SkullcapflavoneⅡsuppresses TGF-β-induced corneal epithelial mesenchymal transition in vitro

AIM:To investigate the effect of skullcapflavone II(SCF-II)on the epithelial-mesenchymal transition(EMT)induced by transforming growth factor beta(TGF-β)in human corneal epithelial cells(HCECs),as well as to identify the signaling pathways that may be involved.METHODS:HCECs were cultured in vitro.At a SCFII(5,10μmol/L)dose,cell viability was analysed with a cell counting kit-8(CCK-8)assay,and cell migration was monitored with wound healing and Transwell migration assays.There were 4 groups:SCF-II,TGF-β,SCF-II+TGF-βand Control.Western blotting and immunofluorescence were performed to show the expression of EMT markers and the translocation of nuclear factor kappa-B(NF-κB)into the nucleus in the 4 groups.RESULTS:Treatment with SCF-II decreased HCEC viability in a dose-dependent manner.A concentration below 10μmol/L did not present obvious cell toxicity,and survival rates were more than 70%at 48h.Treatment with SCF-II(5 and 10μmol/L)significantly impeded migration in wound healing and Transwell migration assays(P<0.05),and EMT markers and NF-κB translocation into the nucleus were inhibited.After both TGF-βand SCF-II treatment,the migration of TGF-β-treated HCECs were suppressed by SCF-II(P<0.05).The expression levels of the mesenchymal markers N-cadherin(P<0.05),α-smooth muscle actin(α-SMA;P<0.05)and NF-κB(P<0.05)in both TGF-β-and SCF-II-treated HCECs were lower than those in the HCECs treated with TGF-βalone and higher than those in HCECs treated with SCF-II alone.Immunofluorescence showed that the entry of NF-κB into the nucleus in both TGF-β-and SCF-IItreated HCECs was less than that in the TGF-β-treated HCECs.CONCLUSION:SCF-II inhibit TGF-β-induced EMT in HCECs by potentially regulating the NF-κB signalling pathway.Thus,SCF-II represents a candidate putative therapeutic agent in corneal fibrotic diseases.

Malignant Transformation and Abnormal Expression of Eukaryotic Initiation Factor in Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To analyze the relationship between malignant transformation and abnormal expression of eukaryotic initiation factor 3 （eIF3 p36） in human bronchial epithelial （16HBE） cells induced by cadmium chloride （CdCl2）. Methods 16HBE cells were treated several times with different concentrations of CdCl2. Tumorigenic potential of transformed cells was identified by assays for anchorage-independent growth in soft agar and for tumorigenicity in nude mice after the 35th passage. Total RNA was isolated from 16HBE cells induced by CdC12, including non-transformed, Cd-transformed, and Cd-tumorigenic cell lines. Special primers for eIF3 p36 were designed and the expression of eIF3 mRNA in different cell lines was detected with fluorescent quantitative-polymerase chain reaction technique （FQ-PCR）. Results The 35th passage of 16HBE cells transformed by CdCl2 exhibited overlapping growth. Compared with the non-transformed cells, colonies of transformed cell lines in soft agar showed statistically significant increases and dose-dependent effects （P〈0.01）. All Cd-induced transformed cell lines formed rumors in nude mice within 2 weeks of inoculation, but none of the mice injected with non-transformed cells showed tumors even after 3 weeks. All tumors were pathologically identified as poorly differentiated squamous cell carcinoma. The eIF3 p36 genes in different stages of 16HBE cells transformed by CdCl2 were elevated as compared with the non-transformed control （P〈0.01）, and the eIF3 expression increased with the degree of cell malignancy. Conclusion CdCl2 is capable of inducing morphological transformation in 16HBE cells and transformed cells are potentially tumorigenic. Over-expression of eIF3 p36 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2 and may be one of the molecular mechanisms potentially responsible for carcinogenesis due to Cd.

Alterative Expression and Sequence of Human Elongation Factor-1δ during Malignant Transformation of Human Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To study the alternative expression and sequence of human elongation factor-1δ （human EF-1δ p31） during malignant transformation of human bronchial epithelial cells induced by cadmium chloride （CdCl2） and its possible mechanism. Methods Total RNA was isolated at different stages of transformed human bronchial epithelial cells （16HBE） induced by CdCl2 at a concentration of 5.0 μM. Special primers and probe for human EF-1δ p31 were designed and expression of human EF-18 mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-18 cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis. Results The expressions of human EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2 was elevated （P〈0.01 or P〈0.05）. Compared with their corresponding non-transformed ceils, the overexpression level of EF-15 p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed ceils and 7.2 folds in Cd-tumorigenic cells. No change was found in the sequence of overexpressed EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2. Conclusion Overexpression of human EF-1δ p31 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2, but is not correlated with DNA mutations.

Alcohol promotes epithelial mesenchymal transformation-mediated premetastatic niche formation of colorectal cancer by activating interaction between laminin-γ2 and integrin-β1

BACKGROUND Colorectal cancer(CRC) is a common malignant tumor. Alcohol consumption is positively correlated with CRC malignant metastasis;however, the mechanism is unclear. The interaction between laminin-γ2(LAMC2) and integrin-β1(ITGB1) plays a role in premetastatic niche signaling, which may induce epithelial mesenchymal transformation(EMT) and lead to metastasis.AIM To investigate the effects of alcohol on CRC metastasis from the molecular mechanism of the premetastatic niche.METHODS The interaction between LAMC2 and ITGB1 was measured by Duolink assay, and the expression levels of LAMC2, ITGB1 and focal adhesion kinase(FAK), snail, fibronectin, N-cadherin and special AT-rich sequence binding protein 1(SATB1) were measured by quantitative real-time polymerase chain reaction, immunohistochemistry and western blotting. Interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α) and IL-6 levels were measured via enzyme-linked immunosorbent assay, histopathological assessment via hematoxylin eosin staining, and determination of aberrant crypt foci via methylene blue.RESULTS The lymph node metastasis rate was higher in the alcohol group than non-alcohol group. There was a significant increase in interaction signals between LAMC2 and ITGB1, and an increase in phosphorylate-FAK/FAK, snail, fibronectin, N-cadherin and SATB1, whereas E-cadherin was reduced in the alcohol group compared to the non-alcohol group in both animal and clinical samples. Serum IL-1β, TNF-α and IL-6 were higher in alcohol group than in non-alcohol group. Alcohol may promote CRC metastasis by influencing the molecular mechanism of the premetastatic niche.CONCLUSION Our study suggests that alcohol promotes EMT-mediated premetastatic niche formation of CRC by activating the early interaction between LAMC2 and ITGB1 and lead to CRC metastasis.

NEOPLASTIC TRANSFORMATION OF HUMAN EMBRYONIC NASOPHARYNGEAL EPITHELIAL CELLS INDUCED BY N,N'-DINITROSOPIPERAZINE (DNP) IN VITRO

This experiment is the first report on N, N'-dini-trosopiperazine (DNF)-induced neoplastic transformation of human embryonic nasopharyngeal (HENPE) cells. The transformed cells showed a prolonged life span, anchorage independent growth, chromosome aberration, tumorigenicity and an altered cell morphological appearance. The results demonstrated that DNP was able to induce not only nasopharyngeal carcinoma (NPC) of rats in vivo, but also neoplastic transformation of HENPE cells in vitro.

Alterations of FHIT Gene and P16 Gene in Nickel Transformed Human Bronchial Epithelial Cells

Objective To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immortal human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis. Methods 16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting. Results NiS-treated cells exhibited overlapping growth. Compared wkh that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases （P〈0.01） and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in pl6 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8. Conclusions The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.

Effects of transforming growth factor β2 and connective tissue growth factor on induction of epithelial mesenchymal transition and extracellular matrix synthesis in human lens epithelial cells

AIM:To Investigate the effects of transforming growth factorβ2(TGF-β2)and connective tissue growth factor(CTGF)on transdifferentiation of human lens epithelial cells(HLECs)cultured in vitro and synthesis of extracellular matrix(ECM).METHODS:HLECs were treated with TGF-β2(0,0.5,1.0,5,10μg/L)and CTGF(0,15,30,60,100μg/L)for different times(0,24,48,72h)in vitro and the expression ofα-smooth muscle actin(α-SMA),the main component of the extracellular matrix typeⅠcollagen(Col-1)and fibronectin(Fn)were measured by using real-time polymerase chain reaction(PCR)and western-blot.RESULTS:TGF-β2 and CTGF significantly increased expression ofα-SMA mRNA and protein(P【0.05,P【0.001),Fn mRNA and protein(P【0.001),Col-1 mRNA and protein(P【0.001).TGF-β2 could induce HLECs expression of CTGF mRNA and protein in dosedependent manner(P【0.05,P【0.001).TGF-β2 and CTGF could induce HLECs to expressα-SMA,Fn and Col-1 in time-dependent manner.Each time of TGF-β2and CTGF induced HELCs expression ofα-SMA,Fn,Col-1 mRNA and protein was significant increase compared with control(P【0.05,P【0.001).CONCLUSION:TGF-β2 and CTGF could induce HLECs epithelial mesenchymal transition and ECM synthesis.

Effect of NF-κB p65 antisense oligodeoxynucleotide on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2

AIM：To study the inhibition of nuclear factor kappa-B p65（NF-κB p65）antisense oligodeoxynucleotide（ASODN）on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2（TGF-β2）.·M ETHODS：NF-κBp65ASODNand NF-κBp65missense oligodeoxynucleotide（MSODN）were designed and synthesized.Human lens epithelial cell line（HLE B-3）cells were prepared for study and divided into 7 groups.Control group was HLE B-3 cells cultured in dulbecco’s modified eagle medium（DMEM）.T1,T2,and T3 group were HLE B-3 cells cultured in DMEM with 10 ng/m L TGF-β2 for 6h,12h,24h respectively.A＋T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2for 24h after transfected by NF-κB p65 ASODN for 24h.M＋T group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h.The negative control group was HLE B-3 cells cultured with 10 ng/m L TGF-β2 for 24h after cultured with transfer agent（Hi Per Fect）for 24h.Cell morphology was observed at different time points using an inverted microscope.The expression of NF-κB p65 m RNA was detected with reverse transcription-polymerase chain reaction（RT-PCR）,and the expression ofα-smooth muscle actin（α-SMA）protein was assayed with ELISA.·RESULTS：With the TGF-β2 stimulation prolongation,the expression of NF-κB p65 m RNA and a-SMA protein increased in T1,T2,T3 groups compared with the control group,and the difference was statistically significant（〈0.05）.NF-κB p65 ASODN lowered the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.NF-κB p65 MSODN and Hi Per Fect did not lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2.The difference between control group and A＋T group was not statistically significant（〉0.05）,but the difference among A＋T group and other groups was statistically significant（〈0.05）.·CONCLUSION：NF-κB p65 ASODN could lower the expression of NF-κB p65 m RNA andα-SMA protein induced by TGF-β2,and antagonized TGF-β2-induced transdifferentiation of HLE B-3.NF-κB p65ASODN could be used as a new biological therapeutic target of posterior capsular opacification.

Bone morphogenetic protein-6 suppresses TGF-β_(2)-induced epithelial-mesenchymal transition in retinal pigment epithelium

AIM:To evaluate the effect of bone morphogenetic protein-6(BMP-6)on transforming growth factor(TGF)-β_(2)-induced epithelial-mesenchymal transition(EMT)in retinal pigment epithelium(RPE).METHODS:Adult retinal pigment epithelial cell line(ARPE-19)were randomly divided into control,TGF-β_(2)(5μg/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-β_(2)+empty plasmid group,BMP-6 overexpression group,and TGF-β_(2)+BMP-6 overexpression group.The EMT-related indexes and extracellular regulated protein kinases(ERK)protein levels were detected.RESULTS:Compared with the control group,the migration of RPE cells in the TGF-β_(2) group was significantly enhanced.TGF-β_(2) increased the protein expression levels ofα-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 ofα-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-β_(2) and prevented TGF-β_(2) from affecting EMT-related biomarkers(P<0.05).CONCLUSION:BMP-6 prevents the EMT in RPE cells induced by TGF-β_(2),which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

EDIL3 depletion suppress epithelial-mesenchymal transition of lens epithelial cells via transforming growth factor β pathway

AIM： To study the effect of discoidin I-like domaincontaining protein 3（EDIL3） depletion on the proliferation and epithelial-mesenchymal transition（EMT） in human lens epithelial cells（LECs）. METHODS： RNA interference was used to inhibit the expression of EDIL3 in human LECs in vitro. The morphology of cells was observed using an inverted microscope. Cell proliferation was assessed using Ed U kit. Cell migration was investigated using Transwell chamber and EMT of LECs was assessed using confocal microscope and Western blotting. The transforming growth factor β（TGFβ） pathway was investigated using Western blotting. RESULTS： The data showed that silencing EDIL3 expression changed LECs morphology and suppressed LECs proliferation（P〈0.05） and migration（P〈0.01）. Furthermore, the result of Western blotting showed that EDIL3 depletion reduced the expression of α-smooth muscle actin（α-SMA）（P〈0.001） and vimentin（P〈0.01）, while increased the expression of E-cadherin（P〈0.001）. EDIL3 depletion could suppress the phosphorylation of Smad2（P〈0.01） and Smad3（P〈0.01） and the activation of exracellular signal regulated kinase（ERK）（P〈0.05）. CONCLUSION： The findings indicate that EDIL3 might participate in the proliferation and EMT in LECs via TGFβ pathway and may be a potential therapeutic target for the treatment of posterior capsule opacification.

Cross-talk between microRNA-let7c and transforming growth factor-β2 during epithelial-to-mesenchymal transition of retinal pigment epithelial cells

AIM: To explore the roles of microRNA-let7 c(miR-let7 c) and transforming growth factor-β2(TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition(EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial(ARPE-19) cells were cultured with no serum for 12 h, and then with recombinant human TGF-β2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7 c mimcs(miR-let7 cM), miR-let7 c mimcs negative control(miR-let7cMNC) and miR-let7 c inhibitor(miR-let7 cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B(NF-κB) signaling pathway was activated after induction by TGF-β2(P<0.05). In turn, overexpressed miR-let7 c significantly inhibited TGF-β2-induced EMT(P<0.05). However, miR-let7 c was unable to inhibit TGF-β2-induced EMT when the NF-κB signaling pathway was inhibited by BAY11-7082(P<0.01). CONCLUSION: The miR-let7 c regulates TGF-β2-induced EMT through the NF-κB signaling pathway in ARPE-19 cells.

Total flavone of Abelmoschus manihot suppresses epithelial-mesenchymal transition via interfering transforming growth factor-β1 signaling in Crohn's disease intestinal fibrosis

AIM To explore the role and mechanism of total flavone of Abelmoschus manihot(TFA) on epithelial-mesenchymal transition(EMT) progress of Crohn's disease(CD) intestinal fibrosis.METHODS First,CCK-8 assay was performed to assess TFA on the viability of intestinal epithelial(IEC-6) cells and select the optimal concentrations of TFA for our further studies.Then cell morphology,wound healing and transwell assays were performed to examine the effect of TFA on morphology,migration and invasion of IEC-6 cells treated with TGF-β1.In addition,immunofluorescence,real-time PCR analysis(q RT-PCR) and western blotting assays were carried out to detect the impact of TFA on EMT progress.Moreover,western blotting assay was performed to evaluate the function of TFA on the Smad and MAPK signaling pathways.Further,the role of co-treatment of TFA and si-Smad or MAPK inhibitors has been examined by q RTPCR,western blotting,morphology,wound healing andtranswell assays.RESULTS In this study,TFA promoted transforming growth factor-β1(TGF-β1)-induced(IEC-6) morphological change,migration and invasion,and increased the expression of epithelial markers and reduced the levels of mesenchymal markers,along with the inactivation of Smad and MAPK signaling pathways.Moreover,we revealed that si-Smad and MAPK inhibitors effectively attenuated TGF-β1-induced EMT in IEC-6 cells.Importantly,co-treatment of TFA and si-Smad or MAPK inhibitors had better inhibitory effects on TGF-β1-induced EMT in IEC-6 cells than either one of them.CONCLUSION These findings could provide new insight into the molecular mechanisms of TFA on TGF-β1-induced EMT in IEC-6 cells and TFA is expected to advance as a new therapy to treat CD intestinal fibrosis.

Hepatic regeneration and the epithelial to mesenchymal transition

Liver injuries are repaired by fibrosis and regeneration. The core stage is the repair response and fibrosis formation as a scar. The cause of overly-responsive scar formation and diminished regeneration, especially in liver fibrosis and cirrhosis, is still unknown. The epithelial to mesenchymal transition (EMT), a previously discovered mechanism, plays an important role in liver fibrosis and tumor metastasis. Recently, EMT has been found to be associated with liver and bile duct cell fibrosis. Analyzing the established models and chronic disease processes, we propose that EMT liver cells may also lose their regenerative capability due to phenotype changes and that the remaining liver cells may quickly lose their regenerative capability in liver fibrosis or cirrhosis. Recognizing these phenotype changes or transition cells may play an important role in targeting therapy to reverse fibrosis not only by disrupting the transition that is necessary to produce the extracellular matrix but also by restoring the regenerative capacity of EMT-like cells.

Telomere and telomerase in the initial stage of immortalization of esophageal epithelial cell

AIM: To search for the biomarker of cellular immortalization, the telomere length, telomerase activity and its subunits in cultured epithelial cells of human fetal esophagus in the process of immortalization. METHODS: The transgenic cell line of human fetal esophageal epithelium (SHEE) was established with E(6)E(7) genes of human papillomavirus (HPV) type 18 in our laboratory. Morphological phenotype of cultured SHEE cells from the 6th to 30th passages, was examined by phase contrast microscopy, the telomere length was assayed by Southern blot method, and the activity of telomerase was analyzed by telomeric repeat amplification protocol (TRAP). Expressions of subunits of telomerase, hTR and hTERT, were assessed by RT-PCR. DNA content in cell cycle was detected by flow cytometry. The cell apoptosis was examined by electron microscopy (EM) and TUNEL label. RESULTS: SHEE cells from the 6th to 10th passages showed cellular proliferation with a good differentiation. From the 12th to the 16th passages, many senescent and apoptotic cells appeared, and the telomere length sharply shortened from 23kb to 17kb without expression of hTERT and telomerase activity. At the 20th passage, SHEE cells overcame the senescence and apoptosis and restored their proliferative activity with expression of telomerase and hTERT at low levels, but the telomere length shortened continuously to the lowest of 3kb. After the 30th passage cells proliferation was restored by increment of cells at S and G2M phase in the cell cycle and telomerase activity expressed at high levels and with maintenance of telomere length. CONCLUSION: At the early stage of SHEE cells, telomeres are shortened without expression of telomerase and hTERT causing cellular senescence and cell death. From the 20th to the 30th passages, the activation of telomerase and maintenance of telomere length show a progressive process for immortalization of esophageal epithelial cells. The expression of telomerase may constitute a biomarker for detection of immortalization of cells.

Expressions of TGF-β2, bFGF and ICAM-1 in lens epithelial cells of complicated cataract with silicone oil tamponade

AIM： To investigate the expression differences of transforming growth factor-β2（TGF-β2）, basic fibroblast growth factor（b FGF） and intercellular cell-adhesion molecule-1（ICAM-1） in lens epithelial cells（LECs） of complicated cataract with silicone oil tamponade and agerelated cataract. METHODS： Totally 150 eyes of 150 patients（aged 35 to 77y） were investigated, including 75 patients with complicated cataract after silicone oil tamponade and 75 patients with age-related cataract. The central piece of anterior capsules was collected during cataract surgery. TGF-β2, b FGF and ICAM-1 were detected in the 60 specimens of the two groups by immunohistochemistry. The expression levels of the three kinds of messenger ribonucleic acid（m RNA） were determined by real-time quantitative reverse transcriptionpolymerase chain reaction in the 90 specimens of the two groups.RESULTS： TGF-β2 was detected in the cytomembrane and cytoplasm of the LECs and b FGF was detected in the nucleus. ICAM-1 was positive in the cytomembrane of the LECs and the distribution of positive cells was uneven. The m RNA genes expression of the TGF-β2, b FGF and ICAM-1 was significant differences between the two groups and markedly increased in complicated cataract group（P〈0.05）.CONCLUSION： The up-regulated TGF-β2, b FGF and ICAM-1 maybe associate with the occurrence and development of complicated cataract with silicone oil tamponade.

Effects of exogenous recombinant human bone morphogenic protein-7 on the corneal epithelial mesenchymal transition and fibrosis

AIM：To evaluate the effect of exogenous recombinant human bone morphogenic protein-7（rhBMP-7）on transforming growth factor-β（TGF-β）-induced epithelial mesenchymal cell transition（EMT）and assessed its antifibrotic effect via topical application.METHODS：The cytotoxic effect of rhBMP-7 was evaluated and the EMT of human corneal epithelial cells（HECEs）was induced by TGF-β. HECEs were then cultured in the presence of rhBMP-7 and/or hyaluronic acid（HA）. EMT markers,fibronectin,E-cadherin,α-smooth muscle actin（α-SMA）,and matrix metaloproteinase-9（MMP-9）,were evaluated. The level of corneal fibrosis and the reepithelization rate were evaluated using a rabbit keratectomy model. Expression of α-SMA in keratocytes were quantified following treatment with different concentrations of rhBMP-7.RESULTS：Treatment with rhBMP-7 attenuated TGF-β-induced EMT in HECEs. It significantly attenuated fibronectin secretion（31.6%; P〈0.05）,the α-SMA protein level（72.2%; P〈0.01）,and MMP-9 expression（23.6%,P〈0.05）in HECEs compared with cells grown in the presence of TGF-β alone. E-cadherin expression was significantly enhanced（289.7%; P〈0.01）in the presence of rhBMP-7. Topical application of rhBMP-7 combined with 0.1% HA significantly reduced the amount of α-SMA~＋ cells by 43.18%（P〈0.05）at a concentration of 2.5 μg/mL and by 47.73%（P〈0.05）at 25 μg/mL,compared with the control group,without disturbing corneal reepithelization.CONCLUSION：rhBMP-7 attenuates TGF-β-induced EMT in vitro,and topical application of rhBMP-7 reduces keratocyte myodifferentiation during the early wound healing stages in vivo without hindering reepithelization. Topical rhBMP-7 application as biological eye drops seems to be feasible in diseases involving TGF-β-related corneal fibrosis with corneal reepithelization disorders.

Mechanisms of fibrogenesis in liver cirrhosis:The molecular aspects of epithelial-mesenchymal transition

Liver injuries are repaired by fibrosis and regeneration.The cause of fibrosis and diminished regeneration,especially in liver cirrhosis,is still unknown.Epithelialmesenchymal transition(EMT) has been found to be associated with liver fibrosis.The possibility that EMT could contribute to hepatic fibrogenesis reinforced the concept that activated hepatic stellate cells are not the only key players in the hepatic fibrogenic process and that other cell types,either hepatic or bone marrow-derived cells could contribute to this process.Following an initial enthusiasm for the discovery of this novel pathway in fibrogenesis,more recent research has started to cast serious doubts upon the real relevance of this phenomenon in human fibrogenetic disorders.The debate on the authenticity of EMT or on its contribution to the fibrogenic process has become very animated.The overall result is a general confusion on the meaning and on the definition of several key aspects.The aim of this article is to describe how EMT participates to hepatic fibrosis and discuss the evidence of supporting this possibility in order to reach reasonable and useful conclusions.

MiR-520f-3p inhibits epithelial-mesenchymal transition of colorectal cancer cells by targeting Yes-associated protein 1

Background:Colorectal cancer(CRC)is one of the most common malignancies.Early diagnosis is the key to effective treatment of CRC.Since microRNAs(miRNAs)can be used as biomarkers of CRC,the objective of this work was to examine the effect of miR-520f-3p,which targets YAP1(Yes-associated protein 1),on the ability of CRC cells to proliferate,invade,migrate,and undergo epithelial-mesenchymal transition(EMT).Methods:A miR-520f-3p mimic was used to overexpress miR-520f-3p in HT29 cells.To establish the tumor-bearing mouse model,transfected HT29 cells were subcutaneously implanted into BALB/c-nu nude mice,and YAP1 and miR-520f-3p levels were determined using qRT‒PCR.The viability,invasion ability,and migration ability of cells were evaluated by CCK-8,Transwell,and wound healing assays.Apoptosis was detected by flow cytometry and TUNEL assays.The regulatory link between miR-520f-3p and the YAP1 gene was examined by dual-luciferase reporter assay.Tumor tissues with positive Ki-67 expression were identified by immunohistochemistry.Vimentin,E-cadherin,and YAP1 expression were evaluated by western blotting.Results:MiR-520f-3p overexpression could inhibit proliferation,invasion,migration,and EMT and induce apoptosis in HT29 cells.YAP1 was found as a target of miR-520f-3p.The inhibitory effects of miR-520f-3p on proliferation,invasion,migration,and EMT may be reversed by overexpressing YAP1.In tumor-bearing mice,miR-520f-3p overexpression reduced the Ki-67 level,increased apoptosis,and prevented tumor development and spread.Conclusion:By targeting YAP1,miR-520f-3p may be capable of suppressing CRC cell proliferation,invasion,migration,and EMT,providing a novel therapeutic target for the disease.

Side Population Cells in Human Gallbladder Cancer Cell Line GBC-SD Regulated by TGF-β-induced Epithelial-mesenchymal Transition

Mounting evidence has shown that side population （SP） cells are enriched for cancer stem cells （CSCs） responsible for cancer malignancy. In this study, SP technology was used to isolate a small subpopulation of SP cells in human gallbladder cancer cell line GBC-SD, and SP cells which had superior potential for proliferation in vitro and tumorigenesis in vivo were identified. Importantly, the abundance of GBC-SD SP cells was increased by a transforming growth factor-β （TGF-β）-induced epithelial-mesenchymal transition （EMT）, and this effect was accompanied with a strong up-regulation of ABCG2 mRNA expression, and a decreased sensitivity to mitoxantrone. SP cells were restored upon the removal of TGF-β and the reversion of the cells to an epithelial phenotype, and smad3-specific siRNA reduced SP abundance in response to TGF-β. In conclusion, TGF-β-induced EMT by smad-dependent signaling pathway promotes cancer development and anti-cancer drug resistant phenotype by augmenting the abundance of GBC-SD SP cells, and a better understanding of mechanisms involved in TGF-β-induced EMT may provide a novel strategy for preventing cancer progression.

Can a fibrotic liver afford epithelial-mesenchymal transition?

The question whether epithelial-mesenchymal transition (EMT) occurs during liver fibrogenesis is a controversial issue. In vitro studies confirm that hepatocytes or cholangiocytes undergo EMT upon transforming growth factor beta (TGF-beta) stimulation, whereas in vivo experiments based on genetic fate mapping of specific cell populations suggest that EMT does not occur in fibrotic animal models. In this review we present current data supporting or opposing EMT in chronic liver disease and discuss conditions for the occurrence of EMT in patients. Based on the available data and our clinical observations we hypothesize that EMT-like alterations in liver cirrhosis are a side effect of high levels of TGF-beta and other pro-fibrotic mediators rather than a biological process converting functional parenchyma, i.e., hepatocytes, into myofibroblasts at a time when essential liver functions are deteriorating.