The Mechanisms Of Bone Morphogenetic Protein 4 Induced Epithelial-mesenchymal Transition And Metastasis In Squamous Cell Carcinoma Of The Head And Neck

Early metastasis has always been an important factor for the poor prognosis of Squamous cell carcinoma of the head and neck (SCCHN), So looking for the prediction of metastasis and studying the molecular mechanism of SCCHN are of great significance. This manuscript will discuss the effects and mechanisms of a novel molecule-BMP4 on the metastasis of SCCHN in separate chapters.Chapter 1 The aberrant expression of BMP4 and its clinical significance in SCCHNObjective. To evaluate the expressions of BMP4 and two related signal pathway proteins, Smadl and p-Smadl in human SCCHN, then to determine their relationships with lymph node metastasis, tumor differentiation, lymphatic metastasis and the prognosis of SCCHN.Methods. Tissue samples of primary tumors from 89 SCCHN, and 20 normal tissue samples from pharynx or larynx were undergoing paraffin imbedding slices. Immunohistochemistry was used to detect the expressions of BMP4, Smadl and p-Smad1. The relationships between the pathological parameters and three proteins were analysed, Sperman correlation test was applied to analysis the relationships between expressions of BMP4 and Smadl, p-Smadl. Log-rank test was employed to analyse the relationships between the three proteins and the prognosis of SCCHN.Results. The expressions of BMP4,Smadl and p-Smadl in SCCHN tissues were significantly higher than those in normal mucosa (P<0.05). The expressions of both BMP4 and p-Smad1 were correlated with clinical stage, pathological grade, lymph node metastasis and recurrence (P<0.05), but were not related to patients'age (P>0.05). The expression of Smad1 was correlated with clinical stage, pathological grade(P<0.05), but was not related to patients'age, lymph node metastasis and recurrence (P>0.05). The expression of BMP4 was not related to Smadl, while was positively correlated with p-Smadl (rs values were 0.013 and 0.548, respectively).The high expressions of BMP4 and p-Smadl were associated with the poor prognosis of SCCHN (P values were 0.01562 and 0.0418, respectively), the expression of Smadl was not related to the prognosis of SCCHN (P=0.6587).Conclusions. All the results suggest that detection of BMP4, Smad1 and p-Smadl will be helpful for the prediction of carcinogenesis, early metastasis and prognosis of SCCHN, it provides new clues for the metastatic studies of SCCHN.Chapter 2 Bone morphogenetic protein 4 induces invasiveness of the SCCHN cells through EMT in vitroObjective. To study the mechanisms of BMP4 induced invasiveness of SCCHN cell in vitro.Methods. Six SCCHN cell lines:Hep-2,Tu686,Tu212,M2,M4,212LN were treated with recombination human BMP4 for 72 hours, MTT test was used to detect the proliferations of each cell line after treatment. Confocal microscopy was applied to observe the phenotypic changes of EMT in both Tu686 and Tu212 after treatment of BMP4; western blot or RT-PCR was applied to detect the expressions of associated genes after treatment by BMP4; Transwell invasive test and scratch test were employed to detect the changes of invasive cell number and migrating cell number of Tu686 and Tu212 after treatment by BMP4, respectively.Results. There was no statistic difference in proliferation between the BMP4 treated groups and there respective control groups (P>0.05) in all six cell lines after treatment for 72 hours. Both Tu686 and Tu212 epithelial cells turned into more narrow, long strip or fusiform shapes, stretch out silipues boundary, undergoing EMT. The expressions of p-Smad1 and Vimentin in Tu686 and Tu212 cells showed dose and time-dependent increases after treatment with BMP4, while the expression of E-Cadherin were dose and time-dependent decreased after treatment with BMP4. The invasive and migratory abilities of both Tu686 and Tu212 cells were increased significantly after treatment with BMP4 for 72 hours(P<0.05).Conclusions. BMP4 doesn't affect the proliferation of SCCHN cells, it induces Tu686 and Tu212 cells undergoing EMT in vitro, and facilitates the invasiveness and migration in two cells, so it gives theorical foundations for the further study of BMP4 induced invasiveness in SCCHN, the latent applied values of BMP4 will be proved in the prevention of metastasis in SCCHN.Chapter 3 The effect of Smadl on the metastasis of SCCHN through BMP4 mediated signal pathwaysObjective. To study the effects of Smadl on the invasiveness and the expressions of EMT associated genes in SCCHN cell lines with the stimulation of BMP4 in vitro.Methods. Tu686 and Tu212 cells were transfected with siRNA Smad1 or pcDNA3.1(+) Smad1, then followed by the treatment of 100ng/ml BMP4 for 72 hours, the efficiencies of transfection were detected on both gene and protein levels. Western blot was applied to detect the changes of p-Smad1, E-cadherin and Vimentin after successfully up or down regulating the expression of Smad1 in two cells. Transwell invasive test or scratch test were employed to detect the invasive and migratory ability after treatment, respectively. Results. After transfection of Tu686 and Tu212 cells with Smadl gene, Smadl was successfully up-regulated, under the stimulation of 100ng/ml BMP4 for 72 hours, the expressions of E-Cadherin were all significantly down-regulated(P<0.05), the expressions of p-Smad1 and Vimentin were significantly up-regulated(P<0.05). The invasive and migratory abilities of two cells were both decreased significantly(P<0.05). While Smadl was successfully knocked down, in Tu686 and Tu212 cells, the expressions of E-Cadherin were significantly up-regulated(P<0.05), the expression of p-Smad1 and Vimentin were significantly down-regulated(P<0.05). The invasive and migratory abilities of two cells were all decreased significantly(P<0.05).Conclusions. Smadl and p-Smadl play important roles in the BMP4 induced EMT and invasiveness in SCCHN cells. It paves the way for the further study of Smadl on the metastasis of SCCHN.