A Molecular Mechanism Study Of Trop2 On Invasiveness, Metastasis And Chemosensitivity In Squamous Cell Carcinoma Of The Head And Neck

Early metastasis and chemoresistance have always been serious barriers to successful treatment in Squamous cell carcinoma of the head and neck (SCCHN), To obtain optimal effect of SCCHN patients, it is urgently needed to reveal effective moleculars for the prediction of metastasis and chemoresistance and study the molecular mechanism of metastasis and chemoresistance in SCCHN. The manuscript will discuss the effects and mechanisms of a novel molecule Trop2 on the metastasis and chemoresistance of SCCHN as follows.Part I The aberrant expression of Trop2 and its clinical significance in SCCHNObjective Detect the expressions of Trop2 in human SCCHN tissues and determine its relationships with clinical significance. Additionally, evaluate the expressions of Trop2 in SCCHN cell lines.Methods Quantitative PCR was used to detect the expressions of Trop2 mRNA in 53 cases of SCCHN tissues biopsies and 25 cases of adjacent tissues biopsies. Immunohistochemistry was used to detect the expressions of Trop2 in 87 cases of SCCHN tissues and 20 cases of adjacent tissues paraffin imbedding sections. Western blot was used to detect the expressions of Trop2 in SCCHN cell lines.Results The expressions of Trop2 mRNA was significantly up-regulated in SCCHN tissues compare with adjacent tissues and positive correlated with tumor size, clinical stage and lymph node metastasis (P<0.05). We also found the expressions of Trop2 protein was significantly up-regulated in SCCHN tissues compare with adjacent tissues and positive correlated with clinical stage and lymph node metastasis (P<0.05) with no relation to tumor size (P>0.05). In addition, the expressions of Trop2 were up-regulated in SCCHN cell lines compare with nomal oropharyngeal epithelial cells.Conclusions All the results suggest that Trop2 correlates with tumor progress and lymph node metastasis in SCCHN and Trop2 may play a role in SCCHN cell metastasis.PartⅡThe Construction of Trop2 shRNA and Trop2 knockdown of SCCHN cell linesObjectives To construct the Trop2 shRNA and Trop2 knockdown SCCHN cell lines.Methods Got the synthesized target genes for Trop2 short hairpin RNA (shRNA) online; conceived Trop2-targeted shRNA particles and no-targeted shRNA particles as control, then they were transformed into E. coli and identification of positive clones to confirm the vectors successfully constructed. The interference sequence was made after producing and purifying. Quantity RT-PCR and Western blot were used to detect the expression of Trop2 in transient transfection cells; CCK-8 test was used to detect the cell proliferation. For stable transfection, cells seeded in 24-well plates were transfected with 1μg of particles using LipofectamineTM LTX according to the manufacturer’s instructions. After no less than 14 days of selection in RIPM-1640 containing 10%FBS and G418 (300μg/ml), individual G418-resistant colonies were isolated and expanded.Results Sequencing results showed that vectors containing short hairpin RNA were successfully constructed. Quantity RT-PCR and Western blot have shown that the expression of Trop2 was significantly reduced in TACSTD2-RNAi-3, which achieved at knockdown rate of 49.7% and 45.0% in Tu686 cells and M4 cells respectively and cell proliferation inhibition rate of 27.84±4.15%and 25.08±3.73%in Tu686 cells and M4 cells respectively. Moreover, we have constructed TACSTD2-RNAi-3 stable transfection Tu686 and M4 cells, which achieved at Trop2 knockdown rate of 81.5%and 76.2%respectively.Conclusions We have successfully constructed effective shRNA vector target for Trop2 gene and established stable Trop2-knockdown Tu686 and M4 cells.PartⅢThe Effect of down-regulation of Trop2 Expression on Migration and Invasiveness Ability in SCCHN and its possible mechanismObjectives To study the effect of Trop2 on migration and invasiveness in SCCHN and to investigate its possible molecular mechanism.Methods Stable transfected trop2-shRNA or no-target-shRNA vector Tu686 cells were used in this study. The cell proliferation was detected by CCK-8. The invasiveness ability was tested by wound-healing assay and migration ability was performed by a matrigel invasiveness assay in transwell culture chambers. The expression of E-cadherin and (3-catenin were examined by Western-blot.Results The CCK-8 detection showed great difference between the experimental group and the control group. The Inhibition rate at 24h、48h、72h in the experimental group was 14.3±2.1%,28.7±3.6%、35.4±3.4%compared to the control group 3.3±1.4%、5.7±1.3%、4.8±1.1%. The result of wound-healing assay showed that the invasiveness capability of the experimental was signifieantly weakenecd compared with control group, the healing rate of trop2-shRNA Tu686 cells were 42.6%%lower at 48 h than no-target-shRNA Tu686 cells. And the result of matrigel invasiveness assay in transwell culture chambers showed that the capability of experimental group invasiveness was significantly weakened, compared with the control group, the cells getting through the basement membrane were 153±20.6 to 247±36.4 at 48 h. Western-blot results showed that the experimental group epithelial marker E-cadherin significantly increased andβ-catenin not changed compared to control group.Conclusions Trop2 affects the proliferation of SCCHN cells, and Trop2 knockdown facilitates the invasiveness and migration in Tu686 cells. Moreover, E-Cadherin might be influenced by Trop2. So it gives theorical foundations for the further study of Trop2 induced invasiveness in SCCHN, the latent applied values of Trop2 will be proved in the prevention of metastasis in SCCHN. Part IV The Effect of Trop2 knockdown on chemosensitivity in SCCHN cellsObjective To detect weather Trop2 knockdown affects the chemosensitivity in SCCHN cells.Methods Stable transfected Trop2-shRNA or no-target-shRNA vector Tu686 and M4 cells were used in this study. Cells were incubated with different concentration of paclitaxel and Cisplatin for 48 h. Then, the cell proliferation was detected by CCK-8. Cell apoptosis was assessed by Tunnel assay and Annexin V Flow cytometry assay after cultured with paclitaxel at a concentration of IC30 for 24 h.Results 1) After treatment of paclitaxel, the survival rate of Trop2-shRNA tu686 is obvious lower than no-target-shRNA tu686, the IC50 of experimental group is (9.931±0.876)×10-8mol/L compared to (9.484±0.744)×10-7 mol/L of control group (P＜.001).2) after treatment of paclitaxel, the survival rate of Trop2-shRNA M4 is obvious lower than no-target-shRNA M4, the IC50 of experimental group is (1.959±0.0176)×10-7 mol/L compared to (1.396±0.174)x10-6 mol/L of the control group (P<0.001).3) After the treatment of Cisplatin, there is no significant difference in the survival rate between experimental group and control group of tu686 and M4 cells.4) After the treatment of paclitaxel, The Tunnel assay showed the apoptosis ratio of Trop2-shRNA tu686 cells is 44.3±3.4%, which is significantly higher than that of no-target-shRNA tu686 cells 28.7±2.6%(P＜.001). Annexin V Flow cytometry assay showed the apoptosis ratio of Trop2-shRNA tu686 is 31.39±2.83%, which is significantly higher than that of no-target-shRNA tu686 cells 14.67±2.77%(P＜0.001).5) After treatment of paclitaxel, Tunnel assay showed the apoptosis ratio of Trop2-shRNA M4 cells is 42.8±4.6%, which is significantly higher than that of no-target-shRNA M4 cells 27.9±3.1%(P<0.01). Annexin V Flow cytometry assay showed the apoptosis ratio of Trop2-shRNA M4 cells is 33.75±3.47%, which is significantly higher than that of no-target-shRNA M4 cells 14.72±1.94%(P<0.001)Conclusions The Inhibition of Trop2 can significantly strengthen chemosensitivity of paclitaxel in SCCHN, while there is no change in that of Cisplatin.