miR-29c-3p represses the angiogenesis of esophageal squamous cell carcinoma by targeting SERPINH1 to regulate the Wnt signaling pathway

Purpose: Esophageal squamous cell carcinoma (ESCC) is characterized by early metastasis and late diagnosis. miR-29c-3p is confirmed to repress angiogenesis in multiple tumor types. Yet, the functions of miR-29c-3p in the mechanism of ESCC angiogenesis, which were not sufficiently explored previously, were exactly what we investigated here at the molecular level. Methods: The mRNA level of miR-29c-3p and Serpin peptidase inhibitor clade H member 1 (SERPINH1) in ESCC tissues were assessed via bioinformatics analysis. Thereafter, miR-29c-3p and SERPINH1 (HSP47) mRNA level in ESCC cell lines was evaluated via quantitative real-time polymerase chain reaction. The effects of abnormal miR-29c-3p and SERPINH1 expression on ESCC cell viability, proliferation, migration, invasion, and HUVEC angiogenesis were examined via CCK8, colony formation, transwell, and angiogenesis assays, respectively. The protein levels of SERPINH1, vascular endothelial growth factor-A (VEGFA), Wnt-1, ?-catenin, and p-?-catenin were evaluated via Western blot. Expression of VEGFA secreted by ESCC cells was measured via enzyme-linked immunosorbent assay. Treatment with the Wnt activator BML-284 further revealed the way miR-29c-3p mediated the Wnt signaling pathway and its effects on angiogenesis. Results: Herein, we revealed a decrease of miR-29c-3p expression in ESCC tissues and cells, while the overexpressed miR-29c-3p could remarkably suppress ESCC cell progression, as well as HUVEC angiogenesis. Meanwhile, overexpressed miR-29c-3p notably downregulated VEGFA and repressed the Wnt signaling pathway. Treatment with the Wnt activator BML-284 could reverse the inhibition of HUVEC angiogenesis caused by miR-29c-3p. SERPINH1 was a downstream target of miR-29c-3p. SERPINH1 knockdown suppressed the malignant phenotypes of ESCC cells and impeded the Wnt signaling activation, while such suppression was reversed through miR-29c-3p inhibitor. Conclusions: We confirmed the mechanism that miR-29c-3p targeted SERPINH1, thus regulating angiogenesis in ESCC through the Wnt signaling pathway. It improves the understanding of angiogenesis in ESCC and offers new ideas for the research of ESCC treatment strategies in the future.

The expression of heat shock protein 47 and transforming growth factor-β2 in vitreous specimens and epiretinal membranes of patients with proliferative vitreoretinopathy diseases / 中华眼底病杂志

Objective To observe the expression of heat shock protein 47 (HSP47) and transforming growth factor-β2 (TGF-β2) in vitreous specimens and epiretinal membranes of patients with proliferative vitreoretinopathy diseases.Methods Vitreous specimens and epiretinal membranes were obtained from 48 patients (48 eyes) with proliferative vitreoretinopathy (PVR) and 50 patients (50 eyes) with proliferative diabetic retinopathy (PDR).Vitreous specimens and internal limiting membranes were collected from 20 patients (20 eyes) with idiopathic macular hole (IMH) as control group.The expression of HSP47 and TGF-β2 in the vitreous specimens was evaluated using enzyme linked immunosorbent assay.The expression of HSP47,TGF-β2,types Ⅰ and Ⅲ collagen in epiretinal membrane and internal limiting membrane specimens were observed for immunohistochemical staining method.The correlation between the positive expression of HSP47 and TGF-β2,types Ⅰ and Ⅲ collagen in epiretinal membrane specimens of patients with PVR and PDR were analyzed.Results The expression of HSP47 in vitreous specimens of patients with PVR,PDR and IMH were (212.35±23.32),(231.30±26.79),(171.06±28.91) pg/ml,respectively.The expression of TGF β2 in vitreous specimens of patients with PVR,PDR and IMH were (1919.96 ± 318.55),(1939.39 ± 177.57),(1194.61 ± 234.20) pg/ml,respectively.The expression of HSP47,TGF-β2 in the vitreous specimens of patients with PVR and PDR were significantly increased compared with patients with IMH and the difference was statistically significant (F=12.952,34.532;P＜0.01).The epiretinal membrane of patients with PVR and PDR showed markedly increased expression of HSP47,TGF β2,types Ⅰ and Ⅲ collagen in the cytoplasm and extracellular matrix.The expression of HSP47 and type Ⅲ collagen was negative and the expression of TGF-β2 was weakly positive and the expression of types Ⅰ collagen was positive in internal limiting membrane of patients with IMH.The expression of HSP47,TGF β2,types Ⅰ and Ⅲ collagen in the epiretinal membrane of patients with PVR and PDR were significantly increased compared with patients with IMH and the difference was statistically significant (F=13.469,18.752,12.875,20.358;P＜0.01).The expression of HSP47 was positively correlated with thepositive expression of TGF-~,types Ⅰ and Ⅲ collagen in epiretinal membrane specimens of patients withPVR (r=0.475,0.556,0.468;P＜0.05) and PDR (r=0.484,0.589,0.512;P＜0.05).Conclusions This study showed increased consistent expression of HSP47 and TGF-β2 in vitreous and epiretinal membrane specimens of patients with PVR and PDR.Both HSP47 and TGF-β2 were expressed in the cytoplasm and extracellular matrix.HSP47 and TGF-β2 may be involved in the pathological process of PDR and PVR by promoting collagen synthesis.

The invivo interference with the plasmid of heat shock protein 47-short hairpin RNA against Schistosoma j aponicum-induced hepatic fibrosis in mice / 中华传染病杂志

Objective To investigate the anti-fibrogenesis property and mechanism of short hairpin RNA (shRNA ) targeting heat shock protein 47 (HSP47) in schistosomiasis liver fibrosis mice model . Methods Sixty female BALB/c mice (SPF level) were infected percutaneously with (16 ± 1) Schistosoma japonicum cercariae . Twelve mice which were not infected with Schistosoma japonicum were set as uninfected control .The 60 mice were randomly divided into five groups including 1 mg/kg HSP47-shRNA intervention group ,2 mg/kg HSP47-shRNA intervention group ,4 mg/kg HSP47-shRNA intervention group ,unrelated shRNA intervention group and infected control group ,with 12 mice each group .From 6 to 14 weeks post infection ,mice in HSP47-shRNA intervention groups were intravenously injected with HSP47-shRNA weekly and those in unrelated shRNA intervention group were intravenously injected with 2 mg/kg of unrelated shRNA vector weekly .Survival rate ,liver and spleen in general and liver histology of mice in each group were observed at week 14 which was considered as end of intervention . The expressions of HSP47 mRNA and protein were determined by real-time polymerase chain reaction (PCR) and Western blot .Collagen type Ⅰ ,collagen type Ⅲ ,tissue inhibitor of metalloproteinase-1 (TIMP-1) , matrix metalloproteinase-9 (MMP-9) ,plasminogen activator inhibitor-1 (PAI-1) ,transforming growth factor-β1 (TGF-β1 ) , connective tissue growth factor (CTGF ) , interleukin (IL )-13 and IL-17 at transcriptional level were measured by real-time PCR .Means among groups were compared using student-t test .Results In vivo intervention of HSP47-shRNA mainly localized in hepatic stellate cells of mice . The survival rate of 1 mg/kg ,2 mg/kg and 4 mg/kg HSP47-shRNA intervention groups were 25 .00% , 25 .00% and 33 .33% , respectively .Mice received 4 mg/kg dose of HSP47-shRNA had significantly higher survival rate than the infected controls (χ2 = 4 .168 ,P= 0 .043) .In both 4 mg/kg HSP47-shRNA intervention group and the infected control group , hematoxylin and eosin staining showed chronic granuloma change ,of which ova were wrapped by the spindle-shaped collagen and inflammatory cell infiltrated .The percentage of positive staining for Masson trichrome and sirius red showed the quantity of the collagen deposition was significantly reduced by the HSP 47-shRNA intervention ,compared to the infected control group (t = 3 .191 ,P = 0 .039) .HSP47-shRNA significantly reduced HSP47 expression , and reduced the transcriptional levels of collagen type Ⅰ ,collagen type Ⅲ ,TIMP-1 ,PAI-1 ,TGF-β1 , CTGF ,IL-13 and IL-17 (all P < 0 .01) ,and increased the expression of MMP-9 mRNA ( P < 0 .01) . Conclusion Delivery of shRNA targeting HSP47 to the hepatic schistosomiasis mice model can silence the expression of HSP47 and improve the liver fibrosis .Collagen degradation and related cytokines release through upregulation of MMP-9 and downregulation of PAI-1 may be one of the anti-fibrotic mechanisms in HSP47-shRNA intervention .

Effect of heat shock protein 47 on epithelial-mesenchymal transdifferentiation induced by transforming growth factor β1 in the renal tubular epithelial cells / 中华肾脏病杂志

Objective To detect the expression of heat shock protein 47(HSP47) in renal proximal epithelial cell lines (HK-2) and to investigate the role of HSP47 in the progress of transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transdifferentiation (EMT) in HK-2 cells.Methods HK-2 cells were exposed to TGF-β1 (0,2.5,5,10 μg/L) for different time (0,12,24,48 h).The expression of HSP47 was examined by Western blotting.Then HK-2 cells were exposed to 10 μg/L TGF-β1,the expressions of vimentin,zona occludens-1 (ZO-1) were examined by Western blotting and real-time PCR.Furthermore,the expressions of p-Smad3 and Smad3 were examined by Western blotting.HK-2 cells were transfected with HSP47 siRNA and siRNA negative control before exposing to TGF-β1.Then the expressions of vimentin,ZO-1 were detected by Western blotting and real-time PCR,meanwhile Western blotting for HSP47,p-Smad3 and Smad3.Results Stimulating HK-2 with TGF-β1 resulted in a significant increased expression of HSP47 in time-and concentration-dependent manner (P ＜ 0.05).Meanwhile,TGF-β1 up-regulated the protein and mRNA expression of vimentin (P ＜ 0.05),and down-regulated the protein and mRNA expression of ZO-1 (P ＜ 0.05),all in time-dependent manner.Stimulating HK-2 with TGF-β1 resulted in phosphorylation of Smad3,which was peaked at 30 min,slightly decreased at 1 h,and then increased again between 24 and 48 h (P ＜ 0.05).Compared to the TGF-β1 group,inhibition of HSP4.7 expression in HK-2up-regulated the protein and mRNA expression of ZO-1,down-regulated the protein and mRNA expression of vimentin (P ＜ 0.05) and down-regulated the ratio of p-Smad3/Smad3.HSP47 siRNA negative control had no significant effect on the expressions of ZO-1,vimentin and p-Smad3/Smad3 (P ＞ 0.05).Conclusion HSP47 can promote the EMT of renal tubular epithelial cell which is possibly via the TGF-β1-Smad3 pathway.

Effect of fractional Er:YAG laser on skin collag en proliferation / 中华皮肤科杂志

Objective To investigate the histological changes of normal human skin after fixed irradiation with multiple-pulse fractional Er:YAG laser.Methods After hair removal,the upper arms of 7 healthy volunteers were consecutively irradiated with multiple-pulse fractional Er:YAG laser.Tissue samples were resected from the irradiated skin at 1,24,48,72 hours,on day 5,7,15,and 30 after the irradiation.Hematoxylin and eosin(HE)staining and Masson staining were performed to observe the histological changes and collagen proliferation respectively.Immunohistochemical staining was used to detect the expression of heat shock protein(HSP)70 and 47.Results The consecutive irradiation with multi-pulse fractional Er:YAG laser generated an array of tapering microscopic treatment zones(MTZs)of gasification and ablation in the upper arm skin.After the irradiation,inflammation developed in the microscopic lesions with the epithelization of epidermal cells within 7 days; local dermal collagen was renewed and remodelled during the 7th to 30th day.HSP70 expression peaked as early as 48 hours after the treatment and maintained until the 7th day,while the high expression of HSP47 persisted from the 15th to 30th day after irradiation.Conclusion The fixed,multiplepulse and fractional Er:YAG laser irradiation can reach the deep dermis,and induce the local proliferation of dermal collagen.