Effects of Allocryptopine on the Proliferation and Epithelial-Mesenchymal Transition of Oral Squamous Cell Carcinoma through m6A Mediated Hedgehog Signaling Pathway.

BACKGROUND: Allocryptopine is an isoquinoline alkaloid extracted from Macleaya cordata. This study aimed to explore the effects of allocryptopine on the growth and metastasis of oral squamous cell carcinoma (OSCC) cells. METHODS: The human OSCC cell line HSC-3 and SAS were selected in this study. MTT assay was performed to measure cell viability. Western blot was used to detect protein expressions. transwell assay was conducted to determine the migrated and invaded cells. M6A modification was confirmed by methylated RNA immunoprecipitation assay. RESULTS: Compared with the NC group, the cell viability, migration and invasion ability of OSCC cells were suppressed after allocryptopine treatment in a dose dependent manner. Allocryptopine upregulated the E-cadherin expression and downregulated N-cadherin and Vimentin expressions in the OSCC cells. In addition, the protein expressions of patched receptor 1 (PTCH1), smoothened co-receptor (SMO) and Gli family (GLI1) were downregulated after allocryptopine treatment. Furthermore, allocryptopine treatment decreased the expression of Methyltransferase like 3 (METTL3) and inhibited N6-methyladenosine (m6A) modification of PTCH1. Moreover, overexpression of PTCH1 reversed the effects of allocryptopine and induced the aggressiveness of OSCC cells. CONCLUSION: Allocryptopine suppressed the proliferation and epithelial-mesenchymal transition (EMT) of OSCC cells via m6A mediated Hedgehog signaling pathway, relieving the carcinogenic behaviors of OSCC.

Mechanical stress promotes biological functions of C2C12 myoblasts by activating PI3K/AKT/mTOR signaling pathway.

The PI3K/AKT signaling pathway regulates cell proliferation and differentiation in multiple types of cells. The present study aimed to investigate the effects of mechanical stress on C2C12 cell proliferation and to explore the associated mechanisms. A cyclic mechanical stress model of C2C12 myoblasts was established. Reverse transcription­quantitative PCR and western blotting assay were used to examine the PI3K signaling pathways involved in the progress of cell differentiation. Cell counting kit­8 (CCK­8) assay was used to evaluate the proliferation of C2C12 cells. Flow cytometry was employed to evaluate apoptosis following mechanical stress. The results demonstrated that mechanical stress activated the PI3K signaling pathway in C2C12 myoblasts. Mechanical stress significantly promoted phosphorylation (p­) of AKT and expression of mammalian target of rapamycin (mTOR) compared with the normal group. Mechanical stress significantly promoted 4E­binding protein 1 (4EBP1) expression in C2C12 cells compared with the normal group. The PI3K specific inhibitor LY294002 significantly decreased 4EBP1 expression and reduced p­AKT and p­mTOR expression compared with the mechanical stress group. Mechanical stress promoted C2C12 cell proliferation. Apoptosis of C2C12 significantly decreased in the mechanical stress group compared with the normal group. Cyclin D levels significantly increased in the mechanical stress group compared with the normal group. In conclusion, mechanical stress promoted biological functions of C2C12 cells by activating the PI3K/AKT signaling pathway. These results may contribute to a better understanding of the effects of mechanical stress on cells.

Effects of mammalian target of rapamycin on proliferation, apoptosis and differentiation of myoblasts undergoing mechanical stress.

Myoblasts characterize by the potential to transform into skeletal muscle, and involve in processes of proliferation, differentiation and apoptosis. Mammalian target of rapamycin (mTOR) is an important protein of PI3K signaling pathway in muscle metabolism and physiology. This study aimed to investigate effects of mTOR on proliferation, apoptosis and differentiation of myoblasts undergoing mechanical stress. We paid much attention on mTOR function undergoing mechanical stress in myoblasts. C2C12 myoblasts were cultured and mTOR gene was knocked down by using Crisper/Cas9 method. Western blot assay and quantitative polymerase chain reaction (Q-PCR) were used to test 4E-binding protein 1 (4EBP1) and p70 ribosomal protein S6 kinase (p70S6k) expression. Cell counting kit 8 (CCK-8) was used to measure cell proliferation, and flow cytometry to used to detect cell apoptosis. Differentiation was counted by using immunofluorescence staining. Rresults showed that the knockdown of mTOR reduced the phosphorylation of 4EBP1 and p70S6k levels undergoing mechanical stress and decreased PI3K signaling pathway proteins synthesis. In addition, proliferation of myoblasts was decelerated by the mTOR knockdown. However, when mTOR knocked down cells treated with mechanical stress, apoptosis rate increased significantly and the differentiation speed was slow down. In conclusion, our study revealed the mTOR function on regulating myoblast proliferation, apoptosis and differentiation undergoing mechanical stress.

[Specific markers and ultrastructure of lymphatic vessel in healthy human dental pulp].

PURPOSE: To explore the existence of lymphatic vessels in healthy human dental pulp. METHODS: Thirty healthy human dental pulps were obtained from non-carious premolars removed for orthodontic reasons. Immunohistochemistry was performed using the antibodies specific for lymphatic endothelium such as D2-40 and LYVE-1, and for vascular endothelial cell such as CD31 and CD34. The expression of D2-40 was detected by Western blotting and ultrastructure was examined by transmission electron microscopy. RESULTS: In healthy human dental pulps, we failed to detect any reactivity for the lymphatic markers D2-40 and LYVE-1 in the observed vessels. These vessels were positive stained by blood endothelial markers CD34 and CD31. Odontoblasts were weakly stained with D2-40. Western blotting performed on collagenase-treated human dental pulps did not show a band at 40 kDa, corresponding to the molecular weight of the lymphatic marker D2-40. Transmission electron microscopy indicated that vessels in dental pulp consisted of endothelial monolayer surrounded by pericytes and complete basement membrane, which were typical ultrastructural characteristics of blood vessels rather than lymphatic vessel. CONCLUSIONS: Human dental pulp does not contain true lymphatic vessels under healthy conditions. Whether lymphatic system is involved in dental pulp interstitial fluid circulation during inflammation deserved further study. Supported by National Natural Science Foundation of China (81100768) and Key Project Supported by Medical Science and Technology Development Foundation, Department of Health of Nanjing City (YKK11040 and QRX11123).

Increased expression of EphA7 in inflamed human dental pulp.

INTRODUCTION: Pulpitis has been associated with abundant inflammatory cells, dilated blood vessels, and thickening nerve fibers histopathologically with or without severe pain clinically. On the basis of EphA7 receptor expression in inflammatory cells, the developing mouse dental pulp, and trigeminal nerve system, EphA7 may possibly be involved in local inflammatory response and sensory innervation of adult dental pulp as well as odontogenic pain conducted through the trigeminal system. The purpose of the study was to analyze the expression of EphA7 gene in healthy and inflamed human dental pulps and to elucidate the roles of EphA7 gene in dental pulp inflammation response and odontogenic pain. METHODS: Twelve healthy controls, 5 acute pulpitis from dental trauma, 21 symptomatic, and 20 asymptomatic irreversible pulpitis human dental pulps were involved in the study. The protein expression, subcellular localization, and mRNA level of EphA7 gene were detected by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction, respectively. RESULTS: In healthy samples, immunohistochemical staining showed positive EphA7 expression only in vascular endothelial cells and odontoblasts with cytoplasm staining. Under inflammatory conditions, in addition to the above cells, EphA7 staining began to occur in fibroblasts, nerve fiber tissues, and inflammatory cells. Compared with healthy samples, EphA7 expressions at both mRNA and protein levels increased significantly in acute and irreversible pulpitis samples. In asymptomatic irreversible pulpitis samples, EphA7 expressions were significantly lower than those in symptomatic ones but still higher than those in healthy ones. There was no significant difference between acute and symptomatic irreversible pulpitis groups. CONCLUSIONS: The results suggest that EphA7 gene may be a marker reflecting inflammatory activity and pain state for human dental pulp.

[Hardness development of self-adhesive resin cement in simulated root canal].

OBJECTIVE: To compare the hardness development of dual-cured self-adhesive and universal resin cement in simulated root canal. METHODS: The light-proof half-cylinder steel slot with one end open were syringed and filled respectively by self-adhesive A (RelyX Unicem), B (BisCem) and universal C (DUOLINK) resin cements, then the open end of slot was irradiated directly by a light unit for 20 s. Specimens were stored in a light-proof box for 0.5 h, Knoop microhardness was measured along the vertical surfaces of specimens from 1 mm to 10mm depth at 1 mm intervals. The same measurements were taken at 24 h and 120 h after irradiation. Data were analyzed by One-way ANOVA. RESULTS: Hardness of each group decreased with the increase of simulated canal depth (P<0.001), however hardness showed no significant change between 5 mm and more depth of group A, between 4 mm and more depth of group B and C. The increase of hardness for each group was more rapid within 0.5 h after irradiation, thereafter the hardness increased gradually to maximum at 24 h. At 120 h after irradiation, hardness of group C was greater than that of other two groups at more than 1 mm depth (P<0.001). CONCLUSION: Under dual-cured condition, hardness has significant difference between self-adhesive and universal resin cements, however their hardness development is similar.