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1.
Article in Chinese | WPRIM | ID: wpr-879092

ABSTRACT

Chemical constituents were isolated and purified from fruiting bodies of Ganoderma calidophilum by various column chromatographic techniques, and their chemical structures were identified through combined analysis of physicochemical properties and spectral data. As a result, 11 compounds were isolated and identified as(24E)-lanosta-8,24-dien-3,11-dione-26-al(1), ganoderone A(2), 3-oxo-15α-acetoxy-lanosta-7,9(11), 24-trien-26-oleic acid(3),(23E)-27-nor-lanosta-8,23-diene-3,7,25-trione(4), ganodecanone B(5), ganoderic aldehyde A(6), 11β-hydroxy-lucidadiol(7), 3,4-dihydroxyacetophenone(8), methyl gentiate(9), ganoleucin C(10), ganotheaecolumol H(11). Among them, compound 1 is a new triterpenoid. The cytotoxic activities of all of the compounds against tumor cell lines were evaluated. The results showed that compounds 1, 3, 4 and 6 showed cytotoxic activity against BEL-7402, with IC_(50) values of 26.55, 11.35, 23.23, 18.66 μmol·L~(-1); compounds 1 and 3-6 showed cytotoxic activity against K562, with IC_(50) values of 5.79, 22.16, 12.16, 35.32, and 5.59 μmol·L~(-1), and compound 4 showed cytotoxic activity against A549, with IC_(50) value of 42.50 μmol·L~(-1).


Subject(s)
Cell Line, Tumor , Fruiting Bodies, Fungal , Ganoderma , Molecular Structure , Triterpenes/pharmacology
2.
Article in Chinese | WPRIM | ID: wpr-879054

ABSTRACT

This study is to observe whether platycodin D has the guiding role in treatment of mouse lung cancer with doxorubicin and explore its guiding mechanism. In vitro, platycodin D and doxorubicin(alone or in combination) were added into Lewis lung cancer(LLC) cells to detect the cell proliferation and doxorubicin uptake. Cell morphological changes were analyzed by cell holographic analysis system; cell gap junctional intercellular communication(GJIC) was tested by fluorescent yellow tracer; lyso-tracker red was used to examine lysosomal function; LC-3 B(Light chain 3 beta)and P62(heat shock 90-like protein)staining were used to test auto-phagy and autophagic degradation respectively; and P-glycoprotein(P-gp) expression was examined by Western blot. In vivo, lung solid tumor was formed in mouse LLC cells via intravenous injection. Platycodin D and doxorubicin(alone or in combination) were used to treat tumor-bearing mice for four weeks, and then the tumor size was examined, mouse survival time was recorded, doxorubicin uptake in lung tissues was tested, and lung tissues were stained for observation by HE(hematoxylin-eosin) and immunohistochemistry. The results showed that platycodin D at the experimental concentration had no effect on LLC cell proliferation but decreased LLC cell volume, promoted the cells to uptake doxorubicin and enhanced the inhibitory action of doxorubicin on cell proliferation. Platycodin D could promote GJIC and lysosomal function, increase autophagy and autophagic degradation and suppress P-gp expression. Platycodin D at the experimental dose in this study had no effect on LLC lung solid tumors in mice, increased doxorubicin uptake in lung tissues and enhanced the therapeutic efficacy of doxorubicin on lung solid tumors. Platycodin D could improve the extracellular matrix deposition in lung solid tumors, decreased the lung mucin 5 AC secretion and pulmonary vessel permeability. In summary, platycodin D had the guiding role in treating mouse lung cancer with doxorubicin, and its guiding mechanism may be associated with the promotion of cell communication, lysosomal function, and improvement of extracellular environment.


Subject(s)
Animals , Cell Line, Tumor , Doxorubicin , Lung Neoplasms/drug therapy , Mice , Saponins , Triterpenes
3.
Article in Chinese | WPRIM | ID: wpr-879025

ABSTRACT

To prove that ursolic acid(UA)could activate the autophagy of colorectal cancer HCT116 cells by inhibiting hedgehog signaling pathway. The effect of UA on the viability of HCT116 cells was determined by MTT assay. The effect of UA on the proliferation and migration of HCT116 cells was detected by crystal violet staining and scratch test. In the study on autophagy, the time points were screened out first: the autophagy fluorescence intensity of UA acting on HCT116 at different time points were detected by Cell Meter~(TM) Autophagy Assay Kit; Western blot was used to detect the expression of autophagy protein P62 at different time points. Then, Cell Meter~(TM) Autophagy Assay Kit was used to detect the effect of UA on autophagy fluorescence intensity of HCT116 cells. The effect of different doses of UA on the expressions of LC3Ⅱ and P62 proteins in HCT116 cells were detected by Western blot. Further, AdPlus-mCherry-GFP-LC3 B adenovirus transfection was used to detect the effects of UA on autophagy flux of HCT116 cells; UA combined with autophagy inhibitor chloroquine(CQ) was used to detect the expression of LC3Ⅱ by Western blot. In terms of mechanism, the effect of UA on hedgehog signaling pathway-related proteins in HCT116 cells was detected by Western blot. The results showed that UA inhibited the activity, proliferation and migration of HCT116 cells. UA enhanced the fluorescence intensity of autophagy in HCT116 cells, while promoting the expression of LC3Ⅱ and inhibiting the expression of P62, in a time and dose dependent manner. UA activated the autophagy in HCT116 cells, which manifested that UA resulted in the accumulation of fluorescence spots and strengthened the fluorescence intensity of autophagosomes; compared with UA alone, UA combined with autophagy inhibitor CQ promoted the expression of LC3Ⅱ. UA reduced the expressions of PTCH1, GLI1, SMO, SHH and c-Myc in hedgehog signaling pathway, while increased the expression of Sufu. In conclusion, our study showed that UA activated autophagy in colorectal cancer HCT116 cells, which was related to the mechanism in inhibiting hedgehog signaling pathway activity.


Subject(s)
Apoptosis , Autophagy , Cell Line, Tumor , Colorectal Neoplasms , Hedgehog Proteins/genetics , Humans , Signal Transduction , Triterpenes
4.
Article in Chinese | WPRIM | ID: wpr-879022

ABSTRACT

To screen the sensitive cell lines of active fraction from clove(AFC) on human colon cancer cells, investigate the effects of AFC on the cells proliferation and apoptosis as well as PI3 K/Akt/mTOR(phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin) signaling pathways involved, and reveal the mechanism of AFC for inducing apoptosis of human colorectal carcinoma cells. Cell counting kit-8(CCK-8) assay was used to detect the cytotoxic effect of different concentrations of AFC. AFC-induced apoptosis was detected by Hoechst 33258 fluorescence staining and Annexin V-FITC/PI double staining. HCT116 cells were treated with AFC with or without pretreatment with insulin-like growth factor-Ⅰ(IGF-Ⅰ), and then the protein expression levels of caspase-3, caspase-9, poly ADP-ribose polymerase(PARP), PI3 K, p-PI3 K, Akt, p-Akt, mTOR and p-mTOR in PI3 K/Akt/mTOR signaling pathway were detected by Western blot. RESULTS:: showed that the most obvious inhibitory effect of AFC was on human colon cancer HCT116 cells, and the optimal AFC treatment time was 48 hours. After AFC treatment, typical apoptotic features such as nuclear chromatin concentration, nuclear fragmentation and apoptotic bodies appeared in a dose-dependent manner. Annexin V-FITC/PI double staining showed that as compared with the control group, 50 and 100 μg·mL~(-1) AFC groups increased the apoptosis rate of HCT116 cells significantly(P<0.001); AFC activated caspase-9, cleaved caspase-3 and cleaved PARP in a concentration-dependent manner. The protein expression levels of cleaved caspase-3/procaspase-3, cleaved PARP/PARP and caspase-9/β-actin after treatment of AFC(100 μg·mL~(-1)) were significantly different from those in the control group(P<0.001). The relative protein expression of p-PI3 K, p-Akt and p-mTOR decreased in a concentration dependent manner, while Akt and mTOR showed no significant differences among groups. The ratios of p-PI3 K/PI3 K, p-Akt/Akt and p-mTOR/mTOR in the AFC groups(50 and 100 μg·mL~(-1)) were significantly lower than those in the control group(P<0.01). Its combination with IGF-Ⅰ weakened the effect of AFC in inhibiting PI3 K/Akt/mTOR signaling pathway. The ratios of p-Akt/Akt and p-mTOR/mTOR in the AFC+IGF-Ⅰ group were significantly enhanced as compared with the AFC group(P<0.05). Apoptosis-related protein expression levels(cleaved caspase-3 and cleaved PARP) in HCT116 cells treated with AFC+IGF-Ⅰ were also down regulated. As compared with the AFC group, the ratios of cleaved caspase-3/procaspase-3 and cleaved PARP/PARP in the AFC+IGF-Ⅰ group were significantly decreased(P<0.01). In summary, AFC activated caspase-mediated cascades and induced HCT116 cells apoptosis in a dose-dependent manner, which may be associated with the inhibition of the PI3 K/Akt/mTOR signaling pathway.


Subject(s)
Apoptosis , Cell Line, Tumor , Cell Proliferation , Colonic Neoplasms/drug therapy , HCT116 Cells , Humans , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Syzygium , TOR Serine-Threonine Kinases/metabolism
5.
Article in Chinese | WPRIM | ID: wpr-878976

ABSTRACT

Breast tumor has become one of the malignant tumors with the highest incidence, and is a serious threat to human health, especially to women. Chemotherapy is an important anti-breast tumor therapy, which can be used in almost every stage of breast tumor therapy alone or in the combination with surgery and radiation therapy. Alkaloids are a kind of ubiquitous natural products, and important active components of various medicinal plants. A large number of studies have shown that alkaloids could exert an anti-breast tumor effect by inhibiting proliferation, metastasis and angiogenesis, resisting mitosis, promoting apoptosis and autophagy, and triggering cell cycle arrest. The extensive anti-breast tumor effect makes alkaloids an important candidate drug source. This paper reviews the anti-breast tumor mechanism of natural products of alkaloids.


Subject(s)
Alkaloids/pharmacology , Apoptosis , Autophagy , Breast Neoplasms/drug therapy , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Proliferation , Female , Humans
6.
Article in Chinese | WPRIM | ID: wpr-878928

ABSTRACT

Based on the PI3K/Akt signaling pathway, this study aimed to observe the proliferation and apoptosis of ovarian cancer SKOV3 cells at different concentrations of icaritin, in order to explore the possible molecular mechanisms. The research object was ovarian cancer SKOV3 cells. The cells were divided into the control group and icaritin groups(5, 10, 20 μmol·L~(-1)), and administrated with drugs for 48 hours. The cell counting kit-8(CCK-8)assay was used to detect the inhibitory effect of icaritin on the proliferation of ovarian cancer SKOV3 cells. The proliferation ability of the SKOV3 cells was detected by EdU assay. Hoechst 33342 fluorescence staining was used to observe the apoptotic morphology of SKOV3 cells in each group. The distribution of cell cycle and the apoptosis rate of each group were detected by flow cytometry. Quantitative Real-time PCR was used to detect mRNA expressions of PTEN, PI3K, Akt in each group of cells. Protein expressions of PTEN, PI3K, Akt and p-Akt were measured by Western blot. The results showed that the cell inhibition rates of icaritin groups were significantly increased compared with the control group(P<0.05). The rates of EdU-positive cells of icaritin groups were significantly decreased(P<0.05). SKOV3 cells in icaritin groups showed morphological changes of apoptosis. Apoptosis rates of icaritin groups were significantly increased(P<0.05). The proportions of cells in G_0/G_1 phase of icaritin groups were decreased(P<0.05), while the proportions of S phase cells were increased(P<0.05). The gene and protein expressions of PTEN in icaritin groups were elevated(P<0.05). The gene expressions of PI3K and Akt in icaritin groups were down-regulated(P<0.05). The protein expression of PI3K and p-Akt in icaritin groups were reduced(P<0.05). These results indicated that icarin may inhibit the proliferation of ovarian cancer SKOV3 cells in vitro, induce cell apoptosis and affect the cycle distribution of cells by inhibiting the PI3K/Akt signaling pathway.


Subject(s)
Apoptosis , Cell Line, Tumor , Cell Proliferation , Female , Flavonoids , Humans , Ovarian Neoplasms/genetics , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-akt/genetics
7.
Article in Chinese | WPRIM | ID: wpr-878875

ABSTRACT

Mechanism study was performed to explore how Shouhui Tongbian Capsules promotes energy metabolism of gastrointestinal stromal cells. In this study, gastrointestinal stromal cells line GIST-882 was used as the model to explore energy metabolism regulation effects of Shouhui Tongbian Capsules extract(10, 20, 50 and 100 μg·mL~(-1)) by measuring the cell proliferation, ATP level, mitochondrial membrane potential, and mitochondrial isocitrate dehydrogenase activity. Meanwhile, Western blot was used to detect the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Our results showed that Shouhui Tongbian Capsules promoted cell proliferation and increased ATP level of gastrointestinal stromal cells. In addition, Shouhui Tongbian Capsules obviously improved mitochondrial structural integrity, and increased mitochondrial membrane potential in GIST-882 cells. Mechanism study revealed that Shouhui Tongbian Capsules increased mitochondrial isocitrate dehydrogenase activity and up-regulated the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Collectively, our study indicated that Shouhui Tongbian Capsules promoted the energy metabolism for gastrointestinal stromal cells proliferation by activating mitochondrial isocitrate dehydrogenase to induce ATP production, as well as activating SCF/c-Kit and CDK2/cyclin A signaling pathways.


Subject(s)
Capsules , Cell Line, Tumor , Energy Metabolism , Gastrointestinal Stromal Tumors , Humans , Proto-Oncogene Proteins c-kit/metabolism , Stromal Cells/metabolism
8.
Chinese Journal of Biotechnology ; (12): 331-341, 2021.
Article in Chinese | WPRIM | ID: wpr-878566

ABSTRACT

Genetic and epigenetic alterations accumulate in the process of hepatocellular carcinogenesis, but the role of genomic spatial organization in HCC is still unknown. Here, we performed in situ Hi-C in HCC cell line PLC/PRF/5 compared with normal liver cell line L02, together with RNA-seq and ChIP-seq of SMC3/CTCF/H3K27ac. The results indicate that there were significant compartment switching, TAD shifting and loop pattern altering in PLC/PRF/5. These spatial changes are correlated with abnormal gene expression and more opening promoter regions of the HCC cell line. Thus, the 3D genome organization alterations in PLC/PRF/5 are important in epigenetic mechanisms of HCC tumorigenesis.


Subject(s)
Carcinoma, Hepatocellular/genetics , Cell Line , Cell Line, Tumor , Genomics , Humans , Liver Neoplasms/genetics
9.
Article in English | WPRIM | ID: wpr-878451

ABSTRACT

OBJECTIVES@#The effect of isoprenylcysteine carboxymethyltransferase (ICMT) silencing on the migration and invasion of tongue squamous cell carcinoma was investigated by constructing the small interfering RNA (siRNA) of ICMT.@*METHODS@#Through liposomal transfection, siRNA was transfected into human tongue squamous cell carcinoma CAL-27 and SCC-4 cells (ICMT-siRNA group) with a negative control group (transfected with NC-siRNA) and a blank control group (transfected with a transfection reagent but not with siRNA). Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of ICMT and RhoA in each group of cells after transfection and to measure the silencing efficiency. Western blot was applied to examine the expression levels of ICMT, total RhoA, membrane RhoA, ROCK1, matrix metalloproteinase (MMP)-2, and MMP-9 proteins in each group. The migration and invasion abilities were evaluated via wound healing and Transwell motility assays.@*RESULTS@#After CAL-27 and SCC-4 cells were transfected with ICMT-siRNA, the expression levels of ICMT genes and proteins decreased significantly in the experimental group compared with those in the negative and blank control groups (@*CONCLUSIONS@#The migration and invasion abilities of CAL-27 and SCC-4 cells were reduced significantly after the transfection of ICMT-siRNA, and the involved mechanism might be related to the RhoA-ROCK signaling pathway.


Subject(s)
Carcinoma, Squamous Cell , Cell Line, Tumor , Cell Movement , Cell Proliferation , Humans , Neoplasm Invasiveness , Protein Methyltransferases , RNA, Small Interfering , Tongue , Tongue Neoplasms , Transfection , rho-Associated Kinases
10.
Article in English | WPRIM | ID: wpr-878450

ABSTRACT

OBJECTIVES@#This study aims to investigate the effect of the regulator of G-protein signaling 2 (RGS2) on the proliferation and invasion of oral squamous cell carcinoma (OSCC) cells and its potential molecular mechanism. Metho⁃ds The expression status and clinical significance of RGS2 in head and neck squamous cell carcinomas and matched adjacent normal tissues were evaluated using TCGA database. Three OSCC cell lines (i.e., SCC-9, Cal27, and Fadu) were overexpressed with RGS2, and the effect of RGS2 on cell proliferation and invasion was determined using the Transwell, clone formation, and cell counting kit (CCK)-8 assays. Moreover, the yeast two-hybrid scree-ning and co-immunoprecipitation (Co-IP) assays were conducted to detect the correlation of RGS2, four and a half LIM domains protein 1 (FHL1), and damage DNA-binding protein 1 (DDB1).@*RESULTS@#The expression level of RGS2 in OSCC was significantly lower than that in matched adjacent normal tissues (@*CONCLUSIONS@#RGS2 plays an important role in the inhibition of OSCC proliferation and invasion. The structure stability of RGS2 is competitively regulated by FHL1 and DDB1.


Subject(s)
Carcinoma, Squamous Cell , Cell Line, Tumor , Cell Movement , Cell Proliferation , GTP-Binding Proteins , Head and Neck Neoplasms , Humans , Intracellular Signaling Peptides and Proteins , LIM Domain Proteins , Mouth Neoplasms , Muscle Proteins , Squamous Cell Carcinoma of Head and Neck
11.
Article in Chinese | WPRIM | ID: wpr-878425

ABSTRACT

OBJECTIVES@#To investigate the expression of cyclophilin A (CyPA) in oral squamous cell carcinoma (OSCC) and explore the effect of downregulating the expression of CyPA gene on the proliferation and invasion of SCC-25 cells.@*METHODS@#A total of 77 cases of patients with OSCC were selected. The expression levels of CyPA proteins in OSCC and adjacent normal tissues were evaluated. SCC-25 cells were cultured and divided into the CyPA interference sequence group, negative control group, and blank group. The expression levels of CyPA mRNA and protein in cells were detected by using real-time fluorescent quantitative polymerase chain reaction and Western blot, respectively. Cell proliferation was detected by using methyl thiazolyl tetrazolium and plate colony formation assays. Cell invasion was detected by using Transwell assay.@*RESULTS@#The positive expression rate of CyPA protein in OSCC tissues was 76.62%, which was higher than that in adjacent tissues (@*CONCLUSIONS@#The CyPA protein is highly expressed in OSCC tissues, and the downregulation of CyPA gene expression in SCC-25 cells can reduce cell proliferation and inhibit cell invasion.


Subject(s)
Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cyclophilin A/genetics , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms , Humans , Mouth Neoplasms/genetics , Squamous Cell Carcinoma of Head and Neck
12.
Article in English | WPRIM | ID: wpr-878413

ABSTRACT

OBJECTIVES@#A study was conducted to investigate the molecular mechanism of chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) influencing the invasion and metastasis of tongue squamous cell carcinoma and to provide a new target for clinical inhibition of invasion and metastasis of tongue squamous cell carcinoma.@*METHODS@#Ualcan website was used to analyze the expression of CHD1L in normal epithelial tissue and primary head and neck squamous cell carcinoma and to analyze the effect of lymph node metastasis on the expression of CHD1L in tissues with head and neck squamous cell carcinoma. The relationship between CHD1L expression and the survival rate of patients with head and neck squamous cell carcinoma was tested by the GEPIA website. Western blot was used to quantify the levels of CHD1L protein in human tongue squamous cell carcinoma CAL27 and immortalized human skin keratinocyte cell HaCaT. After knocking down CAL27 in human tongue squamous cell carcinoma cells with an RNA interference plasmid, the cells were designated as SiCHD1L/CAL27 and Scr/CAL27. Western blot was utilized to detect the expression of CHD1L in each group of cells. The change in CAL27 cell proliferation ability was tested by EdU proliferation test after CHD1L knockdown. The change of cell migration ability of each group cells was tested through the wound healing assay. Western blot was used to detect epithelial-mesenchymal transition (EMT) marker E-cadherin and Vimentin protein expression levels.@*RESULTS@#Ualcan database showed that the expression of CHD1L in primary head and neck squamous cell carcinoma tissues was higher than in normal epithelial tissues and in head and neck squamous cell carcinoma tissues with lymph node metastasis. GEPIA website analysis showed that the overall survival rate of patients with head and neck squamous cell carcinoma with high expression of CHD1L was significantly lower than that of patients with low expression. Western blot results showed that CHD1L expression in human tongue squamous carcinoma cells CAL27 was higher than that of human normal skin cells HaCaT. CHD1L expression in SiCHD1L/CAL27 cells was much lower than that in Scr/CAL27 cells. Results of EdU proliferation experiments showed the significant reduction in the cell proliferation ability of the SiCHD1L/CAL27 cells. Results of the wound healing experiments showed the reduction in the migration capacity of the SiCHD1L/CAL27 cells. The expression of E-cadherin increased, whereas that of Vimentin decreased, in SiCHD1L/CAL27 cells.@*CONCLUSIONS@#CHD1L promoted the EMT, proliferation, migration, and invasion ability of tongue squamous cell carcinoma cells.


Subject(s)
Adenosine Triphosphatases , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , DNA Helicases , DNA-Binding Proteins , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms , Humans , Neoplasm Invasiveness/genetics , Tongue , Tongue Neoplasms/genetics
13.
Article in English | WPRIM | ID: wpr-878412

ABSTRACT

OBJECTIVES@#This study aimed to explore the effect of sex determining region Y-box 9 (SOX9) on the microtubule formation and epithelial-mesenchymal transition (EMT) of human oral squamous cell carcinoma (OSCC) CAL27 and the underlying mechanism.@*METHODS@#SOX9-shRNA1 and SOX9-shRNA2 were designed and synthesized and then transfected into CAL27 cells. The expression of SOX9 was detected by quantitative real-time polymerase chain reaction. Microtubule formation assay was used to detect the change in the number of microtubule nodules after interfering with SOX9. Immunofluorescence was used to detect the Vimentin content. Western blot was used to detect the protein expression of EMT marker molecules and Wnt/β-catenin pathway-related proteins, such as E-cadherin, N-cadherin, Fibronectin, Wnt, β-catenin, T-cell factor-4 (TCF-4).@*RESULTS@#The expression level of SOX9 significantly decreased after transfection with SOX9-shRNA1 and SOX9-shRNA2 in CAL27 cells (@*CONCLUSIONS@#Interference with SOX9 decreased Vimentin content and inhibited the microtubule formation and protein expression of EMT marker molecules, as well as the expression of proteins related to the Wnt/β-catenin pathway. Thus, SOX9 can induce microtubule formation and EMT in CAL27, which was related to the inhibition of the Wnt/β-catenin pathway activation.


Subject(s)
Carcinoma, Squamous Cell , Cell Line, Tumor , Epithelial-Mesenchymal Transition , Head and Neck Neoplasms , Humans , Microtubules/metabolism , Mouth Neoplasms , SOX9 Transcription Factor/metabolism , Squamous Cell Carcinoma of Head and Neck , Wnt Signaling Pathway , beta Catenin/metabolism
14.
Article in English | WPRIM | ID: wpr-878411

ABSTRACT

OBJECTIVES@#This study aimed to explore the effects of silencing isoprenylcysteine carboxyl methyltransfe-rase (Icmt) through small interfering RNA (siRNA) interference on the proliferation and apoptosis of tongue squamous cell carcinoma (TSCC).@*METHODS@#Three siRNA were designed and constructed for the Icmt gene sequence and were then transfected into TSCC cells CAL-27 and SCC-4 to silence Icmt expression. The tested cells were divided as follows: RNA interference groups Icmt-siRNA-1, Icmt-siRNA-2, and Icmt-siRNA-3, negative control group, and blank control group. The transfection efficiency of siRNA was detected by the fluorescent group Cy3-labeled siRNA, and the expression of Icmt mRNA was screened by quantitive real-time polymerase chain reaction (qRT-PCR) selected the experimental group for subsequent experiments. The expression of Icmt, RhoA, Cyclin D1, p21, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) were analyzed by Western blot. The proliferation abilities of TSCC cells were determined by cell counting kit-8 assay. The change in apoptosis was detected by AnnexinV-APC/propidium staining (PI) assay. Cell-cycle analysis was conducted by flow cytometry.@*RESULTS@#The expression of Icmt mRNA and protein in TSCC cells significantly decreased after Icmt-siRNA transfection (@*CONCLUSIONS@#Silencing Icmt can effectively downregulate its expression in TSCC cells, reduce the RhoA membrane targeting localization and cell proliferation, and induce apoptosis. Thus, Icmt may be a potential gene therapy target for TSCC.


Subject(s)
Apoptosis , Carcinoma, Squamous Cell , Cell Line, Tumor , Cell Proliferation , Humans , Protein Methyltransferases , RNA, Small Interfering , Tongue , Tongue Neoplasms
15.
Article in English | WPRIM | ID: wpr-878331

ABSTRACT

Objective@#The underlying mechanism of Ezrin in ovarian cancer (OVCA) is far from being understood. Therefore, this study aimed to assess the role of Ezrin in OVCA cells (SKOV3 and CaOV3) and investigate the associated molecular mechanisms.@*Methods@#We performed Western blotting, reverse transcription-quantitative polymerase chain reaction, MTT, cell colony, cell wound healing, transwell migration and invasion, RhoA and Rac active pull down assays, and confocal immunofluorescence experiments to evaluate the functions and molecular mechanisms of Ezrin overexpression or knockdown in the proliferation and metastasis of OVCA cells.@*Results@#The ectopic expression of Ezrin significantly increased cell proliferation, invasiveness, and epithelial-mesenchymal transition (EMT) in OVCA cells. By contrast, the knockdown of endogenous Ezrin prevented OVCA cell proliferation, invasiveness, and EMT. Lastly, we observed that Ezrin can positively regulate the active forms of RhoA rather than Rac-1 in OVCA cells, thereby promoting robust stress fiber formation.@*Conclusion@#Our results indicated that Ezrin regulates OVCA cell proliferation and invasiveness by modulating EMT and induces actin stress fiber formation by regulating Rho-GTPase activity, which provides novel insights into the treatment of the OVCA.


Subject(s)
Cell Line, Tumor , Cell Movement , Cell Proliferation , Cytoskeletal Proteins/metabolism , Epithelial-Mesenchymal Transition , Female , Gene Expression Regulation, Neoplastic , Humans , Neoplasm Invasiveness , Ovarian Neoplasms/pathology , Stress Fibers/metabolism , rhoA GTP-Binding Protein/metabolism
16.
Article in English | WPRIM | ID: wpr-878318

ABSTRACT

Objective@#Antimony (Sb) has recently been identified as a novel nerve poison, although the cellular and molecular mechanisms underlying its neurotoxicity remain unclear. This study aimed to assess the effects of the nuclear factor kappa B (NF-κB) signaling pathway on antimony-induced astrocyte activation.@*Methods@#Protein expression levels were detected by Western blotting. Immunofluorescence, cytoplasmic and nuclear fractions separation were used to assess the distribution of p65. The expression of protein in brain tissue sections was detected by immunohistochemistry. The levels of mRNAs were detected by Quantitative real-time polymerase chain reaction (qRT-PCR) and reverse transcription-polymerase chain reaction (RT-PCR).@*Results@#Antimony exposure triggered astrocyte proliferation and increased the expression of two critical protein markers of reactive astrogliosis, inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP), indicating that antimony induced astrocyte activation @*Conclusion@#Antimony activated astrocytes by activating the NF-κB signaling pathway.


Subject(s)
Animals , Antimony/toxicity , Astrocytes/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Glial Fibrillary Acidic Protein/metabolism , MAP Kinase Kinase Kinases , Male , Mice, Inbred ICR , NF-kappa B/metabolism , Nitric Oxide Synthase Type II/metabolism , Rats , Signal Transduction/drug effects
17.
Acta Physiologica Sinica ; (6): 233-243, 2021.
Article in English | WPRIM | ID: wpr-878252

ABSTRACT

There is increasing evidence that long non-coding RNA (lncRNA) plays critical roles in cancer progression. However, the role of long non-coding RNA 00665 (LINC00665) in most cancers is poorly understood. The purpose of the present study was to reveal the functional role of LINC00665 in cervical cancer cells. HeLa cells were subjected to LINC00665 short hairpin RNA (shRNA) or control shRNA treatment to investigate the metastasis and proliferation phenotype of cervical cancer cells in vitro and in vivo. Transcriptome sequencing experiments of HeLa cells in LINC00665 silencing or control group were conducted, and the differentially expressed genes (DEGs) were screened. The DEGs were subjected to Metascape database functional analysis and gene set enrichment analysis. Epithelial-mesenchymal transition (EMT) related markers and a key element of WNT/β‑catenin pathway, CTNNB1 (catenin beta 1), were detected by Western blot and immunofluorescence assay. The results showed that silencing LINC00665 reduced cell viability of Hela cells, up-regulated protein expression level of E-cadherin, down-regulated protein expression levels of N-cadherin, Vimentin and CTNNB1, and inhibited cell migration and invasion of HeLa cells. Bioinformatics analysis results showed that LINC00665 might promote EMT by activating WNT-CTNNB1/β‑catenin signaling pathway. These results indicate that LINC00665 has functions in transcriptional EMT regulation via WNT-CTNNB1/β‑catenin signaling pathway and therefore can be developed as a therapeutic target for cervical cancer.


Subject(s)
Cell Line, Tumor , Cell Movement , Cell Proliferation , Epithelial-Mesenchymal Transition , Female , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , Wnt Signaling Pathway , beta Catenin/metabolism
18.
Chinese Medical Journal ; (24): 1345-1355, 2021.
Article in English | WPRIM | ID: wpr-878149

ABSTRACT

BACKGROUND@#Although increasing abnormal expression of circular RNAs (circRNAs) has been revealed in various cancers, there were a small number of studies about circRNAs in gastric cancer (GC). Here, we explored the expression and function of a novel circRNA, circ_0049447, in GC.@*METHODS@#A total of 80 GC tissues and non-tumorous tissues were collected from the First Affiliated Hospital of China Medical University. And all cells were cultured with 10% fetal bovine serum and incubated at 37°C and 5% CO2. The expression of circ_0049447 was quantified by real-time polymerase chain reaction. The biological function of circ_0049447 on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) was evaluated by cell counting kit-8 (CCK-8), colony formation assay, transwell migration and invasion assay, and Western blotting. Luciferase report assay was used to verify the direct binding between circ_0049447 and predicted microRNA (miRNA). Furthermore, a xenograft mouse model was used to validate the function of circ_0049447 in vivo.@*RESULTS@#We demonstrated that circ_0049447 was downregulated in GC (P < 0.001). The area under the receiver operating characteristic curve reached 0.838, while sensitivity was 82.3% and specificity was 77.2%. CCK-8 and colony formation assay showed that overexpression of circ_0049447 could inhibit the proliferation (P < 0.05). Transwell migration and invasion assay showed upregulated circ_0049447 could impede migration in GC cells (P < 0.05). In addition, overexpression of circ_0049447 could impede GC cell EMT. Upregulation of miR-324-5p in GC specimens and direct binding between miR-324-5p with circ_0049447 proven by luciferase reporter assay indicated that circ_0049447 may inhibit GC by sponging certain miRNA.@*CONCLUSION@#Circ_0049447 acts as a tumor suppressor in GC through reducing proliferation, migration, invasion, and EMT, and it is a promising biomarker for diagnosis.


Subject(s)
Animals , Cell Line, Tumor , Cell Proliferation/genetics , China , Epithelial-Mesenchymal Transition/genetics , Mice , Stomach Neoplasms/genetics
19.
Chinese Medical Journal ; (24): 963-970, 2021.
Article in English | WPRIM | ID: wpr-878129

ABSTRACT

BACKGROUND@#Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1β)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/β-catenin signaling pathway.@*METHODS@#Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1β and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1β. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1β, the translocation of β-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/β-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3β) plasmids were assessed for their effects on HDAC4 levels using Western blotting.@*RESULTS@#IL-1β downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1β-induced increases in MMP3 and MMP13. IL-1β upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3β rescued IL-1β-induced downregulation of HDAC4 in SW1353 cells.@*CONCLUSION@#HDAC4 exerted an inhibitory effect on IL-1β-induced extracellular matrix degradation and was regulated partially by the WNT3A/β-catenin signaling pathway.


Subject(s)
Cell Line, Tumor , Cells, Cultured , Chondrocytes/metabolism , Glycogen Synthase Kinase 3 beta/genetics , Histone Deacetylases/genetics , Humans , Interleukin-1beta/pharmacology , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase 3 , Repressor Proteins , Wnt Signaling Pathway , Wnt3A Protein/genetics , beta Catenin/metabolism
20.
Chinese Medical Journal ; (24): 564-572, 2021.
Article in English | WPRIM | ID: wpr-878081

ABSTRACT

BACKGROUND@#The pathogenesis of osteosarcoma (OS) is still unclear, and it is still necessary to find new targets and drugs for anti-OS. This study aimed to investigate the role and mechanism of the anti-OS effects of miR-296-5p.@*METHODS@#We measured the expression of miR-296-5p in human OS cell lines and tissues. The effect of miR-296-5p and its target gene staphylococcal nuclease and tudor domain containing 1 on proliferation, migration, and invasion of human OS lines was examined. The Student's t test was used for statistical analysis.@*RESULTS@#We found that microRNA (miR)-296-5p was significantly downregulated in OS cell lines and tissues (control vs. OS, 1.802 ± 0.313 vs. 0.618 ± 0.235, t = 6.402, P < 0.01). Overexpression of miR-296-5p suppressed proliferation, migration, and invasion of OA cells. SND1 was identified as a target of miR-296-5p by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of SND1 abrogated the effects induced by miR-296-5p upregulation (miRNA-296-5p vs. miRNA-296-5p + SND1, 0.294 ± 0.159 vs. 2.300 ± 0.277, t = 12.68, P = 0.003).@*CONCLUSION@#Our study indicates that miR-296-5p may function as a tumor suppressor by targeting SND1 in OS.


Subject(s)
Bone Neoplasms/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Endonucleases/genetics , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Humans , MicroRNAs/genetics , Osteosarcoma/genetics
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