SPP1 as a key gene in the lymph node metastasis and a potential predictor of poor prognosis in head and neck carcinoma.

BACKGROUND: Lymph node metastasis (LNM) is an important cause leading to recurrence and development of head and neck carcinoma (HNC), with the precise mechanisms unclear. Thus, we aimed to identify the key genes involved in LNM and further evaluate their expressions and roles. METHODS: A cohort of HNC in the TCGA was analyzed. The study involved three phases (one screening and two validation phases). First, the differentially expressed genes regarding LNM were screened, from which a key gene was identified by a series of data mining approaches. Then, the expressions and roles of the key gene were validated in HNC through bioinformatics. Afterward, the expression of the key gene was detected by qPCR, western blot, and Immunohistochemistry based on a cell model and a tissue microarray. Further, colony formation and transwell migration and invasion assays were used to evaluate the roles of the key gene. RESULTS: SPP1 was overexpressed in HNC tissues and was identified as the key gene. Overexpression of SPP1 in HNC was correlated with advanced pathological stages and T-stage, as well as the presence of LNM, which predicted poor prognosis. The expression of SPP1 was closely associated with the infiltration of immune cells in HNC, especially M2 macrophages. Lab experiments confirmed that SPP1 silence in HNC cells resulted in weakened invasive and metastatic abilities. CONCLUSION: This study reveals that SPP1 may be a key gene associated with LNM in HNC, raising the possibility of SPP1 as a target for HNC prevention and treatment.

Fluorimetric analysis of the binding characteristics of 5-enolpyruvylshikimate-3-phosphate synthase with substrates in Dunaliella salina.

A general model of the catalytic mechanism for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) has already been proposed. But whether shikimate-3-phosphate (S3P) alone can cause EPSPs' conformation changes, and whether the binding site of phosphoenolpyruvate (PEP) and glyphosate is the same are still in debate. In this paper, DsaroA gene amplified and cloned from Dunaliella salina (our laboratory's early study) was used for DsEPSPs expression and purification. Then the DsEPSP conformation changes as it bind with different substrates were detected by fluorimetry. The results show that we obtained the DsEPSPs by prokaryotic expression and purification, and the S3P binding with DsEPSPs alone cannot cause DsEPSPs to form "close" conformation directly. However, when S3P exits, DsEPSPs did have a trend to change to the "close" conformation. Then the "close" conformation can be formed completely with the addition of phosphoenolpyruvate (PEP) or glyphosate. The inorganic phosphorus can help S3P to induce two domains of DsEPSPs to form "close" conformation. Besides, when DsEPSPs binds with S3P, in 295 nm, only the intensity of emission peak decreases, however, in 280 nm, not only the peak intensity reduces but also the blue-shift phenomenon takes place. The reason for blue-shift phenomenon was the distribution of aromatic amino acids in EPSPs. EPSPs is a good target for novel antibiotics and herbicides, because of shikimic acid pathway is only present in plants and microorganisms, completely absent in mammals, fish, birds, reptiles, and insects. The results demonstrate that the binding of substrates to EPSPs causes a conformational change from an open form to a closed form, that might be important for designing of novel antimicrobial and herbicidal agents that block closure of the enzyme.