The Expression And Effects Of TLR4 Signaling Pathway On The Origin And Development Of Gallbladder Cancer

BackgroundAs the tumor originated from the gallbladder endothelial cells, gallbladder cancer is highly malignant tumor in gastrointestinal. Early diagnosis is dufficult because its high degree of malignancy, local invasion and distant spread along the lymphatic. Surgery is the only way of healing for current treatments of gallbladder cancer. In fact, the resection rate is low and prognosis is unpleasant, because the tumor is hard to recognized. Chemotherapy can not improve survival of patients with gallbladder cancer. Anyhow, it is still lacking of effective treatments for gallbladder. Cancer is a complex network system, intervention or termination of a link can change the whole process. Epidemiological survey proved the risk factors of gallbladder cancer was chronic inflammation. As the member of Toll-like receptors family, TLR4 plays an important role not only in the immune response and inflammation, but also in the development of tumor cells. The function of TLR4 in tumor cells in the part may be through COX-2. It has been confirmed as the important prognostic indicators in some of tumors. This topic is based on the relationship of gallbladder and chronic inflammation. We focused on TLR4 signaling pathways in inflammatory system and tumor development through the important role in vivo and in vitro. Then observed the inhibition of gallbladder cancer cells, using the TLR4 target-based little interference RNA technology to block TLR4 signaling pathway. We hope to find new effective targets gene for gallbladder cancer.ObjectivesResearching expression of TLR4 and COX-2 in the gallbladder and analysing the relationship, revealing the function in development of the gallbladder cancer. Applicating small interfering RNA inhibition expression of TLR4 in gallbladder cancer cells.After blocking TLR4 signaling pathway, searching TLR4 and COX-2 gene expression and inhibitory effect of gallbladder cancer cell proliferation in vivo. To TLR4 as potential target for gene therapy in gallbladder feasible for clinical exploration of new methods of treatment of gallbladder carcinoma. MethodsCollecting clinical gallbladder cancer tissue samples from 13 cases,chronic cholecystitis from 30 cases and normal tissue samples gallbladder 10. Immunohistochemical, polymerase chain reaction and immune imprinting technique were used in the above samples with COX-2 and TLR4 expression. Analysis of TLR4 expression and COX-2 expression and the relevance of gallbladder carcinoma. In vitro, using RNA interference suppression TLR4 expression of gallbladder cancer cells SGC996, Immunofluorescence and cell count analysis TLR4 on the cell proliferation of gallbladder carcinoma. Subsequently, using the Western blot, and polymerase chain reaction study the impact of TLR4 on the role of downstream genes. Last, SGC996 cells were injected subcutaneously into nude mice, treated by small target TLR4 RNA interference experiment, observing body circumstance and anti-tumor effect of TLR4.ResultsAfter gallbladder tissue immunohistochemistry results showed that TLR4 was mainly expressed in the gallbladder and glandular epithelial cells. TLR4 expression in gallbladder carcinoma was significantly lower than normal gallbladder tissues and chronic cholecystitis (P<0.05), between normal gallbladder and chronic cholecystitis with no significant difference (P> 0.05). Immunofluorescence, polymerase chain reaction and Western blot results showed that in vitro synthesized small interfering RNA targeting of TLR4 transfected gallbladder SGC996 cells can inhibit the expression of TLR4, Inhibiting the proliferation of cancer cells. With the inhibition of TLR4, COX-2 has also come down. We successfully established the growth of human gallbladder carcinoma cell SGC996 subcutaneous tumor model in vivo. Ability in tumor formation by RNA interference targeting TLR4 after the tumor volume and weight were significantly lower than the control group (P<0.05). In the treatment experiment, the group of TLR4 small interfering RNA was injected, the average size and weight of tumor were not significantly difference with the negative control group and blank control group (P>0.05). ConclusionsTLR4 and COX-2 expression in human gallbladder tissue and SGC996 cells, and is closely related with the development of gallbladder carcinoma. In SGC996 cells, a small interfering RNA targeting TLR4 can inhibit SGC996 cell proliferation and lead to COX-2 expression reduced. In gallbladder cancer cell SGC996 nude mice model, small interfering RNA targeting TLR4 can significantly inhibit tumor growth. Therefore, We believe that TLR4 may be the gallbladder cancer potentially effective target for gene therapy. Small interfering RNA targeting TLR4 technology may be a clinical potential treatment for gallbladder cancer related to inflammation.