Expression Of TLR3 In Laryngeal Squamous Cell Carcinoma And Apoptosis Of (LSCC) Cell Induced By TLR3 Knockdown

Objective:To investigate the expression of toll like rceptors-3 and clinical implications.Small interfering RNA trigger apoptosis of in laryngeal squamous cell carcinoma(LSCC)through Caspase and bcl-2 gene family.Methods:(1)53 specimens of surgically resected LSCC tissues and 30 specimens of adjacent normal tissue were examined for the expressions of TLR3 using immunohistochemistry.The correlation of TLR3 expressions with the clinicopathological factors and prognosis of LSCC patients.(2)3 pairs siRNA targeted sequences of TLR3 were designed and synthesized according to the Hep-2 and the scrambled control sequenceswith no gene homology.Q-PCR Western blot were detected their efficiency of suppression.(3)Scarification test and transwell invasion assay were used to test the metastasis ability of Hep-2 cells after gene silencing of TLR3.(4)Cell tunel assy was used to evaluate the cell proliferation of Hep-2 cells.Cell cycle and apoptosis was analyzed by flow cytometry.(5)The mRNA and protein expressions of the caspase/bcl-2 gene family were tested by reverse transcription polymerase chain reaction(RT-PCR)and Western blot.Results:(1)The positive expression rates of TLR3 in LSCC were 68%(38/53),significantly different from those in the adjacent tissue(11%;P<0.05).The expression of TLR3 in LSCC was positively correlated with tumor classification and lymph node metastasis(P<0.05).The expression of TLR3 was negatively correlated with gender?ages ?smok(P>0.05).(2)The relative expression levels of TLR3 mRNA and protein in TLR3siRNA-3 group were significantly lower than NC control group(P <0.05).(3)Scarification test and transwell invasion assay showed that compare with NC control group,the migration and invasive cells decreased significantly in siTLR3 group(t=3.255,P<0.05;t=2.942,P<0.05).(4)CCK-8 assay was used to evaluate the influence of siTLR3 on the proliferation of Hep-2 cells.Cell-cycle and apoptosis was analyzed by flow cytometry.Compared with the NC control group,the proportion of cells in S phase was significantly increased in siTLR3 group(P<0.01),and the proportion in G2/M phase was inhibited(P<0.05).Cell apoptosis assay showed that the apoptotic rate in siTLR3 group((25±1.28)% was increased in NC group(15±2.04)%.(5)The mRNA and Western blot showed that cleaved-Caspase3 ?cleaved-Caspase8 and cleaved-Caspase9 upregulated the protein upregulated by siRNA mediated gene TLR3 silencing while bcl-2 was downregulated.Conclusion.TLR3 expression is an important factor associated with the tumor classification and lymph node metastasis of LSCC patients,Small interfering RNA trigger apoptosis of in laryngeal squamous cell carcinoma(LSCC)through Caspase and bcl-2.