| Objects:Tissue transglutaminase (tTG), as a multifunctional protease in transglutaminasefamily, is an80-kDa protein. tTG can catalyze Ca2+-dependent cross-linking betweenthe γ-carboxamide group of a glutamine and ε-amino group of a peptidebound lysine.It is also involved in signal transduction by combining or hydrolyzing GTP. As ascaffold protein, it binds to integrins, fibronectin and so on. Besides these, tTG playsthe role as protein disulfide isomerase or protein kinase. In turn, the impact of tTG onthese processes implicates this protein in various physiological responses andpathological states, contributing to wound healing, apoptosis, inflammation, cell cyclecontrol, tissue fibrosis and tumor growth, metastasis and drug resistance. Thisresearch aims to explore the roles of tTG in hepatic fibrosis induced bySchistosoma japonicum (Sj) infection and lung cancer with drug resistance andmetastasis.Methods:(1)70six to eight-week-old female BABL/c mice were randomly devided into2groups:10mice as normal control group and60mice as experiment group.50mice inexperiment group were infected cutaneously with203Sj cercariae.40infected micewere sacrificed at week5,6,8,12post infection. And starting at Day3post-infection,another10infected mice and10uninfectd mice were administered with100ulcystamine(CTM)(10-2mM) in PBS through intra-peritoneal injection once per day for7days. The CTM treatment mice were sacrificed at week8after Sj infection. (2)The mice livers and sera were collected. Sera were stored at-20°C. A part ofliver tissue was used for RNA and protein extraction, another part of liver was usedfor paraffin sections preparation.(3) A549cell line, A549cell line with cisplatin-resistance (A549-CDDPR), andA549-CDDPR with tTG-siRNA or NC-siRNA transferring were collected for RNAextraction or cell lysate preparation.(4)Liver pathological changes were measured with Masson staining, the expressionof hepatic α-smooth muscle actin (α-SMA), tTG, tranfroming growth factor-β1(TGF-β1) and interleukin-13(IL-13) were detected by immunohistochemistry (IHC),reverse transcription-PCR (RT-PCR), quantitative PCR(Q-PCR) and Western Blot.Serum concentration of TGF-β1and IL-13were detected by ELISA. tTG expressionin lung cancer cell lines were detected with quantitative PCR, Western Blot andimmunofluorescence assay. tTG expression in lung cancer tissue microarray ofpatients were detected with IHC. Cell migration and invasion capacity of lung cancercells were evaluated by Transwell (with or without matrigel) assay. MTS assay wasused for detecting lung cancer cell survival.Results:(1) After5weeks post Schistosoma japonicum(Sj) infection, mice liver collagenarea and α-SMA level were increased gradually, and were highest at week8postinfection. Compared with week8，at week12liver collagen area and α-SMA levelwere decreased. After Sj infection, the expression of tTG in mice liver wasupregulated, and tTG’s expression obviously increased in the hepatic cells around thehepatic sinusoids, around&in the liver granuloma&fibrosis areas where Sj eggsdeposited or inflammatory cell infiltration area; after be treated with tTGinhibitor-CTM, infected mice liver collagen area and α-SMA level were reduced. Atweek5,6,8and12post-infection, TGF-β1level in serum and liver tissue wereincreased. RNA expression level of Sj-derived TGF-β1was increased, butmouse-derived TGF-β1in liver was reduced; After CTM treatment, TGF-β1level inserum and liver were decreased. At week5,6,8and12post-infection, IL-13level in serum and liver were increased, after CTM treatment, IL-13RNA expression level ininfected mice were downregulated, and IL-13protein expression level in infectedmice liver and serum were also reduced.(2) IC50of A549and A549-CDDPR were1.40and36.67ug/μl respectively;Average cell migration number of A549and A549-CDDPR were (44.6±5.36),(229.67±27.34) respectively, and average cell invasion number of them were (55.33±5.96),(359.0±36.87) respectively. The difference of the numbers is statisticallysignificant (P<0.05). tTG level of A549-CDDPR was significantly higher than that ofA549(P<0.05). Immunohistochemical staining of human lung tissue microarrayshowed that lung cancer tissue tTG positive expression rate of patients with lymphnode or distant metastases was72.4%, and that of patients without lymph node anddistant metastases was37.6%. The difference was statistically significant (P<0.01).After down-regulating tTG exprssion by siRNA interference, cell survival rate ofA549-CDDPR with CDDP treatment was50.94±5.59%, and that of A549-CDDPRwith NC-siRNA interference was92.60±5.32%. The difference was statisticallysignificant (P<0.01). Average cell migration number of A549-CDDPR cell withtTG-siRNA or NC-siRNA interference were (103±13.10),(200.8±24.43) respectively,and average cell invasion number of them were (114.6±15.24),(337±22.2)respectively, and the difference was statistically significant (P<0.01).Conclusions:Part1(1)5weeks after Schistosoma japonicum(Sj) infection, the extent of fibrosis in miceliver was low, and it gradually increased at week6and8. It was the most serious atweek8, while at week12chronic hepatic fibrosis appeared.(2)Consistent with the extent of fibrosis, tissue transglutaminase (tTG) level wasincreased in infected mice compared with non infection control mice. tTG inhibitor-CTM alleviated the extent of hepatic fibrosis. So, tTG contributed to liver fibrosisinduced by Sj infection.(3)TGF-β1protein level showed a positive correlation with hepatic fibrosis formationresulted by Sj infection, and the high transcription level of Sj-derived TGF-β1was the source of the high expression level of TGF-β1protein in Sj-infected mice’s liver andserum.(4) IL-13level was positively correlated with the extent of hepatic fibrosis of micewith Sj infection.(5) tTG was involved in the devolpment of mice liver fibrosis induced by Schistosomajaponicum infection, and the mechanism may be associated with tTG-regulatedTGF-β1and IL-13.Part2(1) Compared with A549, A549-CDDPR showed higher CDDP-resistance, migtarionand invasion capacity.(2) tTG level was positively correlated with lung cancer patient metastasis.(3) tTG expression level in A549-CDDPR lung cancer cell was high, and after tTGdown-regulation by tTG-siRNA transferring, drug resistance, migration and invasioncapacity of A549-CDDPR were decreased.(4) tTG contributed to lung cancer metastasis and the drug-resistance and highmigration&invasion of lung cancer cell. |
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