Objective:To investigate the effect of phosphorylated HSP27 on the proliferation and metastasis of nasopharyngeal carcinoma and its molecular mechanism. Methods:①Western blot assay was used to detect the expression levels of HSP27 and p-HSP27 in CNE1 and CNE2 cells of nasopharyngeal carcinoma. Inhibited the phosphorylation of HSP27, Transwell assay detected the metastasis ability of nasopharyngeal carcinoma cells. ②Nasopharyngeal carcinoma cell lines（CNE2-OE1 and CNE2-OE2） overexpressing phosphorylated and dephosphorylated mutants of HSP27 were synthesized, empty vector transfected CNE2 cells（CNE2-NC） were used as controls. The proliferation ability of the three groups of cells was detected by CCK8, and the expression levels of CyclinD1, Bax and Bcl-2 were detected by Western blot. Transwell was used to detect the migration and invasion ability of cells, and Western blot was used to detect the expression levels of E-cadherin, Vimentin, MMP2 and MMP9. Results:The expression level of HSP27 in CNE2 was higher than that of CNE1 cells, while the expression level of p-HSP27 was opposite. After inhibition of HSP27 phosphorylation, the invasion and migration ability of CNE1 cells decreased significantly, with no significant change in CNE2 cells. Compared with CNE2-NC, the growth rate of CNE2-OE1 decreased, and the growth rate of CNE2-OE2 increased. The expression level of CyclinD1 was down-regulated in CNE2-OE1 and higher in CNE2-OE2. The expression level of Bax in CNE2-OE1 was increased, while the expression of Bcl-2 was decreased. There was no significant change in the expression of Bax and Bcl-2 in CNE2-OE2. Compared with CNE2-NC, the migration ability of CNE2-OE1 was enhanced and the invasion ability was weakened, while the migration ability of CNE2-OE2 was weakened and the invasion ability was enhanced. There was no significant difference in the expression levels of E-cadherin was decreased in CNE2-OE1 and increased in CNE2-OE2. There was no significant difference in the expression levels of Vimentin among the three groups. The expression levels of MMP2 and MMP9 were up-regulated in CNE2-OE2, and slightly down-regulated in CNE2-OE1. Conclusion:HSP27 and p-HSP27 were differentially expressed in different nasopharyngeal carcinoma cells, and the metastasis ability of nasopharyngeal carcinoma was not only related to the expression level of HSP27, but also related to the level of p-HSP27. The p-HSP27 inhibited CNE 2 cell proliferation and promoted their apoptosis. As p-HSP27 plays different roles in different stages of nasopharyngeal carcinoma metastasis, it can increase the migration ability of CNE2 cells and reduce its invasion ability. p-HSP27 may induce EMT changes in CNE2 by down-regulating E-cadherin, thus promoting CNE2 migration, and may inhibit CNE2 invasion by down-regulating MMPs expression.
Humans ; Cell Proliferation ; Cell Line, Tumor ; Nasopharyngeal Carcinoma/pathology* ; Nasopharyngeal Neoplasms/pathology* ; Cell Movement ; HSP27 Heat-Shock Proteins/metabolism* ; Phosphorylation ; Neoplasm Metastasis ; Matrix Metalloproteinase 9/metabolism* ; Neoplasm Invasiveness ; Matrix Metalloproteinase 2/metabolism* ; Heat-Shock Proteins/metabolism* ; Cyclin D1/metabolism* ; Molecular Chaperones/metabolism* ; Cadherins/metabolism*

OBJECTIVES: To investigate the protective effect of the probiotic bacterium Streptococcus salivarius K12 (K12) against Mycoplasma pneumoniae (Mp) infection in mice. METHODS: Forty male BALB/c mice were randomized into normal control group, K12 treatment group, Mp infection group, and K12 pretreatment prior to Mp infection group. The probiotic K12 was administered daily by gavage for 14 days before Mp infection induced by intranasal instillation of Mp. Three days after Mp infection, the mice were euthanized for analysis of bronchoalveolar lavage fluid (BALF) cell counts and serum levels of secretory immunoglobulin A (sIgA), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). RT-qPCR was performed to detect the P1 and community-acquired respiratory distress syndrome ( CARDS ) toxin of Mp in the lung tissues and the mRNA expressions of TNF-α, IL-6, chemokine 1 (CXCL1), matrix metalloproteinase 9 (MMP9), mucin 5ac (MUC5ac), collagen 3a1 (Col3a1), Toll-like receptor 2 (TLR2) and TLR4; the protein expressions of TLR2 and TLR4 in the lung tissue were detected using Western blotting. Pathological changes in the lung tissue and airway remodeling were examined with HE staining and AB/PAS staining. RESULTS: Compared with the Mp-infected mice with PBS treatment, the infected mice with K12 treatment showed significantly lowered mRNA levels of P1 and CARDS in the lung tissue and reduced white blood cell counts in the BALF (P<0.05). In spite of the absence of significant differences in serum levels of inflammatory factors between the two groups, the mRNA expressions of TNF‑α, IL-6, CXCL1, MMP9, MUC5ac and COL3A1 and the mRNA and protein levels of TLR2 and TLR4 in the lung tissues were significantly lower in K12-treated mice, in which AB/PAS staining showed obviously decreased mucus secretion. CONCLUSIONS K12 pretreatment can effectively reduce pulmonary inflammatory responses, improve airway remodeling and alleviate lung injury in Mp-infected mice.
Animals ; Mice ; Pneumonia, Mycoplasma/metabolism* ; Mice, Inbred BALB C ; Toll-Like Receptor 2/metabolism* ; Mycoplasma pneumoniae ; Male ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Lung/microbiology* ; Toll-Like Receptor 4/metabolism* ; Streptococcus salivarius ; Probiotics/administration & dosage* ; Bronchoalveolar Lavage Fluid ; Matrix Metalloproteinase 9/metabolism* ; Mucin 5AC/metabolism* ; Chemokine CXCL1/metabolism* ; Immunoglobulin A, Secretory/metabolism* ; Bacterial Toxins ; Bacterial Proteins

OBJECTIVE: To investigate the effects and underlying mechanisms of VX765 on osteoarthritis (OA) and chondrocytes inflammation in rats. METHODS: Chondrocytes were isolated from the knee joints of 4-week-old Sprague Dawley (SD) rats. The third-generation cells were subjected to cell counting kit 8 (CCK-8) analysis to assess the impact of various concentrations (0, 1, 5, 10, 20, 50, 100 μmol/L) of VX765 on rat chondrocyte activity. An in vitro lipopolysaccharide (LPS) induced cell inflammation model was employed, dividing cells into control group, LPS group, VX765 concentration 1 group and VX765 concentration 2 group without obvious cytotoxicity. Western blot, real-time fluorescence quantitative PCR, and ELISA were conducted to measure the expression levels of inflammatory factors-transforming growth factor β ₁ (TGF-β ₁), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Additionally, Western blot and immunofluorescence staining were employed to assess the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Thirty-two SD rats were randomly assigned to sham surgery group (group A), OA group (group B), OA+VX765 (50 mg/kg) group (group C), and OA+VX765 (100 mg/kg) group (group D), with 8 rats in each group. Group A underwent a sham operation with a medial incision, while groups B to D underwent additional transverse incisions to the medial collateral ligament and anterior cruciate ligament, with removal of the medial meniscus. One week post-surgery, groups C and D were orally administered 50 mg/kg and 100 mg/kg VX765, respectively, while groups A and B received an equivalent volume of saline. Histopathological examination using HE and safranin-fast green staining was performed, and Mankin scoring was utilized for evaluation. Immunohistochemical staining technique was employed to analyze the expressions of matrix metalloproteinase 13 (MMP-13) and collagen type Ⅱ. RESULTS: The CCK-8 assay indicated a significant decrease in cell viability at VX765 concentrations exceeding 10 μmol/L ( P<0.05), so 4 μmol/L and 8 μmol/L VX765 without obvious cytotoxicity were selected for subsequent experiments. Following LPS induction, the expressions of TGF-β ₁, IL-6, and TNF-α in cells significantly increased when compared with the control group ( P<0.05). However, intervention with 4 μmol/L and 8 μmol/L VX765 led to a significant decrease in expression compared to the LPS group ( P<0.05). Western blot and immunofluorescence staining demonstrated a significant upregulation of Nrf2 pathway-related molecules Nrf2 and HO-1 protein expressions by VX765 ( P<0.05), indicating Nrf2 pathway activation. Histopathological examination of rat knee joint tissues and immunohistochemical staining revealed that, compared to group B, treatment with VX765 in groups C and D improved joint structural damage in rat OA, alleviated inflammatory reactions, downregulated MMP-13 expression, and increased collagen type Ⅱ expression. CONCLUSION VX765 can improve rat OA and reduce chondrocyte inflammation, possibly through the activation of the Nrf2 pathway.
Rats ; Animals ; Chondrocytes/metabolism* ; Matrix Metalloproteinase 13/metabolism* ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha/metabolism* ; Collagen Type II/metabolism* ; Interleukin-6 ; Lipopolysaccharides/pharmacology* ; NF-E2-Related Factor 2/pharmacology* ; Inflammation/drug therapy* ; Osteoarthritis/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Dipeptides ; para-Aminobenzoates

OBJECTIVE: To investigate the relationship between serum matrix metalloproteinase-1(MMP-1) and matrix metalloproteinase-2(MMP-2) and the formation of deep venous thrombosis(LDVT) in lower extremity patients after surgery for lower extremity fracture, and to analyze the value of MMP-1 and MMP-2 in predicting the occurrence of LDVT after lower extremity fracture. METHODS: From June 2018 to December 2021, 352 patients who planned to receive surgical treatment of lower limb fracture in our hospital were selected as the research objects. Venous blood was collected at 1, 2 and 3 days after surgery, respectively, and serum MMP-1 and MMP-2 levels were detected. The incidence of LDVT during hospitalization was analyzed, and the risk factors of postoperative LDVT in patients with lower limb fracture surgery and the predictive value of MMP-1 and MMP-2 for LDVT were analyzed. RESULTS: LDVT occurred in 40 patients (LDVT group), the incidence of LDVT was 11.36%, and 312 patients did not occurred(no occurred group). The serum levels of MMP-1 and MMP-2 in LDVT group increased gradually after surgery; the serum levels of MMP-1 and MMP-2 in the no occurred group increased slightly after surgery at 2 days and then decreased at 3 days after surgery (P<0.01);the serum levels of MMP-1 and MMP-2 in LDVT group were higher than those in the no occurred group at 2 days and 3 days after surgery (P<0.05). Serum levels of MMP-1 and MMP-2 were positively correlated with serum levels of interleukin-6 (IL-6), IL-8 and tumor necrosis factor -α (TNF-α) in LDVT patients at 2 days and 3 days postoperatively (P<0.05). Operative time, MMP-1 and MMP-2 postoperative 3 days were related to the occurrence of LDVT after lower limb fracture (P<0.01). The area under the curve(AUC) predicted by MMP-1 and MMP-2 postoperative 3 days for LDVT after lower limb fracture was 0.738 and 0.744 respectively, and the AUC predicted by combined MMP-1 and MMP-2 was 0.910, which was higher than that predicted by single indicator(Z=2.819 and 2.025, P<0.05). CONCLUSION High levels of MMP-1 and MMP-2 after lower extremity fracture are closely related to the occurrence of LDVT, and 3 d mMP-1 and MMP-2 after surgery maybe used as evaluation indexes for LDVT risk prediction.
Humans ; Lower Extremity/surgery* ; Matrix Metalloproteinase 1/blood* ; Matrix Metalloproteinase 2/blood* ; Risk Factors ; Venous Thrombosis/etiology* ; Fractures, Bone/surgery*

OBJECTIVE: To explore the potential molecular mechanism of tetrahydropalmatine (THP) on acute myocardial ischemia (AMI). METHODS: First, the target genes of THP and AMI were collected from SymMap Database, Traditional Chinese Medicine Database and Analysis Platform, and Swiss Target Prediction, respectively. Then, the overlapping target genes between THP and AMI were evaluated for Grene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction network analysis. The binding affinity between the protein and THP was assessed by molecular docking. Finally, the protective effects of THP on AMI model and oxygen and glucose deprivation (OGD) model of H9C2 cardiomyocyte were explored and the expression levels of target genes were detected by RT-qPCR in vivo and in vitro. RESULTS: MMP9, PPARG, PTGS2, SLC6A4, ESR1, JAK2, GSK3B, NOS2 and AR were recognized as hub genes. The KEGG enrichment analysis results revealed that the potential target genes of THP were involved in the regulation of PPAR and hormone pathways. THP improved the cardiac function, as well as alleviated myocardial cell damage. Furthermore, THP significantly decreased the RNA expression levels of MMP9, PTGS2, SLC6A4, GSK3B and ESR1 (P<0.05, P<0.01) after AMI. In vitro, THP significantly increased H9C2 cardiomyocyte viability (P<0.05, P<0.01) and inhibited the RNA expression levels of PPARG, ESR1 and AR (P<0.05, P<0.01) in OGD model. CONCLUSIONS THP could improve cardiac function and alleviate myocardial injury in AMI. The underlying mechanism may be inhibition of inflammation, the improvement of energy metabolism and the regulation of hormones.
Humans ; Matrix Metalloproteinase 9 ; Network Pharmacology ; Cyclooxygenase 2 ; Molecular Docking Simulation ; PPAR gamma ; Myocardial Ischemia/genetics* ; Glucose ; RNA ; Drugs, Chinese Herbal/therapeutic use* ; Serotonin Plasma Membrane Transport Proteins

Objective To investigate how the neutrophil extracellular traps (NETs) affect the proliferation and migration of mouse MC38 colorectal cancer cells and its mechanism. Methods Spleen neutrophils were extracted in mouse, followed by collection of NETs after ionomycin stimulation in vitro. The proliferation of MC38 cell was detected by CCK-8 assay, and migration ability were detected by Transwell^TM and cell scratch assay, after co-incubation with MC38 cells. The mRNA expression of cellular matrix metalloproteinase 2 (MMP2) and MMP9 were detected by real-time fluorescence quantitative PCR, and the expression of MMP2, MMP9 and focal adhesion kinase (FAK), phosphorylated FAK protein were detected by Western blot. After silencing MMP9 using small interfering RNA (siRNA), the effect of NETs on the proliferation and migration ability of MC38 cells and the altered expression of related molecules were examined by previous approach. Results NETs promoted the proliferation and migration of MC38 cells and up-regulated the MMP9 expression and FAK phosphorylation. Silencing MMP9 inhibited the promotion of MC38 proliferation and migration by NETs and suppressed FAK phosphorylation. Conclusion NETs up-regulates MMP9 expression in MC38 cells, activates FAK signaling pathway and promotes tumor cell proliferation and migration.
Animals ; Mice ; Focal Adhesion Protein-Tyrosine Kinases/metabolism* ; Matrix Metalloproteinase 2/metabolism* ; Matrix Metalloproteinase 9/metabolism* ; Extracellular Traps/metabolism* ; Cell Movement ; Cell Proliferation ; RNA, Small Interfering/genetics* ; Colorectal Neoplasms/genetics* ; Cell Line, Tumor

The present study observed the regulatory effect of total flavonoids of Ziziphora clinopodioides on autophagy and the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathways in ApoE~(-/-) mice and explored the mechanism of total flavonoids of Z. clinopodioides against atherosclerosis(AS). ApoE~(-/-) mice were fed on a high-fat diet for eight weeks to induce an AS model. The model mice were randomly divided into a model group, a positive control group, and low-, medium-and high-dose groups of total flavonoids of Z. clinopodioides, while C57BL/6J mice fed on a common diet were assigned to the blank group. The serum and aorta samples were collected after intragastric administration for 12 weeks, and the serum levels of total cholesterol(TC), triglyceride(TG), low density lipoprotein-cholesterol(LDL-C), and high density lipoprotein-cholesterol(HDL-C) were detected by an automatic biochemical analyzer. The serum expression levels of intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), matrix metalloproteinase-2(MMP-2), and matrix metalloprotei-nase-9(MMP-9) were detected by enzyme-linked immunosorbent assay(ELISA). Oil red O staining was used to observe the aortic plaque area in mice. Hematoxylin-eosin(HE) staining was used to observe the aortic plaque and pathological changes in mice. The expression of P62 and LC3 in the aorta was detected by the immunofluorescence method. The protein expression of LC3Ⅱ/Ⅰ, Beclin-1, P62, p-PI3K, p-Akt, and p-mTOR in the aorta of mice was detected by Western blot. The results showed that compared with the blank group, the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2 and MMP-9 in the model group were significantly increased(P<0.01 or P<0.05), the content of HDL-C was decreased(P<0.05), intra-aortic plaque area was enlarged(P<0.01), the expression of LC3 in the aorta was significantly down-regulated, P62 expression was up-regulated(P<0.01 or P<0.05), the expressions of LC3Ⅱ/Ⅰ and Beclin-1 in the aortic lysate were significantly down-regulated, and the expressions of p-PI3K, p-Akt, p-mTOR and P62 were significantly increased(P<0.01). The medium-and high-dose groups of total flavonoids of Z. clinopodioides could reduce the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2, and MMP-9 in AS model mice(P<0.01 or P<0.05), and increase the content of HDL-C(P<0.01 or P<0.05). The aortic plaque area of mice after middle and high doses of total flavonoids of Z. clinopodioides was significantly reduced(P<0.01), the content of foam cells decrease, and the narrowing of the lumen decreased. The total flavonoids of Z. clinopodioides significantly increased the expression of LC3 in the aorta and the expression of LC3Ⅱ/Ⅰ and Beclin-1 in the lysate, and decreased the expression of P62 in the aorta and the expression of p-PI3K, p-Akt, p-mTOR and P62 in the lysate(P<0.01 or P<0.05). The results showed that the total flavonoids of Z. clinopodioides could improve the content of blood lipids and inflammatory factors, and reduce the generation of foam cells and plaques in aortic tissue, and the mechanism may be related to the regulation of PI3K/Akt/mTOR signaling pathway.
Animals ; Mice ; Apolipoproteins E ; Atherosclerosis/genetics* ; Beclin-1 ; Cholesterol, LDL ; Intercellular Adhesion Molecule-1 ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases/metabolism* ; Plaque, Atherosclerotic ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/genetics* ; Vascular Cell Adhesion Molecule-1/genetics*

This study aims to investigate the effect of Astragali Radix-Curcumae Rhizoma(AC) combination on the proliferation, migration, and invasion of colon cancer HT-29 cells based on epithelial-mesenchymal transition(EMT). HT-29 cells were respectively treated with 0, 3, 6 and 12 g·kg~(-1) AC-containing serum for 48 h. The survival and growth of cells were measured by thiazole blue(MTT) colorimetry, and the proliferation, migration, and invasion of cells were detected by 5-ethynyl-2'-deoxyuridine(EdU) test and Transwell assay. Cell apoptosis was examined by flow cytometry. The BALB/c nude mouse model of subcutaneous colon cancer xenograft was established, and then model mice were classified into blank control group, 6 g·kg~(-1) AC group, and 12 g·kg~(-1) AC group. The tumor weight and volume of mice were recorded, and the histopathological morphology of the tumor was observed based on hematoxylin-eosin(HE) staining. The expression of apoptosis-associated proteins B-cell lymphoma-2-associated X protein(Bax), cysteine-aspartic acid protease-3(caspase-3), and cleaved caspase-3, and EMT-associated proteins E-cadherin, MMP9, MMP2 and vimentin in HT-29 cells and mouse tumor tissues after the treatment of AC was determined by Western blot. The results showed that cell survival rate and the number of cells at proliferation stage decreased compared with those in the blank control group. The number of migrating and invading cells reduced and the number of apoptotic cells increased in the administration groups compared with those in the blank control group. As for the in vivo experiment, compared with the blank control group, the administration groups had small tumors with low mass and shrinkage of cells and karyopycnosis in the tumor tissue, indicating that the AC combination may improve EMT. In addition, the expression of Bcl2 and E-cadherin increased and the expression of Bax, caspase-3, cleaved caspase-3, MMP9, MMP2, and vimentin decreased in HT-29 cells and tumor tissues in each administration group. In summary, the AC combination can significantly inhibit the proliferation, invasion, migration, and EMT of HT-29 cells in vivo and in vitro and promote the apoptosis of colon cancer cells.
Humans ; Animals ; Mice ; Caspase 3 ; Matrix Metalloproteinase 2 ; Matrix Metalloproteinase 9 ; Vimentin ; HT29 Cells ; bcl-2-Associated X Protein ; Colonic Neoplasms ; Cell Proliferation

Objective: To investigate the effect of nuclear factor erythroid 2-related factor 2 (Nrf2) in the alteration of tight junction protein expression in choroid plexus epithelial cells created by lanthanum-activated matrix metalloproteinase 9 (MMP9) . Methods: In October 2020, immortalized rat choroid plexus epithelial cell line (Z310) cells were used as the blood-cerebrospinal fluid barrier in vitro, and were divided into control group and 0.125, 0.25, 0.5 mmol/L lanthanum chloride (LaCl(3)) treatment group. After treating Z310 cells with different concentrations of LaCl(3) for 24 hours, the morphological changes of Z310 cells were observed under inverted microscope, the protein expression levels of MMP9, occludin and zonula occludens-1 (ZO-1) were observed by cellular immunofluorescence method, and the protein expression levels of MMP9, tissue inhibitors of metalloproteinase1 (TIMP1) , occludin, ZO-1 and Nrf2 were detected by Western blotting. The level of reactive oxygen species (ROS) in cells was detected by flow cytometry. Results: Compared with the control group, Z310 cells in the LaCl(3) treatment group were smaller in size, with fewer intercellular junctions, and more dead cells and cell fragments. The expression level of MMP9 protein in cells treated with 0.25 and 0.5 mmol/L LaCl(3) was significantly higher than that in the control group (P<0.05) , and the expression level of TIMP1 and tight junction proteins occudin and ZO-1 was significantly lower than that in the control group (P<0.05) . Compared with the control group, the ROS production level in the 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly increased (P<0.05) , and the Nrf2 protein expression level in the 0.125, 0.25, 0.5 mmol/L LaCl(3) treatment group was significantly decreased (P<0.05) . Conclusion: Lanthanum may increase the level of ROS in cells by down regulating the expression of Nrf2, thus activating MMP9 to reduce the expression level of intercellular tight junction proteins occludin and ZO-1.
Rats ; Animals ; Matrix Metalloproteinase 9/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Tight Junction Proteins/metabolism* ; Occludin/pharmacology* ; Choroid Plexus/metabolism* ; Reactive Oxygen Species/metabolism* ; Lanthanum/pharmacology* ; Epithelial Cells ; Zonula Occludens-1 Protein/metabolism* ; Phosphoproteins/pharmacology*

This study aims to investigate the effect and mechanism of hydnocarpin(HC) in treating triple negative breast cancer(TNBC). Cell counting kit-8(CCK-8), xCELLigence real-time cellular analysis(RTCA), and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines: MDA-MB-231 and MDA-MB-436. The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis, wound-healing assay, and Transwell assay. The changes in the epithelial-mesenchymal transition(EMT) and the expression of invasion-and migration-associated proteins [E-cadherin, vimentin, Snail, matrix metalloproteinase-2(MMP-2), and MMP-9] were detected by Western blot. Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP) and downstream targets(CTGF and Cyr61). TNBC cells were transfected with Flag-YAP for the overexpression of YAP, and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay, Transwell assay, and wound-healing assay. The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay. The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4) was detected by microscale thermophoresis(MST) assay and drug affinity responsive target stability(DARTS) assay. The results showed that HC significantly inhibited the proliferation, colony formation, invasion, and EMT of TNBC cells. HC down-regulated the protein and mRNA levels of CTGF and Cyr61. HC down-regulated the total protein level of YAP, while it had no effect on the mRNA level of YAP. The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation, migration, and invasion of TNBC cells. HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP. The results of MST and DARTS demonstrated direct binding between HC, YAP, and CNOT4. The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.
Humans ; Triple Negative Breast Neoplasms/metabolism* ; Matrix Metalloproteinase 2/metabolism* ; Cell Line, Tumor ; Cell Proliferation ; Cell Movement ; Ubiquitination ; RNA, Messenger/metabolism* ; Epithelial-Mesenchymal Transition ; Transcription Factors/metabolism*