ERK1/2 Signaling Pathway Activated by EGF Promotes Proliferation, Transdifferentiation, and Migration of Cultured Primary Newborn Rat Lung Fibroblasts

Background. Bronchopulmonary dysplasia (BPD) is a common and serious complication in premature infants. Lung fibroblasts (LFs) are present in the extracellular matrix and participate in pulmonary development in response to BPD. The aim of this study was to investigate the effect of extracellular signal-regulated kinase (ERK) on LFs cultured from newborn rats. Material and Methods. Primary LFs were isolated and treated with epidermal growth factor (EGF, 20 ng/mL) in the presence or absence of an ERK inhibitor, PD98059 (10 μmol/L). Phosphorylated ERK1/2 (p-ERK1/2) protein levels were determined using immunocytochemistry, western blotting, and real-time reverse transcription quantitative (RT–q)PCR. LF proliferation was examined by flow cytometry and a cell counting kit-8 assay. LF transdifferentiation was examined by protein and mRNA expression of α-smooth muscle actin (α-SMA) by immunocytochemistry, western blotting, and RT–qPCR. LF migration was examined by the transwell method. Results. Phosphorylated ERK1/2, which was activated by EGF, promoted LF proliferation by accelerating cell-cycle progression from the G1 to S phase. After treatment with PD98059, the expression of p-ERK1/2 in LFs, cellular proliferation, and the percentage of cells in S phase were significantly decreased. Phosphorylated ERK1/2 also promoted the differentiation of LFs into myofibroblasts through increased α-SMA synthesis and migration. Conclusion. The activation of ERK promotes proliferation, transdifferentiation, and migration of lung fibroblasts from newborn rats.

Download Full-text

Loss of PR55α promotes proliferation and metastasis by activating MAPK/AKT signaling in hepatocellular carcinoma

Cancer Cell International ◽

10.1186/s12935-021-01796-0 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

JiangSheng Zhao ◽

GuoFeng Chen ◽

Jingqi Li ◽

Shiqi Liu ◽

Quan Jin ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle Progression ◽

Lung Metastases ◽

Cell Counting ◽

Migration And Invasion ◽

Signal Pathways ◽

And Migration ◽

Healthy Liver

Abstract Background PR55α plays important roles in oncogenesis and progression of numerous malignancies. However, its role in hepatocellular carcinoma (HCC) is unclear. This study aims to characterize the functions of PR55α in HCC. Methods PR55α expressions in HCC tissues and paired healthy liver samples were evaluated using Western blot and tissue microarray immunohistochemistry. We knocked down the expression of PR55α in SMMC-7721 and LM3 cell lines via small interfering and lentivirus. In vitro cell counting, colony formation, migration and invasion assays were performed along with in vivo xenograft implantation and lung metastases experiments. The potential mechanisms involving target signal pathways were investigated by RNA-sequencing. Results PR55α expression level was suppressed in HCC tissues in comparison to healthy liver samples. Decreased PR55α levels were correlated with poorer prognosis (P = 0.0059). Knockdown of PR55α significantly promoted cell proliferation and migration, induced repression of the cell cycle progression and apoptosis in vitro while accelerating in vivo HCC growth and metastasis. Mechanistic analysis indicated that PR55α silencing was involved with MAPK/AKT signal pathway activation and resulted in increased phosphorylation of both AKT and ERK1/2. Conclusions This study identifies PR55α to be a candidate novel therapeutic target in the treatment of HCC.

Download Full-text

Serum miR-375 Levels Are Closely Related to Disease Progression from HBV Infection to HBV-Related Hepatocellular Carcinoma

BioMed Research International ◽

10.1155/2020/5819385 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Weilu Zhang ◽

Ting Fu ◽

Zhenjun Guo ◽

Ye Zhang ◽

Lei Zhang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Hepatitis B ◽

Disease Progression ◽

Cellular Proliferation ◽

Hbv Infection ◽

Cell Counting ◽

Specificity And Sensitivity ◽

And Migration ◽

Proliferation And Migration

Background. There is an urgent need to identify ideal serological biomarkers that not only are closely related to disease progression from hepatitis B virus (HBV) infection to hepatocellular carcinoma (HCC) but also have high specificity and sensitivity. We conducted this study to analyze whether miR-375 has a potential value in the early prediction of the progression from HBV-related hepatitis or cirrhosis to HCC. Methods. A total of 177 participants were enrolled. Receiver operating characteristic (ROC) curve was used to evaluate the predictive capability of selected miR-375 for HBV-HCC. We upregulated the miR-375 expression in HepG2, HepG2.2.15, and HepAD38 cells to determine its effect on cellular proliferation and migration, in vitro using Cell Counting Kit-8 (CCK-8) assays. Results. Serum miR-375 levels decreased in order from healthy controls to chronic hepatitis B (CHB) without cirrhosis, followed by cirrhosis, and finally, HBV-HCC patients. miR-375 levels were significantly lower in HBeAg-positive and HBV DNA-positive patients than negative (P<0.05) and significantly lower in patients with elevated alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) than normal levels (P<0.05). miR-375 might be a biomarker for HBV-HCC, with a high area under the curve (AUC) of 0.838 (95% confidence interval (CI) 0.780 to 0.897; sensitivity: 73.9%; specificity: 93.0%). The AUC (0.768 vs. 0.584) and sensitivity (93.8% vs. 75.0%) for miR-375 were higher than those for AFP. The overexpression of miR-375 noticeably inhibited proliferation and migration in HepG2, HepG2.2.15, and HepAD38, especially in HepG2.2.15 and HepAD38, which are stably infected with HBV. Conclusions. Serum miR-375 levels are closely related to disease progression from HBV-related hepatitis or cirrhosis to HCC.

Download Full-text

TBK1 regulates YAP/TAZ and fibrogenic fibroblast activation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00324.2019 ◽

2020 ◽

Vol 318 (5) ◽

pp. L852-L863 ◽

Cited By ~ 1

Author(s):

Aja Aravamudhan ◽

Andrew J. Haak ◽

Kyoung Moo Choi ◽

Jeffrey A. Meridew ◽

Nunzia Caporarello ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Large Scale ◽

Smooth Muscle Actin ◽

Lung Fibroblasts ◽

Cell Contact ◽

Actin Stress Fiber ◽

Fibroblast Activation ◽

Protein Levels ◽

Major Cell Type ◽

Interfering Rna

Idiopathic pulmonary fibrosis (IPF) results in scarring of the lungs by excessive extracellular matrix (ECM) production. Resident fibroblasts are the major cell type involved in ECM deposition. The biochemical pathways that facilitate pathological fibroblast activation leading to aberrant ECM deposition are not fully understood. Tank binding protein kinase-1 (TBK1) is a kinase that regulates multiple signaling pathways and was recently identified as a candidate regulator of fibroblast activation in a large-scale small-interfering RNA (siRNA) screen. To determine the effect of TBK1 on fibroblast activation, TBK1 was inhibited pharmacologically (MRT-68601) and genetically (siRNA) in normal and IPF human lung fibroblasts. Reducing the activity or expression of TBK1 led to reduction in α-smooth muscle actin stress fiber levels by 40–60% and deposition of ECM components collagen I and fibronectin by 50% in TGF-β-stimulated normal and IPF fibroblasts. YAP and TAZ are homologous mechanoregulatory profibrotic transcription cofactors known to regulate fibroblast activation. TBK1 knockdown or inhibition decreased the total and nuclear protein levels of YAP/TAZ. Additionally, low cell-cell contact and increased ECM substrate stiffness augmented the phosphorylation and activation of TBK1, consistent with cues that regulate YAP/TAZ. The action of TBK1 toward YAP/TAZ activation was independent of LATS1/2 and canonical downstream TBK1 signaling mediator IRF3 but dependent on proteasomal machinery of the cell. This study identifies TBK1 as a fibrogenic activator of human pulmonary fibroblasts, suggesting TBK1 may be a novel therapeutic target in pulmonary fibrosis.

Download Full-text

Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

Frontiers in Genetics ◽

10.3389/fgene.2021.667724 ◽

2021 ◽

Vol 12 ◽

Author(s):

Juan Tan ◽

Weinan Pan ◽

Huilin Chen ◽

Yafang Du ◽

Peiyong Jiang ◽

...

Keyword(s):

Oxidative Stress ◽

Cell Cycle ◽

Cell Cycle Progression ◽

Circular Rna ◽

Suppressive Effect ◽

Cell Counting ◽

Luciferase Reporter ◽

Protein Levels ◽

Stress Markers ◽

Transcription Factor 4

Circular RNA (circRNA) is an important factor for regulating the progression of many cardiovascular diseases, including acute myocardial infarction (AMI). However, the role of circ_0124644 in AMI progression remains unclear. Hypoxia was used to induce cardiomyocytes injury. The expression of circ_0124644, microRNA (miR)-590-3p, and SRY-box transcription factor 4 (SOX4) mRNA was measured by qRT-PCR. Cell counting kit 8 (CCK8) assay and flow cytometry were utilized to detect cell viability, cell cycle progression, and apoptosis. The protein levels of apoptosis markers and SOX4 were determined by western blot (WB) analysis, and the levels of oxidative stress markers were assessed using commercial Assay Kits. Dual-luciferase reporter assay, RIP assay, and RNA pull-down assay were employed to confirm the interaction between miR-590-3p and circ_0124644 or SOX4. Circ_0124644 was upregulated in AMI patients and hypoxia-induced cardiomyocytes. Hypoxia could inhibit cardiomyocytes viability, cell cycle process, and promote apoptosis and oxidative stress, while silencing circ_0124644 could alleviate hypoxia-induced cardiomyocytes injury. In terms of mechanism, circ_0124644 could target miR-590-3p. MiR-590-3p overexpression could relieve hypoxia-induced cardiomyocytes injury. Also, the suppressive effect of circ_0124644 knockdown on hypoxia-induced cardiomyocytes injury could be reversed by miR-590-3p inhibitor. Moreover, SOX4 was found to be a target of miR-590-3p, and its overexpression also could reverse the regulation of miR-590-3p on hypoxia-induced cardiomyocytes injury. Circ_0124644 silencing could alleviate hypoxia-induced cardiomyocytes injury by regulating the miR-590-3p/SOX4 axis, suggesting that it might be a target for alleviating AMI.

Download Full-text

TGF-β1/SMOC2/AKT and ERK axis regulates proliferation, migration, and fibroblast to myofibroblast transformation in lung fibroblast, contributing with the asthma progression

Hereditas ◽

10.1186/s41065-021-00213-w ◽

2021 ◽

Vol 158 (1) ◽

Author(s):

Yuebin Wang ◽

Huike Yang ◽

Xian Su ◽

Anqiang Cao ◽

Feng Chen ◽

...

Keyword(s):

Calcium Binding ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Transforming Growth Factor Β ◽

Chronic Respiratory Disease ◽

Lung Fibroblasts ◽

Promoting Effect ◽

And Migration ◽

Tgf Β1 ◽

Proliferation And Migration

Abstract Background Asthma is a common chronic respiratory disease that influences 300 million people all over the world. However, the pathogenesis of asthma has not been fully elucidated. It has been reported that transforming growth factor-β (TGF-β) can activate myofibroblasts. Moreover, the fibroblast to myofibroblast transformation (FMT) can be triggered by TGF-β, which is a major mediator of subepithelial fibrosis. Secreted modular calcium-binding protein 2 (SMOC2) is a member of cysteine (SPARC) family and is involved in the progression of multiple diseases. However, its role in asthma remains poorly understood. RT-qPCR evaluated the expression of SMOC2. Bromodeoxyuridine assay and wound-healing assay detected the proliferation and migration of lung fibroblasts, respectively. IF staining was performed to assess the expression of α-smooth muscle actin (α-SMA). Western blot analysis detected the levels of proteins. Flow cytometry was utilized for determination of the number of myofibroblasts. Results We found the expression of SMOC2 was upregulated by the treatment of TGF-β1 in lung fibroblasts. In addition, SMOC2 promoted the proliferation and migration of lung fibroblasts. More importantly, SMOC2 accelerated FMT of lung fibroblasts. Furthermore, SMOC2 was verified to control the activation of AKT and ERK. Rescue assays showed that the inhibition of AKT and ERK pathway reversed the promoting effect of SMOC2 overexpression on proliferation, migration and FMT in lung fibroblasts. Conclusions This work demonstrated that SMOC2 modulated TGF-β1-induced proliferation, migration and FMT in lung fibroblasts and may promote asthma, which potentially provided a novel therapeutic target for the management of asthma.

Download Full-text

Long Non-coding RNA CRNDE Exacerbates NPC Advancement Mediated by the miR-545-5p/CCND2 Axis

10.21203/rs.3.rs-882684/v1 ◽

2021 ◽

Author(s):

Sichen Ge ◽

Chengyi Jiang ◽

Min Li ◽

Zhongqiang Cheng ◽

Xiaojia Feng

Keyword(s):

Cell Cycle ◽

Regulatory Networks ◽

Cell Cycle Progression ◽

Cellular Proliferation ◽

Xenograft Model ◽

Cell Counting ◽

Luciferase Reporter ◽

Related Protein ◽

Model Studies

Abstract Background: Previous studies indicated CRNDE to have a pivotal part within tumorigenesis. Notwithstanding, precise details on CRNDE activities within NPC are still uncertain. The investigation described in this article served to focus in greater depth on the mechanistics regarding CRNDE, together with all associated regulatory networks, on nasopharyngeal carcinoma (NPC) and its treatment possibilities.Methods：Quantitative real-time polymerase chain reaction (RT-qPCR) analyzed CRNDE, miR-545-5p and CCND2 expression within NPCs and representative cell lineages. CCK-8 cell counting-, EdU-, wound-healing- / transwell-assays analyzed cellular proliferation, migrative, together with invasive properties. Apoptosis / cell cycle progression were scrutinized through flow cytometry. Dual-luciferase reporter assays validated CRNDE / miR-545-5p / CCND2 interplay. Proteomic expression of apoptosis-related protein, EMT-related protein and CCND2 protein were evaluated through Western blotting. In addition, Ki67 expression was evaluated through immunohistochemical staining. The effect of CRNDE in vivo was assessed by nude murine xenograft model studies.Results: This study demonstrated up-regulated expression of CRNDE and CCND2 within NPC tissues /cell lines. Meanwhile, miR-545-5p was downregulated. CRNDE knockdown or miR-545-5p over-expression drastically reduced NPC proliferative, migrative and invasive properties, promoted apoptosis / altered cell cycle, and inhibited the expression of CCND2. However, miR-545-5p downregulation had opposing effects. All inhibiting functions generated by CRNDE downregulation upon NPC progression could be counterbalanced or synergistically exacerbated, depending on miR-545-5p downregulation or upregulation, respectively. Multiple-level investigations revealed CRNDE to serve as a sponge for miR-545-5p and can target CCND2 within NPCs.Conclusions：CRNDE increases CCND2 expression by competitive binding with miR-545-5p, thus accelerating the development of NPC. This provides potential therapeutic targets and prognostic markers against NPC.

Download Full-text

Inhibition of PLOD3 Suppresses Proliferation, Invasion and Migration of Breast Cancer Cells

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2019.2171 ◽

2019 ◽

Vol 9 (11) ◽

pp. 1528-1534

Author(s):

Shiqiong Su ◽

Qing Ni ◽

Jing Hou

Keyword(s):

Breast Cancer ◽

Cell Lines ◽

Western Blotting ◽

Molecular Mechanisms ◽

Treatment Strategies ◽

Cell Counting ◽

Matrix Metalloproteinase 2 ◽

Invasion And Migration ◽

And Migration ◽

Interfering Rna

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) has been reported to be involved in various human cancers. However, the function of PLOD3 in breast cancer (BC) has not been addressed. This research attempted to probe the effects and molecular mechanisms of PLOD3 in BC. The expression of PLOD3 was examined by Western blotting and RT-qPCR in several BC cell lines and nontumorigenic breast MCF-10A cells. Then, PLOD3 was silenced by transfecting with small interfering RNA (siRNA). Cell proliferation was measured by Cell Counting Kit-8 assay and cell cycle was evaluated by flow cytometry assay after transfection. Subsequently, wound healing assay and Transwell assay were exploited for detecting the abilities of cell invasion and migration, respectively. In addition, the expression of proliferation- and migration-related genes were examined by Western blotting. The results revealed that the expression of PLOD3 was upregulated in BC cell lines compared with MCF-10A cells. PLOD3 silencing suppressed the proliferative ability of BC cells, enhanced the ratio of cells in the G1 and G2 phases and reduced those in the S phase. Moreover, the expression of Ki67 and cyclinD1 were significantly downregulated, accompanied by an upregulation in p27 expression after transfection with PLOD3 siRNA. Furthermore, inhibition of PLOD3 restrained invasion and migration of BC cells coupled with a reduced expression of matrix metalloproteinase 2 (MMP2) and MMP9. The explorations unveiled that PLOD3 silencing restrained proliferation, invasion and migration of BC cells, which provides theoretical basis and treatment strategies for the treatment of BC.

Download Full-text

Sirtuin 7 is decreased in pulmonary fibrosis and regulates the fibrotic phenotype of lung fibroblasts

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00473.2016 ◽

2017 ◽

Vol 312 (6) ◽

pp. L945-L958 ◽

Cited By ~ 23

Author(s):

Anne E. Wyman ◽

Zahid Noor ◽

Rita Fishelevich ◽

Virginia Lockatell ◽

Nirav G. Shah ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Smooth Muscle Actin ◽

Accelerated Aging ◽

Lung Fibroblasts ◽

Mrna Levels ◽

Muscle Actin ◽

Limited Data ◽

Severe Condition ◽

Protein Levels ◽

Induced Changes

Pulmonary fibrosis is a severe condition with no cure and limited therapeutic options. A better understanding of its pathophysiology is needed. Recent studies have suggested that pulmonary fibrosis may be driven by accelerated aging-related mechanisms. Sirtuins (SIRTs), particularly SIRT1, SIRT3, and SIRT6, are well-known mediators of aging; however, limited data exist on the contribution of sirtuins to lung fibrosis. We assessed the mRNA and protein levels of all seven known sirtuins in primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) in comparison with lung fibroblasts from healthy controls. These unbiased tests revealed a tendency for all sirtuins to be expressed at lower levels in fibroblasts from patients compared with controls, but the greatest decrease was observed with SIRT7. Similarly, SIRT7 was decreased in lung tissues of bleomycin-challenged mice. Inhibition of SIRT7 with siRNA in cultured lung fibroblasts resulted in an increase in collagen and α-smooth muscle actin (α-SMA). Reciprocally, overexpression of SIRT7 resulted in lower basal and TGF-β-induced levels of COL1A1, COL1A2, COL3A1, and α-SMA mRNAs, as well as collagen and α-SMA proteins. Induced changes in SIRT7 had no effect on endogenous TGF-β mRNA levels or latent TGF-β activation, but overexpression of SIRT7 reduced the levels of Smad3 mRNA and protein. In conclusion, the decline in SIRT7 in lung fibroblasts has a profibrotic effect, which is mediated by changes in Smad3 levels.

Download Full-text

Phospholipase Cδ1 regulates cell proliferation and cell-cycle progression from G1- to S-phase by control of cyclin E–CDK2 activity

Biochemical Journal ◽

10.1042/bj20080233 ◽

2008 ◽

Vol 415 (3) ◽

pp. 439-448 ◽

Cited By ~ 14

Author(s):

Katherine A. Kaproth-Joslin ◽

Xiangquan Li ◽

Sarah E. Reks ◽

Grant G. Kelley

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Cellular Proliferation ◽

Cyclin E ◽

Critical Role ◽

S Phase ◽

G1 Phase ◽

Serum Starvation ◽

Cycle Progression ◽

Cdk2 Activity

In the present study, we examined the role of PLCδ1 (phospholipase C δ1) in the regulation of cellular proliferation. We demonstrate that RNAi (RNA interference)-mediated knockdown of endogenous PLCδ1, but not PLCβ3 or PLCϵ, induces a proliferation defect in Rat-1 and NIH 3T3 fibroblasts. The decreased proliferation was not due to an induction of apoptosis or senescence, but was associated with an approx. 60% inhibition of [3H]thymidine incorporation. Analysis of the cell cycle with BrdU (bromodeoxyuridine)/propidium iodide-labelled FACS (fluorescence-activated cell sorting) demonstrated an accumulation of cells in G0/G1-phase and a corresponding decrease in cells in S-phase. Further examination of the cell cycle after synchronization by serum-starvation demonstrated normal movement through G1-phase but delayed entry into S-phase. Consistent with these findings, G1 cyclin (D2 and D3) and CDK4 (cyclin-dependent kinase 4) levels and associated kinase activity were not affected. However, cyclin E-associated CDK2 activity, responsible for G1-to-S-phase progression, was inhibited. This decreased activity was accompanied by unchanged CDK2 protein levels and paradoxically elevated cyclin E and cyclin E-associated CDK2 levels, suggesting inhibition of the cyclin E–CDK2 complex. This inhibition was not due to altered stimulatory or inhibitory phosphorylation of CDK2. However, p27, a Cip/Kip family CKI (CDK inhibitor)-binding partner, was elevated and showed increased association with CDK2 in PLCδ1-knockdown cells. The result of the present study demonstrate a novel and critical role for PLCδ1 in cell-cycle progression from G1-to-S-phase through regulation of cyclin E–CDK2 activity and p27 levels.

Download Full-text

NDRG1 Inhibition of Tumor Progression in Caco2 Cells

10.21203/rs.3.rs-141427/v1 ◽

2021 ◽

Author(s):

Yixiao He ◽

Yuzhu Ji ◽

Hairong Hua ◽

Yu Zhu ◽

Peng Yu ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Cycle ◽

Tumor Progression ◽

Early Stage ◽

Strong Relationship ◽

S Phase ◽

Cell Counting ◽

Invasion And Migration ◽

Caco2 Cells ◽

And Migration

Abstract Background: Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Invasiveness and migration are the main cause of death, and so there is a need to find a sensitive, reliable molecular marker that can predict the migration of colorectal cancer at an early stage. NDRG1 (N-myc Downstream Regulated Gene 1) has been reported to be a multifunctional gene that has a strong relationship with tumor invasion and migration, but theories about the current role of NDRG1 in colorectal cancer remains to be conclusively determined. Methods and Results: Through lentivirus infection and CRISPR/Cas9 methods, respectively, we established that NDRG1 stably overexpressed and knocked out Caco2 cell lines. CCK8(Cell Counting kit 8) data showed that NDRG1 inhibited Caco2 proliferation. Flow cytometry further confirmed that the cell cycle can be arrested at the G1/S phase when NDRG1 overexpresses, while the number of G2 phase cells significantly increased after NDRG1 was knocked out. This means that NDRG1 inhibited the proliferation of Caco2 cells by arresting the cell cycle in the G1/S phase. Our data also demonstrated that NDRG1 promotes early cell apoptosis. The strength of invasion and migration was decreased when NDRG1 overexpressed. Conclusions: Our results underline that NDRG1 inhibits tumor progression in Caco2 cells. These findings may provide a new potential therapeutic strategy for the treatment of CRC.

Download Full-text