Protein Expression Levels Of E-cadherin Research Articles

Study on the protection of gingival epithelial barrier by interleukin-22 through regulating microbiota and E-cadherin expression

Objective: To investigate the regulatory effect and mechanism of interleukin-22 (IL-22) on the gingival epithelial barrier in the context of periodontal inflammation. Methods: IL-22 knockout (IL-22 KO) mice were constructed, and periodontitis mice models were established through oral gavage with polymicrobial inoculation. DNAs were extracted from the oral plaques of IL-22 KO periodontitis mice group (n=7) and their wild-type littermates periodontitis group (n=7) to establish a periodontitis-related oral microbiota database"PD-RiskMicroDB", determining the relationship between changes in oral microbiota and microbial function in two groups using 16S rRNA sequencing results. Gingival epithelial cells (GEC) were cultured by modified trypsinization method, and were stimulated with 100 μg/L IL-22, Porphyromonas gingivalis (Pg) (multiplicity of infection:100), separately or together for 3 and 12 hours. The experimental groups were as follows: control group (no stimulation), IL-22 group, Pg group and Pg+IL-22 group. The expression of barrier protein E-cadherin in each group at 3 h was detected by immunofluorescence, real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting. Fluorescein isothiocyanate-dextran-mediated epithelial cell permeability experiment was conducted to clarify the changes in permeability of GEC in each group at 3 and 12 h. The mRNA expressions of E-cadherin in the gingival epithelium of wild-type littermates periodontitis group and IL-22 KO periodontitis group were detected by RT-qPCR. Fifteen C57BL/6 wild-type mice were randomly divided into control group (n=5), periodontitis group (n=5) and periodontitis+IL-22 treatment group (n=5). RT-qPCR and immunohistochemistry (IHC) staining were used to detect the expression level of E-cadherin in the gingival epithelium of each group. Results: 16S rRNA sequencing results showed that the composition of oral microbiota changed in IL-22 KO periodontitis group, of which the abundance of bacterial genera related to periodontal tissue invasion was significantly increased (linear discriminant analysis score: 2.22, P=0.009), compared with wild-type littermates periodontitis group. In vitro cell experiments showed that after Pg infection for 3 hours, the cell connections of GEC in Pg group were interrupted, and the fluorescence intensity of E-cadherin was reduced in Pg group compared with the control group. Meanwhile, the mRNA and protein expression levels of E-cadherin (mRNA: 0.69±0.12; protein: 0.60±0.12) were downregulated compared with the control group [mRNA: 1.00±0.00 (P=0.043); protein: 1.04±0.08 (P=0.003)], respectively. The fluorescence intensity of E-cadherin in the Pg+IL-22 group was enhanced compared with Pg group, and expression levels of E-cadherin mRNA (1.16±0.10) and protein (0.98±0.07) in Pg+IL-22 group showed a significant increase compared with Pg group [mRNA: 0.69±0.12 (P=0.005); protein: 0.60±0.12 (P=0.007)]. The result of epithelial permeability test showed that there was no statistical difference in epithelial permeability among control group, Pg group, IL-22 group and Pg+IL-22 group with treatment for 3 hours (F=0.20, P=0.893). While when the treatment time turned to be 12 hours, the epithelial barrier permeability showed a significant increase in Pg group (1.39±0.15) compared with control group (1.00±0.00, P=0.027), and a decrease in Pg+IL-22 group (1.02±0.18) compared with Pg group (1.39±0.15, P=0.034). In vivo, the mRNA expression of E-cadherin in the gingival epithelium of IL-22 KO periodontitis group decreased significantly (0.32±0.21) compared with wild-type littermates periodontitis group (1.01±0.01) (t=5.70, P=0.005). Moreover, RT-qPCR and IHC staining results showed that the mRNA expression level of E-cadherin (0.40±0.07) and absorbance value of E-cadherin positive expression (0.02±0.00) in gingival epithelial tissue of periodontitis group were both significantly down-regulated compared with control group [mRNA: 1.00±0.00 (P=0.005); absorbance value of E-cadherin positive expression: 0.04±0.01 (P=0.006)]. Meanwhile, the mRNA expression level of E-cadherin (1.06±0.24) and the absorbance value of E-cadherin positive expression (0.03±0.01) were both observed increase in periodontitis+IL-22 treatment group compared with periodontitis group (P=0.003, P=0.039). Conclusions: IL-22 may exert a protective effect on the gingival epithelial barrier in an inflammatory environment by regulating the invasiveness of oral microbiota and the expression of host barrier protein.

Lathyrol reduces the RCC invasion and incidence of EMT via affecting the expression of AR and SPHK2 in RCC mice

ObjectiveTo investigate the effects of Lathyrol on the expression of androgen receptor (AR) and sphingosine kinase 2 (SPHK2) in renal cell carcinoma (RCC) mice and to further explore the mechanism by which Lathyrol inhibits the invasion and incidence of epithelial-mesenchymal transition (EMT).MethodsAn RCC xenograft mouse model was constructed, and the mice were randomly divided into a model group, an experiment group and a negative control group. The experiment group was intragastrically gavaged with Lathyrol solution (20 mg/kg), the model group was intragastrically gavaged with 0.9% NaCl (same volume as that used in the experiment group), and the negative control group was injected intraperitoneally with 2 mg/kg cisplatin aqueous solution. Changes in the body weight and tumor volume of the mice were recorded. Western blot (WB) was used to assess the protein expression levels of AR, p-AR, CYP17A1, PARP1, E-cadherin, N-cadherin, vimentin, α-SMA, β-catenin, and ZO-1. Protein expression levels of SPHK2, metal matrix protease 2 (MMP2), MMP9 and urokinase-type plasminogen activator (uPA) in tumor tissues were assessed by immunohistochemistry (IHC). AR expression in tumor tissues was assessed after immunofluorescence (IF) staining.ResultsAfter 14 days of drug administration, compared with that in the model group, the tumor volumes in the negative control and experiment groups were lower; the difference in tumor volume among the model, control and experiment groups was statistically significant (P < 0.05). The differences in body weight among the three groups were not statistically significant (P > 0.05). In the model group, the protein expression levels of AR, p-AR, CYP17A1, SPHK2, and PARP1 were relatively increased, the protein expression levels of E-cadherin and ZO-1 were relatively reduced (P < 0.05), and the protein expression levels of N-cadherin, β-catenin, vimentin, and α-SMA were relatively increased (P < 0.05). In the negative control and experiment groups, the protein expression levels of AR, p-AR, CYP17A1, SPHK2, and PARP1 were relatively decreased (P < 0.05), the protein expression levels of E-cadherin and ZO-1 were relatively increased (P < 0.05), and the protein expression levels of N-cadherin, β-catenin, vimentin and α-SMA were relatively decreased (P < 0.05).ConclusionLathyrol and cisplatin inhibit the proliferation of RCC xenografts, reduce the protein expression levels of AR, CYP17A1, SPHK2, PARP1, E-cadherin, and ZO-1 in tumor tissues (P < 0.05), and promote the protein expression levels of N-cadherin, β-catenin, vimentin and α-SMA (P < 0.05). Therefore, Lathyrol reduces RCC invasion and EMT by affecting the expression of AR and SPHK2 in RCC mice.

Role of CTGF and PI3K/Akt signaling pathway in paraquat-induced mesenchymal changes in alveolar epithelial cells

Objective: To investigate the role of connective tissue growth factor (CTGF) and PI3K/Akt signaling pathways in paraquat (PQ) -induced alterations in alveolar epithelial cell mesenchymalization (EMT) . Methods: In February 2023, RLE-6TN cells were divided into 2 groups, which were set as uncontaminated group and contaminated group (200 μmol/L PQ), and cellular EMT alteration, CTGF and PI3K/Akt signaling pathway related molecules expression were detected by cell scratch assay, qRT-PCR and western-blot assay. Using shRNA interference technology to specifically inhibit the expression of CTGF, RLE-6TN cells were divided into four groups: control group, PQ group (200 μmol/L PQ), interference group (transfected with a plasmid with shRNA-CTGF+200 μmol/L PQ), and null-loaded group (transfected with a plasmid with scramble- CTGF+200 μmol/L PQ), qRT-PCR and western blot were used to examine the alteration of the cellular EMT and the expression of molecules related to the activity of PI3K/Akt pathway. The PI3K/Akt signaling pathway was blocked by the PI3K inhibitor LY294002, and the expression of EMT-related molecules in cells of the control group, PQ group (200 μmol/L PQ), and inhibitor group (200 μmol/L PQ+20 μmol/L LY294002) was examined by qRT-PCR and western blot.The t-test was used to compare the differences between the two groups, while the analysis of variance (ANOVA) was applied to compare the differences among multiple groups. For further pairwise comparisons, the Bonferroni method was adopted. Results: The results of cell scratch test showed that compared with the uncontaminated group, RLE-6TN cells in the contaminated group had faster migration rate, lower mRNA and protein expression levels of E-Cadherin, and higher mRNA and protein expression levels of α-SMA, CTGF, PI3K and Akt, with statistical significance (P<0.05). After specific inhibition of CTGF expression, the mRNA and protein expression of CTGF, PI3K, Akt, and α-SMA in the cells of the interference group were significantly lower than that of the PQ group and the null-loaded group (P<0.05/6), whereas that of E-Cadherin was higher than that of the PQ group and the null-loaded group (P<0.05/6). Specifically blocking the PI3K/Akt signaling pathway, the mRNA and protein expression of PI3K, Akt and α-SMA in the cells of the inhibitor group was decreased compared with that of the PQ group (P<0.05/3), while the expression of E-Cadherin was elevated compared with that of the PQ group (P<0.05/3) . Conclusion: CTGF may promote PQ-induced alveolar epithelial cell EMT through activation of the PI3K/Akt signaling pathway. Inhibition of CTGF expression or blockade of PI3K/Akt signaling pathway activity can alleviate the extent of PQ-induced alveolar epithelial cell EMT.

Bone morphogenetic protein-6 suppresses TGF-β2-induced epithelial-mesenchymal transition in retinal pigment epithelium.

To evaluate the effect of bone morphogenetic protein-6 (BMP-6) on transforming growth factor (TGF)-β2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE). Adult retinal pigment epithelial cell line (ARPE-19) were randomly divided into control, TGF-β2 (5 µg/L), and BMP-6 small interfering RNA (siRNA) group. The cell morphology was observed by microscopy, and the cell migration ability were detected by Transwell chamber. The EMT-related indexes and BMP-6 protein levels were detected by Western blotting. Furthermore, a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group, TGF-β2+empty plasmid group, BMP-6 overexpression group, and TGF-β2+BMP-6 overexpression group. The EMT-related indexes and extracellular regulated protein kinases (ERK) protein levels were detected. Compared with the control group, the migration of RPE cells in the TGF-β2 group was significantly enhanced. TGF-β2 increased the protein expression levels of α-smooth muscle actin (α-SMA), fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6 (P<0.05) in RPE. Similarly, the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced. BMP-6 siRNA increased the protein expression levels of α-SMA, fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin (P<0.05). Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-β2 and prevented TGF-β2 from affecting EMT-related biomarkers (P<0.05). BMP-6 prevents the EMT in RPE cells induced by TGF-β2, which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

Systematic analysis of the role of LDHs subtype in pan-cancer demonstrates the importance of LDHD in the prognosis of hepatocellular carcinoma patients

BackgroundLactate dehydrogenase (LDHs) is an enzyme involved in anaerobic glycolysis, including LDHA, LDHB, LDHC and LDHD. Given the regulatory role in the biological progression of certain tumors, we analyzed the role of LDHs in pan-cancers.MethodsCox regression, Kaplan–Meier curves, Receiver Operating Characteristic (ROC) curves, and correlation of clinical indicators in tumor patients were used to assess the prognostic significance of LDHs in pan-cancer. The TCGA, HPA, TIMER, UALCAN, TISIDB, and Cellminer databases were used to investigate the correlation between the expression of LDHs and immune subtypes, immune checkpoint genes, methylation levels, tumor mutational load, microsatellite instability, tumor-infiltrating immune cells and drug sensitivity. The cBioPortal database was also used to identify genomic abnormalities of LDHs in pan-cancer. A comprehensive assessment of the biological functions of LDHs was performed using GSEA. In vitro, HepG2 and Huh7 cells were transfected with LDHD siRNA and GFP-LDHD, the proliferation capacity of cells was examined using CCK-8, EdU, and colony formation assays; the migration and invasion of cells was detected by wound healing and transwell assays; western blotting was used to detect the levels of MMP-2, MMP-9, E-cadherin, N-cadherin and Akt phosphorylation.ResultsLDHs were differentially expressed in a variety of human tumor tissues. LDHs subtypes can act as pro-oncogenes or anti-oncogenes in different types of cancer and have an impact on the prognosis of patients with tumors by influencing their clinicopathological characteristics. LDHs were differentially expressed in tumor immune subtypes and molecular subtypes. In addition, LDHs expression correlated with immune checkpoint genes, tumor mutational load, and microsatellite instability. LDHD was identified to play an important role in the prognosis of HCC patients, according to a comprehensive analysis of LDHs in pan-cancer. In HepG2 and Huh7 cells, knockdown of LDHD promoted cell proliferation, migration, and invasion, promoted the protein expression levels of MMP-2, MMP-9, N-cadherin, and Akt phosphorylation, but inhibited the protein expression level of E-cadherin. In addition, LDHD overexpression showed the opposite changes.ConclusionLDHs subtypes can be used as potential prognostic markers for certain cancers. Prognostic and immunotherapeutic analysis indicated that LDHD plays an important role in the prognosis of HCC patients. In vitro experiments revealed that LDHD can affect HCC proliferation, migration, and invasion by regulating MMPs expression and EMT via Akt signaling pathway, which provides a new perspective on the anti-cancer molecular mechanism of LDHD in HCC.

Analysis of m6A-related lncRNAs for prognostic and immunotherapeutic response in hepatocellular carcinoma.

Background: RNA methylation modifications are important post-translational modifications that are regulated in an epigenetic manner. Recently, N6-methyladenosine (m6A) RNA modifications have emerged as potential epigenetic markers in tumor biology. Methods: Gene expression and clinicopathological data of LIHC were obtained from the cancer genome atlas (TCGA) database. The relationship between long non-coding RNAs (lncRNAs) and m6A-related genes was determined by gene expression analysis using Perl and R software. Co-expression network of m6A-lncRNA was constructed, and the relevant lncRNAs associated with prognosis were identified using univariate Cox regression analysis. These lncRNAs were then divided into two clusters (cluster 1 and cluster 2) to determine the differences in survival, pathoclinical parameters, and immune cell infiltration between the different lncRNA subtypes. The least absolute shrinkage and selection operator (LASSO) was carried out for regression analysis and prognostic model. The HCC patients were randomly divided into a train group and a test group. According to the median risk score of the model, HCC patients were divided into high-risk and low-risk groups. We built models using the train group and confirmed them through the test group. The m6A-lncRNAs derived from the models were analyzed for the tumor mutational burden (TMB), immune evasion and immune function using R software. AL355574.1 was identified as an important m6A-associated lncRNA and selected for further investigation. Finally, in vitro experiments were conducted to confirm the effect of AL355574.1 on the biological function of HCC and the possible biological mechanisms. Huh7 and HepG2 cells were transfected with AL355574.1 siRNA and cell proliferation ability was measured by CCK-8, EdU and colony formation assays. Wound healing and transwell assays were used to determine the cell migration capacity. The expression levels of MMP-2, MMP-9, E-cadherin, N-cadherin and Akt/mTOR phosphorylation were all determined by Western blotting. Results: The lncRNAs with significant prognostic value were classified into two subtypes by a consistent clustering analysis. We found that the clinical features, immune cell infiltration and tumor microenvironment (TME) were significantly different between the lncRNA subtypes. Our analysis revealed significant correlations between these different lncRNA subtypes and immune infiltrating and stromal cells. We created the final risk profile using LASSO regression, which notably included three lncRNAs (AL355574.1, AL158166.1, TMCC1-AS1). A prognostic signature consisting of the three lncRNAs was constructed, and the model showed excellent prognostic predictive ability. The overall survival (OS) of the low-risk cohort was significantly higher than that of the high-risk cohort in both the train and test group. Both risk score [hazard ratio (HR)=1.062; P<0.001] and stage (HR=1.647; P< 0.001) were considered independent indicators of HCC prognosis by univariate and multivariate Cox regression analysis. In Huh7 and HepG2 cells, AL355574.1 knockdown inhibited cell proliferation and migration, suppressed the protein expression levels of MMP-2, MMP-9, N-cadherin and Akt/mTOR phosphorylation, but promoted the protein expression levels of E-cadherin. Conclusions: This study established a predictive model for the OS of HCC patients, and these OS-related m6A-lncRNAs, especially AL355574.1 may play a potential role in the progression of HCC. In vitro experiments also showed that AL355574.1 could enhance the expression of MMPs and EMT through the Akt/mTOR signaling pathway, thereby affected the proliferation and migration of HCC. This provides a new perspective on the anticancer molecular mechanism of AL355574.1 in HCC.

Dapagliflozin can alleviate renal fibrosis in rats with streptozotocin‑induced type2 diabetes mellitus.

The aim of the present study was to explore the effects of Dapagliflozin on renal fibrosis in streptozotocin (STZ)-induced type 2 diabetes mellitus (T2DM) rats, and to determine the underlying mechanism of action. A total of 24 SPF male SD rats were randomly divided into 4 groups: A normal (Control) group, model group (STZ-induced T2DM rats), Dapagliflozin group (STZ-induced T2DM rats treated with 1 mg/kg Dapagliflozin), and a metformin group (STZ-induced T2DM rats treated with 200 mg/kg metformin), with 6 rats per a group. Peripheral blood and renal tissues were collected from these rats, and the renal indices of each group were examined. The fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c), blood urea nitrogen (BUN), and serum creatinine (SCr) of rats were detected. After 24 h, the urine was collected and the urine protein levels were measured. Hematoxylin and eosin staining was used to detect histological changes in the rat kidney; Masson staining was used to observe the degree of fibrosis in rat renal tissues; and western blot was performed to determine the expression levels of α-smooth muscle actin (SMA), vimentin, E-cadherin, TGF-β1, Smad7, and p-Smad3 in rat renal tissues. Dapagliflozin effectively inhibited the increase in FBG and HbA1c levels in diabetic mice, reduced renal tissue damage, reduced the renal index values, reduced collagen deposition in the glomerulus and interstitial area, and reduced the proliferation of glomerular mesangial cells. In addition, Dapagliflozin significantly lowered the levels of BUN, SCr, and 24-h urine protein, decreased the protein expression of α-SMA, vimentin, TGF-β1, and p-Smad3, and increased the protein expression levels of E-cadherin and Smad7. Together, these results showed that Dapagliflozin alleviated renal fibrosis in STZ-induced T2DM rats, and its mechanism of action may be related to the inhibition of the TGF-β1/Smad pathway.

Liuwei Dihuang Decoction Drug-containing Serum Attenuates Transforming Growth Factor-β1-induced Epithelial-Mesenchymal Transition in HK-2 cells by Inhibiting NF-κB/Snail Signaling Pathway.

The nuclear factor-κB (NF-κB) signaling pathway plays an important role in regulating tubular epithelial-mesenchymal transition (EMT), an indispensable cellular programme for driving organ fibrosis and tumor progression. Liuwei Dihuang Decoction (LWD) is an effective Chinese formula for treating chronic renal failure. First, by using morphological examination, immunofluorescence staining assay, RT-qPCR, and Western blot analysis, in vitro experiments were designed to analyze NF-κB and EMT markers (including Snail, α-SMA, and E-cadherin) in transforming growth factor-β1 (TGF-β1) induced renal tubular epithelial cells (HK-2) and to detect the expression levels of LWD-CS co-treatment. Then, the recombinant lentiviral vector was overexpressed and knocked down by NF-κB and transfected into HK-2 cells. Cells were treated with TGF-β1 (10ng/ml) with blank serum or LWD-containing serum, respectively, and the expression of these molecules in the NF-κB/Snail signaling pathway was further evaluated. Our results confirmed that TGF-β1 could induce EMT, nuclear translocation of NF-κB p65, and activate the NF-κB/Snail signaling pathway in HK-2 cells. Furthermore, NF-κB knocked-down dramatically increases the TGF-β1-induced mRNA and protein expression level of E-cadherin and reduces the level of Snail and α-SMA; this is reversed by NF-κB overexpression. LWD can decrease the EMT levels through the NF-κB/Snail signaling activation in TGF-β1-induced EMT of HK-2 cells. The present study provides evidence suggesting a novel mechanism that LWD exerts anti-fibrosis effects through inhibiting activation of the NF-κB/Snail signaling pathway and consequently downregulating the TGF-β1-induced EMT in renal tubular epithelial cell.

EXOSC10 is a novel hepatocellular carcinoma prognostic biomarker: a comprehensive bioinformatics analysis and experiment verification.

Hepatocellular carcinoma (HCC) is a common malignant tumor. There are few studies on EXOSC10 (exosome component 10) in HCC; however, the importance of EXOSC10 for HCC remains unclear. In the study, the prognosis value of EXOSC10 and the immune correlation were explored by bioinformatics. The expression of EXOSC10 was verified by tissue samples from clinical patients and in vitro experiment (liver cancer cell lines HepG2, MHCC97H and Huh-7; normal human liver cell line LO2). Immunohistochemistry (IHC) was used to detect EXOSC10 protein expression in clinical tissue from HCC. Huh-7 cells with siEXOSC10 were constructed using lipofectamine 3000. Cell counting kit 8 (CCK-8) and colony formation were used to test cell proliferation. The wound healing and transwell were used to analyze the cell migration capacity. Mitochondrial membrane potential, Hoechst 33342 dye, and flow cytometer were used to detect the change in cell apoptosis, respectively. Differential expression genes (DEGs) analysis and gene set enrichment analysis (GSEA) were used to investigate the potential mechanism of EXOSC10 and were verified by western blotting. EXOSC10 was highly expressed in tissues from patients with HCC and was an independent prognostic factor for overall survival (OS) in HCC. Increased expression of EXOSC10 was significantly related to histological grade, T stage, and pathological stage. Multivariate analysis indicated that the high expression level of EXOSC10 was correlated with poor overall survival (OS) in HCC. GO and GSEA analysis showed enrichment of the cell cycle and p53-related signaling pathway. Immune analysis showed that EXOSC10 expression was a significant positive correlation with immune infiltration in HCC. In vitro experiments, cell proliferation and migration were inhibited by the elimination of EXOSC10. Furthermore, the elimination of EXOSC10 induced cell apoptosis, suppressed PARP, N-cadherin and Bcl-2 protein expression levels, while increasing Bax, p21, p53, p-p53, and E-cadherin protein expression levels. EXOSC10 had a predictive value for the prognosis of HCC and may regulate the progression of HCC through the p53-related signaling pathway.

MiR-30a inhibits silica dust-induced epithelial-mesenchymal transition by targeting Snail

The mechanism of action of MicroRNA-30a(miR-30a) and Snail, a transcription factor, in silica(SiO2) dust-induced pulmonary EMT and secondary pulmonary fibrosis remains elusive. In this study, the cellular EMT model induced by the stimulation of A549 cells with SiO2 was established. A549 cells were transfected with miR-30a mimic and miR-30a inhibitor and the SNAIL gene was silenced to examine the mechanism of miR-30a targeting Snail to regulate silica dust-induced EMT. The results showed that 50 μg/mL SiO2 stained A549 cells for 24 h could induce EMT in A549 cells. Exposure of A549 cells to SiO2 dust decreased miR-30a expression, as well as mRNA and protein expression levels of E-cad. Conversely, SiO2 exposure increased mRNA and protein expression levels of α-SMA, vimentin, and Snail. The miR-30a mimic upregulated mRNA and protein expression levels of E-cadherin in SiO2-induced A549 cells, while downregulating mRNA and protein expression levels of α-SMA, vimentin and Snail. MiR-30a inhibitors have the opposite effect. Silencing the SNAIL gene, followed by SiO2 dust-induced stimulation of A549 cells, could enhance mRNA and protein expression levels of E-cad, whereas those of α-SMA and vimentin were reduced. Altogether, we found that miR-30a directly targeted Snail and inhibited its expression, thereby delaying silica induced pulmonary EMT.

Sinularin Exerts Anti-cancer Effects by Inducing Oxidative Stress-mediated Ferroptosis, Apoptosis, and Autophagy in Prostate Cancer Cells.

Prostate cancer is the second-leading cause of cancer death in men. Sinularin is a soft coralsderived natural compound that has anticancer activity in many cancer cells. However, the pharmacological action of sinularin in prostate cancer is unclear. The aim of the study is to examine the anticancer effects of sinularin in prostate cancer cells. We explored the anticancer effects of sinularin on the prostate cancer cell lines, PC3, DU145, and LNCaP, by MTT, Transwell assay, wound healing, flow cytometry, and western blotting. Sinularin inhibited the cell viability and colony formation of these cancer cells. Furthermore, sinularin inhibited testosterone-induced cell growth in LNCaP cells by downregulating the protein expression levels of androgen receptor (AR), type Ⅱ 5α-reductase, and prostate-specific antigen (PSA). Sinularin significantly attenuated the invasion and migration ability of PC3 and DU145 cells, with or without TGF-β1 treatment. Sinularin inhibited epithelialmesenchymal transition (EMT) in DU145 cells after 48 h of treatment by regulating the protein expression levels of Ecadherin, N-cadherin, and vimentin. Sinularin induced apoptosis, autophagy, and ferroptosis by regulating the protein expression levels of Beclin-1, LC3B, NRF2, GPX4, PARP, caspase-3, caspase-7, caspase-9, cleaved-PARP, Bcl-2, and Bax. Moreover, intracellular reactive oxygen species (ROS) were increased but glutathione was decreased after sinularin treatment in PC3, DU145 and LNCaP cells. Sinularin regulated the androgen receptor signaling pathway and triggered apoptosis, autophagy, and ferroptosis in prostate cancer cells. In conclusion, the results indicated that sinularin may be a candidate agent for human prostate cancer and need further study for being applied to human.

Knockdown of ACC1 promotes migration of esophageal cancer cell

Objective: To investigate the effect of acetyl-CoA carboxylase 1 (ACC1) knockdown on the migration of esophageal squamous cell carcinoma (ESCC) KYSE-450 cell and underlying mechanism. Methods: Lentiviral transfection was conducted to establish sh-NC control cell and ACC1 knocking down cell (sh-ACC1). Human siRNA HSP27 and control were transfected by Lipo2000 to get si-HSP27 and si-NC. The selective acetyltransferase P300/CBP inhibitor C646 was used to inhibit histone acetylation and DMSO was used as vehicle control. Transwell assay was performed to detect cell migration. The expression of HSP27 mRNA was examined by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and the expressions of ACC1, H3K9ac, HSP27 and epithelial-mesenchymal transition-related proteins E-cadherin and Vimentin were detected by western blot. Results: The expression level of ACC1 in sh-NC group was higher than that in sh-ACC1 group (P<0.01). The number of cell migration in sh-NC group was (159.00±24.38), lower than (361.80±26.81) in sh-ACC1 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC group were statistically significant compared with sh-AAC1 group (P<0.05). The migrated cell number in sh-NC+ si-NC group was (189.20±16.02), lower than (371.60±38.40) in sh-ACC1+ si-NC group (P<0.01). The migrated cell number in sh-NC+ si-NC group was higher than that in sh-NC+ si-HSP27 group (152.40±24.30, P<0.01), and the migrated cell number in sh-ACC1+ si-NC group was higher than that in sh-ACC1+ si-HSP27 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC+ si-NC group were significantly different from those in sh-ACC1+ si-NC and sh-NC+ si-HSP27 groups (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-ACC1+ si-NC group were significantly different from those in sh-ACC1+ si-HSP27 group (P<0.01). After 24 h treatment with C646 at 20 μmmo/L, the migrated cell number in sh-NC+ DMSO group was (190.80±11.95), lower than (395.80±17.10) in sh-ACC1+ DMSO group (P<0.01). The migrated cell number in sh-NC+ DMSO group was lower than that in sh-NC+ C646 group (256.20±23.32, P<0.01). The migrated cell number in sh-ACC1+ DMSO group was higher than that in sh-ACC1+ C646 group (87.80±11.23, P<0.01). The protein expressions of H3K9ac, HSP27, E-cadherin and Vimentin in sh-NC+ DMSO group were significantly different from those in sh-ACC1+ DMSO group and sh-NC+ C646 group (P<0.01). The protein expression levels of H3K9ac, HSP27, E-cadherin and Vimentin in sh-ACC1+ DMSO group were significantly different from those in sh-ACC1+ C646 group (P<0.01). Conclusion: Knockdown of ACC1 promotes the migration of KYSE-450 cell by up-regulating HSP27 and increasing histone acetylation.

Abstract P5-11-03: Knockdown of TMEM45A regulates malignant progression of triple-negative breast cancer by inhibiting TGF-β/Smad signaling pathway-mediated EMT

Abstract Objectives: The incidence of breast cancer has jumped to first place in the global malignant tumor. As an essential component of the TMEM family, TMEM45A is abnormally highly expressed in some malignant tumors, and plays a role in promoting and regulating the occurrence and development of tumors. This study analyzed the expression of TMEM45A in breast cancer tissues and breast cancer cell lines, and explored the relationship with clinical subtypes. To examine the effects of TMEM45A on TNBC cell proliferation, migration, invasion and other biological functions, and to preliminarily clarify the molecular mechanism of TMEM45A regulating the epithelial-mesenchymal transition of TNBC cell lines. Methods: 1. Download and analyze the TCGA database, analyze the difference in the expression of TMEM45A in normal breast tissue and different subtypes of breast cancer tissue, and detect the protein expression level of TMEM45A in 8 pairs of cancer tissues and adjacent tissues by western blot experiment. 2. Detection of TMEM45A protein expression in normal breast epithelial cell line MCF10A and five breast cancer cell lines (MCF-7, MDA-MB-231, BT-549, MDA-MB-468, SK-BR-3) by western blot experiment. 3. Using small interfering RNA technology to down-regulate the protein expression levels of MDA-MB-231 and BT-549 TNBC cell lines with relatively high TMEM45A expression. The changes in cell proliferation ability were detected by CCK8 assay and clone formation assay, and the changes in cell migration ability and invasion ability were detected by wound healing assay and transwell assay, respectively. 4. After down-regulating TMEM45A in two TNBC cell lines, the expression changes of epithelial cell phenotype marker E-cadherin, mesenchymal phenotype marker N-cadherin, Vimentin, and critical molecules in TGF-β/Smad signal transduction pathway (TGFβ1, Smad2, Smad3, p-Smad2, p-Smad3) were detected by western blot experiment. Results: 1. Based on the TCGA database, the results showed that the expression level of TMEM45A mRNA in breast cancer was significantly higher than in normal breast tissue. The expression level of TMEM45A mRNA in any breast cancer subtype was higher than that in normal breast tissue, and it was the highest in the TNBC subtype. In the results of western blot experiments of clinical breast cancer specimens, the expression of TMEM45A in breast cancer tissues was significantly higher than that in the corresponding adjacent tissues, which further confirmed the reliability of the results of the TCGA database. 2. In normal breast epithelial cell line MCF10A and five breast cancer cell lines, MCF10A has the lowest expression level of TMEM45A protein. Among the five breast cancer cell lines, MDA-MB-231 had the highest expression of TMEM45A, followed by BT-549. 3. CCK8 assay, clone formation assay, wound healing assay and transwell assay showed that down-regulating TMEM45A protein levels in the two TNBC cell lines could significantly reduce cell proliferation, migration and invasion abilities. 4. After down-regulating TMEM45A protein levels in two TNBC cell lines, EMT-related indicators E-cadherin protein expression levels increased, N-cadherin and Vimentin protein expressions decreased, and the expression levels of essential protein molecules Smad2 and Smad3 in the TGF-β/Smad signal transduction pathway did not change significantly, while p-Smad2, p-Smad3 and TGF-β1 protein expressions decreased. Conclusions: TMEM45A is relatively highly expressed in breast cancer tissues and cell lines, especially in the subtype of TNBC. Down-regulation of TMEM45A expression can inhibit the proliferation, migration and invasion of TNBC cell lines. Mechanistically, TMEM45A may reverse EMT by inhibiting the activity of the TGF-β/Smad pathway. Citation Format: Yingkun Xu, Shengchun Liu. Knockdown of TMEM45A regulates malignant progression of triple-negative breast cancer by inhibiting TGF-β/Smad signaling pathway-mediated EMT [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-11-03.

Influence of E-cadherin methylation on prognosis in children with acute lymphoblastic leukemia

To study the significance of E-cadherin and the association between E-cadherin methylation status and prognosis in children with acute lymphoblastic leukemia (ALL) by examining the mRNA and protein expression of E-cadherin and its gene methylation status in bone marrow mononuclear cells of children with ALL. The samples of 5 mL bone marrow blood were collected from 42 children with ALL who were diagnosed for the first time at diagnosis (pre-treatment group) and on day 33 of induction chemotherapy (post-treatment group). RT-qPCR, Western blot, and methylation-specific PCR were used to measure the mRNA and protein expression of E-cadherin and the methylation level of the E-cadherin gene. The changes in each index after induction chemotherapy were compared. The mRNA and protein expression levels of E-cadherin in the post-treatment group were significantly higher than those in the pre-treatment group (P<0.05), while the positive rate of E-cadherin gene methylation in the post-treatment group was significantly lower than that in the pre-treatment group (P<0.05). At the end of the test, the children with negative methylation had significantly higher overall survival rate and event-free survival rate than those with positive methylation (P<0.05). E-cadherin expression is associated with the development of ALL in children, and its decreased expression and increased methylation level may indicate a poor prognosis.

Silencing MARCH2 Inhibits the Invasion, Migration, and EMT Transformation of Human Colorectal Cancer SW480 Cells.

Aim: This study aimed to investigate the role of MARCH2 (membrane-associated RING-CH2) in the progression, invasion, and migration of colorectal cancer (CRC). Methods: In this study, the expression levels of MARCH2 and E-cadherin in CRC tissues were detected by immunohistochemistry through retrospective study, and their correlation was analyzed. After silencing the MARCH2 gene using SiRNA MARCH2-1/-2, the invasion and migration abilities of SW480 cells were detected using Transwell and Scratch assay, respectively. Quantitative real-time PCR (qRT-PCR) and Western blotting assays were performed to detect the expression levels of epithelial-mesenchymal transition (EMT) related markers. Results: As compared to adjacent tissues, the MARCH2 expression level was significantly overexpressed in the CRC tissues, and correlated with tumor size, pathological grade, lymph node metastasis, and survival time. MARCH2 was negatively correlated with E-cadherin. MARCH2 silencing significantly restrained the invasion and migration abilities of SW480 cells in vitro. Meanwhile, the MARCH2 silencing also upregulated the mRNA and protein expression levels of E-cadherin and downregulated those of Vimentin. Conclusions: The high expression of MARCH2 was unfavorable for patients' survival. Thus, MARCH2 might be an independent predictor for CRC patients, affecting the invasion and metastasis of CRC through EMT.

Effect of epithelial-mesenchymal transition on cardiac fibrosis induced by oil mist particulate matter

Objective: To investigate the effects of oil-mist particulate matter (OMPM) on cardiac tissue structure fibrosis in rats and the role of epithelial-mesenchymal transition (EMT). Methods: Six-week-old Wistar rats (half male and half female) were randomly divided into 3 groups: control group (without OMPM exposure), low-dose exposure group (50 mg/m3) and high-dose exposure group (100 mg/m3), 18 rats in each group, with 6.5 hours per day of dynamic inhalation exposure. After 42 days of continuous exposure, cardiac tissues were collected for morphological observation; Western blot was used to detect fibrosis markers collagen I and collagen III levels, epithelial marker E-cadherin levels, interstitial markers N-cadherin, fibronectin, vimentin, alpha-smooth muscle actin (α-SMA) levels, and EMT transcription factor Twist protein levels; Real-time polymerase chain reaction (RT-qPCR) was used to detect collagen I and collagen III mRNA levels. Results: After OMPM exposure, myocardial cell edema and collagen fiber deposition were increased gradually with increasing exposure dose. Western blot results showed that compared with the control group, the expression levels of collagen I, collagen III, N-Cadherin, fibronectin, vimentin, α-SMA, and Twist protein were increased significantly in the low-dose exposure group and the high-dose exposure group (P＜0.01), and protein expression levels were higher in the high-dose exposure group than those in the low-dose exposure group (P＜0.01). In contrast, E-Cadherin protein expression levels were decreased significantly, and lower in the high-dose exposure group (P＜0.01). RT-qPCR results showed that compared with the control group, collagen I and collagen III mRNA levels were increased significantly in the low-dose exposure group and the high-dose exposure group (P＜0.01), and were increased with increasing exposure dose. (P＜0.01). Conclusion: OMPM may induce cardiac fibrosis in rats by promoting EMT process.

Biological function and regulation mechanism of MTA2 expression in bladder cancer

Bladder cancer that occurs on the bladder mucosa is the most common malignant tumor in the urinary system. Exposure to aromatic amine chemicals and cigarette smoking are the two risk factors which causes bladder cancer. The mechanism of complex molecular signaling during its progress is still not understood clearly. High expression of metastasis associated gene 2 (MTA2) in malignant tumors such as ovarian cancer and hepatocellular carcinoma indicates its possible relationship with migration and invasion. However, expression level and biological function of MTA2 in bladder cancer tissues have not been studied. Here, we investigated the biological function, regulation mechanism and clinical application of metastasisrelated genes 2 (MTA2). The expression level of MTA2 in various bladder tumor cells was determined by Western Blot. T24 and EJ cells were selected for in vitro study. T24 cells and EJ cells were transfected with viral vector and SiMTA2, respectively, to induce MTA2 over- or low- expression. MTS colorimetric assay was used to explore the proliferative ability of bladder cancer cells in vitro under MTA2 over- or low-expression. Matrigel invasion assay and Transwell migration assay were used to detect the invasion and migration of cancer cells in vitro under different MTA2 expression conditions. The results of Western Blot assay on bladder cancer cell lines have shown that the MTA2expression level to be significantly lower in T24 cells compared to that of EJ and J82 cells. MTA2 level was significantly higher in MTA2 overexpressed T24 cells than those in the non-transfected and CD511B transfected bladder cancer cells, while the level of MTA2 was much lower in the MTA2-knockdown EJ cells than that in the non-transfected and si-NC transfected. The MTS colorimetric assay significant increase in proliferation of T24 cells with MTA2 overexpression, while the proliferation of EJ cells with MTA2 knockdown was decreased. Results of Transwell migration assay showed that MTA2 overexpression could significantly increase the migration ability of T24 cells, and MTA2 knockdown could significantly reduce the migration ability of EJ cells. Matrigel invasion assay showed that MTA2 overexpression could enhance the invasive ability of T24 cells significantly, while MTA2 knockdown weaken the invasive ability of EJ cells. The expression level of E-cadherin was lower in the T24 cells with MTA2 overexpression than that in the non-transfected group and CD511B transfected group, but the expression level of N-cadherin protein was significantly higher in the T24 cells with MTA2 overexpression compared to the other two groups. The expression level of E-cadherin protein was significantly higher in the EJ cells with MTA2 knockdown than that of the non-transfected group and si-NC transfected group, while the expression level of N-cadherin protein was significantly lower than that in the non-transfected group and si-NC transfected group.

Effects of transient receptor potential vanilloid type 4-specific activator on human vascular endothelial cell functions and blood supply of rat perforator flap and its mechanism

Effects of transient receptor potential vanilloid type 4-specific activator on human vascular endothelial cell functions and blood supply of rat perforator flap and its mechanism

CRISPR-Cas9-Mediated NPC1 Gene Deletion Enhances HEK 293T Cell Adhesion by Regulating E-Cadherin.

NPC1 gene encodes a transmembrane glycoprotein on the late endosome/lysosomal membrane. Its mutation leads to a rare and aggravated autosomal recessive neurovisceral condition, termed Niemann-Pick disease type C1 (NPC1), which is characterized by progressive neurodegeneration, visceral symptoms, and premature death. To investigate the influence of NPC1 gene deletion on cell morphology, adhesion, proliferation, and apoptosis, CRISPR-Cas9 technology was used to knockout the NPC1 gene in HEK 293T cells. Sanger sequencing, western blotting, and immunofluorescence were used to confirm successful NPC1 ablation. Filipin staining results indicated that deletion of NPC1 gene led to accumulation of unesterified cholesterol in HEK 293T cells. Phalloidin staining results revealed cell aggregation, synapse shortening, nuclear enlargement, and cytoskeleton filamentous actin thinning in HEK 293T cells with NPC1 gene mutation. Furthermore, NPC1 gene mutated HEK 293T cell showed enhanced cell adhesion, inhibited cell proliferation, and increased cell apoptosis. In addition, NPC1 gene mutations significantly increased the protein expression levels of E-cadherin and γ-catenin and significantly decreased the protein expression levels of Wnt 3a, c-Myc, and cyclin D1. These results suggest that NPC1 may regulate cell adhesion by affecting the cadherin-catenin complex through E-cadherin, and that the classical Wnt signaling pathway may be inhibited by restricting β-catenin from entering the nucleus to inhibit cell proliferation.

Simvastatin alleviates epithelial-mesenchymal transition and oxidative stress of high glucose-induced lens epithelial cells in vitro by inhibiting RhoA/ROCK signaling.

Diabetic cataracts (DC) is one of the main causes of blindness among patients with diabetes mellitus. The aim of the present study was to examine the effect of simvastatin on lens epithelial cells in DC and the underlying mechanism. The viability of SRA01/04 cells treated with different concentrations of simvastatin was detected using a Cell Counting Kit-8 assay before and after high glucose (HG) treatment. The expression levels of E-cadherin, N-cadherin, Vimentin and α-smooth muscle actin (α-SMA), proteins associated with epithelial-mesenchymal transition (EMT), in addition to RhoA, Rho-associated kinases (ROCK)1 and ROCK2, proteins related to RhoA/ROCK signaling, were also measured in SRA01/04 cells treated with HG and simvastatin, with or without U46619, using western blot analysis. DCFH-DA dyes, superoxide dismutase (SOD) and glutathione (GSH)/glutathione disulfide (GSSG) kits were used to measure the levels of oxidative stress parameters in SRA01/04 cells treated with HG and simvastatin with or without U46619. The cell viability of SRA01/04 cells treated with simvastatin was found to be significantly elevated after HG treatment. The protein expression levels of E-cadherin were increased but those of N-cadherin, Vimentin and α-SMA decreased after HG and simvastatin treatment, and this was reversed by U46619. The levels of SOD and GSH-GSSG were found to be increased whereas reactive oxygen species levels were decreased, effects that were reversed by U46619. Additionally, the protein expression levels of RhoA, ROCK1 and ROCK2 were markedly decreased. These findings provided evidence that simvastatin increased HG-induced SRA01/04 cell viability and exerted inhibitory effects on EMT and oxidative stress that occurs during DC.