| BackgroudBladder cancer is the 7th most common malignant tumor and the 17 th in men amd women respectively worldwide. As the most common subtypes of bladder cancer, bladder transitional cell carcinoma accounts for about 90% of all bladder cancer. Invasion and metastasis are the main cause of death in patients. Although the diagnosis and treatment of bladder cancer has made great progress, but the mechanism of its recurrence, invasion and metastasis is still not clear. Tumor metastasis occurs via a complicated cascade of biological events: invade the adjacent tissues, penetrate the lymphatic/vascular systems, detach from the primary tumor,and spread to near/distant specific sites where they stop, invade and proliferate to form new metastatic lesions. Numerous studies have found that, TGFβ1 and TLR4 signaling pathways are associated with tumor invasion and metastasis. It is reported that the activation of TLR4 signaling pathway in some tumor cells can upregulate the expression of TGFβ1, but studies about the regulation of TGFβ1 on TLR4 signaling are rare. This research aimed to explore the expression and interaction of TGFβ1 and TLR4 signaling pathways in bladder transitional cell carcinoma, that are both closely related with tumor invasion and metastasis.ObjectiveThis study on the expression and interaction of TGFβ1 and TLR4 signaling pathway in bladder transitional cell carcinoma T24 cells aims at providing new ideas for the research on invasion and metastasis mechanisms of bladder transitional cell carcinoma.Methods1. The expression of TLR4 mRNA in T24 cells were detected by RT-PCR before and after the treatment of LPS、rhTGFβ1.2. The expression of TLR4 protein on membranes of T24 cells were detected by flow cytometry before and after the treatment of LPS、rhTGFβ1.3. The expression of TGFβ1 mRNA were detected by RT-PCR before and after the treatment of LPS.4. The expression changes of TGFβ1 protein were detected by ELISA method before and after the treatment of LPS.Results1. In mRNA and protein levels the expression of TLR4 and TGFβ1 in T24 cells was detected.2. After 12 h stimulation of three different concentrations of LPS(1μg/ml,5μg/ml, 10μg/ ml), the expression of TLR4 mRNA in T24 cells was increased as LPS concentration rises, and the relative expression difference within groups was statistically significant(χ2= 12.26, P=0.007). The increasing trend appeared when LPS concentration was 1μg/ml, while 10μg/ml achieved the strongest effect(P<0.001). After the stimμlation of 10μg/ml LPS at different times, TLR4 mRNA relative expression was different(χ2=13.78, P=0.02) and reached the highest level(P<0.001) at 12 h. FACS analysis also revealed that the expression of TLR4 protein was increased with statistical significance(t =﹣3.93, P = 0.02).3. After 12 h stimulation of three different concentrations of LPS(1μg/ml,5μg/ml, 10μg/ ml), the expression of TGFβ1 m RNA in T24 cells was increased with LPS concentration rising, and the relative expression difference within groups was statistically significant(χ2=9.28, P=0.03). When the concentration of LPS is 5μg/ml,its increase began to have statistical significance(P= 0.04), 10μg/ml effect was most obvious(P = 0.003). After the stimulation of 10μg/ml LPS at different times, TGFβ1mRNA relative expression was different(χ2=15.12,P=0.01) and reached the highest level(P <0.001) at 12 h. The elevated level of TGFβ1 protein was also detected byELISA method(t =﹣2.47, P=0.03).4. After 12 h stimulation of three different concentrations of rhTGFβ1(1ng/ml、5ng/ml、10ng/ml), the expression of TLR4 mRNA in T24 cells was increased and reached maximum level at 5ng/ml without statistically significant,(χ2=3.88, P=0.28).And the TLR4 mRNA expression did not change at different times under the treatment of 5ng/ml rhTGFβ1 with statistical significance(χ2=9.69, P=0.08). But FACS analysis revealed that the expression of TLR4 protein were increased with statistical significance after the 24 h treatment of 5ng/ml rhTGFβ1(t= ﹣ 14.92,P<0.001).Conclusions1. T24 cells constitutively express TGFβ1 and TLR4.2. In T24 cells TGFβ1 and TLR4 signaling pathway could promote the expression mutually at the protein level. LPS could activate TLR4 signaling pathway and promote the expression of TGFβ1; TGFβ1 could promote the expression of TLR4 protein, but could not affect the expression of TLR4 mRNA. |
