years. MicroRNAs (miRNAs) as an important non-coding RNA in cells have been proven to possess a regulatory role specific to the Hh signaling pathway. Here, in vitro and in vivo cellular/molecular experiments were adopted to clarify the regulatory mechanism linking miR-636 to the Hh signaling pathway in ovarian cancer (OVC). Methods Protein–protein interaction analysis was performed to identify the hub gene in the Hh pathway. TargetScan database was used to predict the potential upstream regulators for Gli2. qRT-PCR was performed to test the expression of miR-636, while Western blot was conducted to detect the expression of proteins related to the Hh pathway and epithelial-mesenchymal transition (EMT). For cell functional experiments, HO-8910PM OVC cell line was used. MTT assay and wound healing assay were used to measure the effect of miR-636 on cell proliferation and migration. Flow cytometry was carried out to examine the effect of miR-636 on cell cycle, and Western blot was used to identify the change in expression of Hh and EMT-related proteins. Dual-luciferase reporter gene assay was implemented to detect the targeting relationship between miR-636 and Gli2. Xenotransplantation models were established for in vivo examination. Results Gli2 was identified as the hub gene of the Hh pathway and it was validated to be regulated by miR-636 based on the data from TargetScan and GEO databases. In vitro experiments discovered that miR-636 was significantly lowly expressed in OVC cell lines, and overexpressing miR-636 significantly inhibited HO-8910PM cell proliferation, migration and induced cell cycle arrest in G0/G1 phase, while the inhibition of miR-636 caused opposite results. Dual-luciferase reporter gene assay revealed that Gli2 was the target gene of miR-636 in OVC. Besides, overexpressed miR-636 decreased protein expression of Gli2, and affected the expression of proteins related to the Hh signaling pathway and EMT. Rescue experiments verified that overexpression of Gli2 reversed the inhibitory effect of miR-636 on HO-8910PM cell proliferation and migration, and attenuated the blocking effect of miR-636 on cell cycle. The xenotransplantation experiment suggested that miR-636 inhibited cell growth of OVC by decreasing Gli2 expression. Besides, overexpressing Gli2 potentiated the EMT process of OVC cells via decreasing E-cadherin protein expression and increasing Vimentin protein expression, and it reversed the inhibitory effect of miR-636 on OVC cell proliferation in vivo. Conclusion miR-636 mediates the activation of the Hh pathway via binding to Gli2, thus inhibiting EMT, suppressing cell proliferation and migration of OVC. Trial registration: The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of The Second Affiliated hospital of Zhejiang University School of Medicine (IR2019001235). Written informed consent was obtained from individual or guardian participants.

scholarly journals The silencing of long non-coding RNA ANRIL suppresses invasion, and promotes apoptosis of retinoblastoma cells through the ATM-E2F1 signaling pathway

2018 ◽  
Vol 38 (6) ◽  
Author(s):  
Yang Yang ◽  
Xiao-Wei Peng
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Inhibitory Effect ◽  
Down Regulation ◽  
Reading Frame ◽  
Non Coding Rna ◽  
Retinoblastoma Cells ◽  
Human Retinoblastoma ◽  
Y79 Cells ◽  
Long Non Coding Rna

As one of the most common primary intraocular carcinomas, retinoblastoma generally stems from the inactivation of the retinoblastoma RB1 gene in retinal cells. Antisense non-coding RNA in the INK4 locus (ANRIL), a long non-coding RNA (lncRNA), has been reported to affect tumorigenesis and progression of various cancers, including gastric cancer and non-small cell lung cancer. However, limited investigations emphasized the role of ANRIL in human retinoblastoma. Hence, the current study was intended to investigate the effects of ANRIL on the proliferation, apoptosis, and invasion of retinoblastoma HXO-RB44 and Y79 cells. The lentivirus-based packaging system was designed to aid the up-regulation of ANRIL and ATM expressions or employed for the down-regulation of ANRIL in human retinoblastoma cells. Afterward, ANRIL expression, mRNA and protein expression of ATM and E2F1, and protein expression of INK4b, INK4a, alternate reading frame (ARF), p53 and retinoblastoma protein (pRB) were determined in order to elucidate the regulation effect associated with ANRIL on the ATM-E2F1 signaling pathway. In addition, cell viability, apoptosis, and invasion were detected accordingly. The results indicated that the down-regulation of ANRIL or up-regulation of ATM led to an increase in the expressions of ATM, E2F1, INK4b, INK4a, ARF, p53, and pRB. The silencing of ANRIL or up-regulation of ATM exerted an inhibitory effect on the proliferation and invasion while improving the apoptosis of HXO-RB44 and Y79 cells. In conclusion, the key observations of our study demonstrated that ANRIL depletion could act to suppress retinoblastoma progression by activating the ATM-E2F1 signaling pathway. These results provide a potentially promising basis for the targetted intervention treatment of human retinoblastoma.

Xihuang Pill inhibits the development of DMBA combined estrogen and progesterone induced breast precancerous lesions rats by PI3K/AKT/mTOR signaling pathway

2021 ◽  
Author(s):  
Huanfang Fan ◽  
Dehui Li ◽  
Na Guo ◽  
Chunxia Sun ◽  
Jingfei Dong ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathway ◽  
Breast Tissue ◽  
Precancerous Lesions ◽  
Precancerous Lesion ◽  
Mtor Signaling ◽  
Mtor Signaling Pathway ◽  
Vegf Mrna ◽  
Expression Levels ◽  
Pten Mrna

Abstract Objective. To study the inhibitory effect of Xihuang Pill on the development of DMBA combined estrogen and progesterone induced breast precancerous lesions rats by PI3K/AKT/mTOR signaling pathway, and to explore the effect of Xihuang Pill in preventing and treating breast cancer. Method. Establishment of a rat model of breast precancerous lesion with DMBA combined estrogen and progesterone sequential induction for 10 weeks. Xihuang Pill was administered by gavage continuously for 4 weeks. Take rat breast tissue and stain with hematoxylin- eosin (HE). The pathomorphological changes were observed with light microscope; TUNEL staining to detect cell apoptosis in breast tissue; Western blot was used to detect the protein expression of P-PI3K, P-AKT (S473), P-AKT (T308), PTEN, P-Tuberin/TSC2, P-Tuberin (p-S939), p-mTOR, P-4E-BP1 in breast tissues. The qRT-PCR was used to detect the gene expression of PTEN mRNA and VEGF mRNA. Immunohistochemistry was used to detect the protein expression of P-S6, p-p70s6k and VEGF. Result. Compared with the disease model group, the low, middle and high dose Xihuang Pill groups could significantly reduce the degree of breast pathology, and the number of apoptosis of breast precancerous lesions cells increased with the increase of Xihuang Pill dose; The expression levels of P-PI3K, P-AKT (S473), P-AKT (T308), p-mTOR, P-4E-BP1, p-S6, p-p70S6K, VEGF protein and VEGF mRNA dropped with the increase of Xihuang Pill dose. The expression levels of PTEN, P-Tuberin/TSC2, P-Tuberin (p-S939) protein and PTEN mRNA elevated with the increase of Xihuang Pill dose. Conclusion. Xihuang Pill can promote the apoptosis of breast precancerous lesion cells and reduce the proliferation of vascular endothelial cells, and then inhibit the progression of breast precancerous lesions. Its mechanism probably associated with the regulation of PI3K/AKT/mTOR pathway related gene protein expression.

Overexpressed miR-375-Loaded Restrains Development of Cervical Cancer Through Down-Regulation of Frizzled Class Receptor 4 (FZD4) with Liposome Nanoparticle as a Carrier

2021 ◽  
Vol 17 (9) ◽  
pp. 1882-1889
Author(s):  
Suqin Wang ◽  
Lina Xu ◽  
Zhiqiang Zhang ◽  
Ping Wang ◽  
Rong Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
M Phase ◽  
The Impact

Dysregulation expression of miR-375 is noted to correlate with progression of cervical cancer. This study attempted to investigate the impact of overexpressed miR-375-loaded liposome nanoparticles on proliferation of cervical cancer (CC), to provide an insight on pathogenesis of CC disorder. CC cells were co-cultured with pure liposome nanoparticles (empty vector group), miR-375 agonist-loaded liposome nanoparticles, or transfected with miR-375 antagonist. Besides, some cells were exposed to TGF-β/Smads signaling pathway inhibitor or activator whilst cell proliferation was assessed by MTT assay, and expressions of FZD4 and miR-375 were determined. Western blot analysis was carried out to detect the expression of TGF-β pathway factors (TGF-β, Smad2, Smad7, p-Smad2) and its downstream Smads pathway. The interaction between miR-375 and FZD4 was evaluated by dual-luciferase reporter gene assay. Overexpression of miR-375 induced arrest at the G0/G1 phase of cell cycle and elevation of Smad2 protein expression (P <0.05), with lower expressions of TGF-β, Smad7, p-Smad2, and FZD4, while transfection with miR-375 inhibitor exhibited opposite activity. Presence of miR-375 agonist-loaded liposome nanoparticles induced decreased cell proliferation. There was a targeting relationship between miR-375 and FZD4, and administration with TGF-β/Smads agonist resulted in increased miR-375 and Smad2 expressions, as well as decreased TGF-β, Smad7, p-Smad2, FZD4 protein expression, and the number of S phase and G2/M phase cells (P < 0.05). The signaling inhibitor oppositely suppressed cell proliferation decreasing miR-375 expression. miR-375-loaded liposome nanoparticles activated TGF-β/Smads signaling pathway to restrain cell cycle and suppress cell division, and proliferation through targeting FZD4 in CC. Its molecular mechanism is related to activation of TGF-β/Smads signaling pathway.

Sign in / Sign up

Export Citation Format

Share Document