The Experiment Research On Tissue Transglutaminase Mediating The Mechanism Of Drug Resistance In Breast Cancer

Part 1 The clinical significance of tTG in evaluating the efficacy of new adjuvant chemotherapy for breast cancerObjective:The clinical significance of tTG was explored to evaluate the efficacy of new adjuvant chemotherapy for breast cancer.It may be hoped to provide a new theoretical basis for tTG's participation in drug resistance of breast cancer from the clinical level.Methods:(1)71 female patients given new adjuvant chemotherapy with single-sided breast cancer and archived by the Department of Pathology were collected retrospectively from January 2014 to June 2019.Well-document clinical pathology data;(2)The expression of tTG were detected in breast cancer tissue before and after the new adjuvant chemotherapy by immunological chemistry.(3)According to RECIST1.1 standard and Miller-Payne classification on clinical evaluation and pathology evaluation for the efficacy of new adjuvant chemotherapy,the patients were divided into new adjuvant chemotherapy sensitive group and new adjuvant chemotherapy resistance group.Statistical analysis was performed between the tTG expression level before as well as after new adjuvant chemotherapy and the efficacy of new adjuvant chemotherapy,the expression of tTG after new adjuvant chemotherapy and the application of chemotherapy drug regiments in the new adjuvant chemotherapy resistance group,the expression of tTG and clinical pathology parameters of breast cancer.Results:(1)The expression of tTG in breast cancer tissue after new adjuvant chemotherapy was higher than that of tTG in breast cancer tissue before the new adjuvant chemotherapy.(2)The expression of tTG before the new adjuvant chemotherapy was not significantly related to the efficacy of the new adjuvant chemotherapy.However,the expression of tTG after the new adjuvant chemotherapy increased,there was significant between the expression of tTG and the evaluation of the efficacy of the new adjuvant chemotherapy(p<0.05).(3)The difference between the expression of tTG and the application of new adjuvant chemotherapy drug regiments was not statistically significant in patients with drug resistance.(4)The expression of tTG before new adjuvant chemotherapy was significantly associated with lymph node metastasis,Ki67 proliferation index,and there was no significant correlation with age,menstruation,tumor diameter,clinical stage,histological classification as well as ER,PR,Her-2 expression.The expression of tTG after new adjuvant chemotherapy was significantly associated with tumor diameter,clinical stage,lymph node metastasis,and there was no significant correlation with age,menstruation,histological classification Ki67 as well as ER,PR,Her-2 expression.Conclusion:(1)There was no significance between the expression of tTG before the new adjuvant chemotherapy and the evaluation of the efficacy of the new adjuvant chemotherapy,indicating that tTG could not be used as a predictor of the efficacy of the new adjuvant chemotherapy.The expression of tTG was increased after chemotherapy,and there was significant between the expression of tTG after the new adjuvant chemotherapy and the efficacy of the new adjuvant chemotherapy.It may be an important factor in the evaluation of the efficacy of subsequent new adjuvant chemotherapy;(3)By analyzing the correlation between the expression of tTG and the application of the new adjuvant chemotherapy drug regiments,it can be seen that tTG is involved in drug resistance of new adjuvant chemotherapy,independent of the application of the new adjuvant chemotherapy drug.(4)tTG may play an important role in the cell proliferation and metastasis of breast cancer.Innovation:After literature review and retrieval,no studies have been reported on the clinical significance of tTG in evaluating the efficacy of new adjuvant chemotherapy for breast cancer.Because the new adjuvant chemotherapy could predict the sensitivity of cancer cells to the drug,from the perspective of the new adjuvant chemotherapy,it is hoped to provide a more reliable theoretical basis for drug resistance in breast cancer.Part 2 The research on the mechanism of tTG mediating the drug resistant of MCF-7 to adriamycinObjective:To explore the possible mechanism of tTG mediating drug resistance to adriamycin in MCF-7 in vitro.It may be hoped to provide a new theoretical basis for solving the problem of drug resistance in breast cancer.Methods:(1)the small interference RNA sequences were designed,and fluorescent microscopes were used to observe the transfecting rate of green transfected proteins.RT-PCR and Western-blot were used to screen the best sequence of silencing TGM2.(2)The cells were divided into MCF-7,MCF-7/ADR,MCF-7+tTG siRNA.RT-PCR was used to detect the mRNA levels of tTG,P-gp,MRP,LRP,and Western-blot was used to detect the protein expression of tTG,P-gp,MRP,LRP before and after silencing TGM2.The mRNA and protein expression levels of tTG,P-gp,MRP,LRP were statistically analyzed.(3)The MTT method detected the IC50 of MCF-7,MCF-7/ADR,and the proper concentration of adriamycin was determined on MCF-7/ADR.(4)Cells are divided into MCF-7,MCF/ADR,MCF-7/ADR+tTG siRNA,MCF-7/ADR+adriamycin,MCF-7/ADR+tTG siRNA+adriamycin.The biological characteristics of breast cancer cells were detected.The cell growth was detected by MTT,and the apoptosis was detected by flow cytometry.Results:(1)The transfecting rate of green transfecting protein was observed more than 90%under the fluorescent microscope,and the results of RT-PCR and Western-blot showed that the siRNA 3 sequence was the best sequence of silencing TGM2.(2)RT-PCR and Western-blot detected mRNA and protein expression levels of tTG respectively,and the results demonstrated that the mRNA and protein expression levels of tTG significantly increased in MCF-7/ADR compared to MCF-7,and the mRNA and protein expression levels of tTG decreased significantly after silencing TGM2,indicating successful transfection.(3)RT-PCR and Western-blot detected mRNA and protein expression levels of P-gp,MRP,LRP,respectively,and the results demonstrated that the mRNA and protein expression levels of P-gp,MRP,LRP in MCF-7/ADR increased significantly compared to MCF-7,and the mRNA and protein expression levels of P-gp,MRP,LRP decreased significantly after silencing TGM2.(4)The MTT determined that the IC50s of MCF-7 and MCF-7/ADR are 2.43ug/ml and 89.44ug/ml,respectively.(5)The cell growth results showed that MCF-7 and MCF-7/ADR had the lowest cell suppression rate,and MCF-7/ADR tTG siRNA+adriamycin group had the highest rate of cell suppression.Compared to the MCF-7/ADR group,the cell suppression rate of the MCF-7/ADR tTG siRNA group increased significantly(p<0.05)and showed in a time-dependent manner.(6)The results of flow cytometry demonstrated that the apoptosis rate of MCF-7/ADR+tTG siRNA+ adrimycin group was the highest.The apoptosis rate of MCF-7/ADR+adrimycin group was lower than MCF-7/ADR+ tTG siRNA + adrimycin group(p<0.05)Conclusions:(1)tTG and P-gp,MRP,LRP involved in drug resistance to adriamycin in MCF-7;(2)After silencing TGM2,the mRNA and protein expression levels of P-gp,MRP,LRP decreased,indicating that tTG may mediate the drug resistance to adriamycin in MCF-7 by regulating the expression of P-gp,MRP,LRP.(3)In the silencing TGM2 combined with adriamycin group,cell growth inhibition rate and apoptosis rate was the highest.It is concluded that gene silencing TGM2 combined with adriamycin could inhibit cell growth,promote apoptosis,and improve chemotherapy sensitivity to adriamycin in MCF-7/ADR.Innovation:Drug-resistant protein is one of the main mechanisms of tumor resistance In this part,from the point of drug-resistant protein,the research of tTG on mediating drug resistance in breast cancer by regulating P-gp,MRP,LRP was performed.After reviewing the literature and retrieval,it was found that there was individual research on "tTG and P-gp" in the field of cancer.however,it has not yet been found the relevant research on tTG and MRP,LRPPart 3 The research of silencing TGM2 on inhibiting the growth of MCF-7/ADR xenograft in nude miceObjective:From the animal level,further explore the influence of silencing TGM2 on the MCF-7/ADR xenograft in nude mice.Methods:(1)The MCF-7/ADR cells was injected into the fat pad of the axilla of nude mice to build an animal model of xenograft in nude mice;(2)According to whether to inject gene-silencing TGM2 vector and adriamycin,the nude mice were divided into five groups:Blank control group,ADR group,ADR+tTG siRNA group,ADR+adriamycin group,ADR+tTG siRNA+ adriamycin group.According to the divided group,gene-silencing vector and adriamycin were injected;(3)After 28 days,all mice were killed.All tumors were weighed,and immunohistochemistry was used to detect the expression of tTG,P-gp,MRP,LRP.Results:(1)The effect of tumor growth inhibition in the ADR+tTG siRNA group was more obvious compared with the ADR group(p<0.05).Compared to ADR+adriamycin group,the tumor growth inhibition was significant in MDR+tTG siRNA+adriamycin group(p<0.05),of which the tumor growth inhibition rate was the highest.(2)The expression of tTG,P-gp,MRP,LRP was decreased in the ADR+tTG siRNA group.And the expression of tTG,P-gp,MRP and LRP was significantly decreased in the ADR+tTG siRNA+adriamycin group compared with ADR+adriamycin group.Conclusion:Silencing TGM2 combined with adriamycin could significantly inhibit tumor growth,and its mechanism may be related to the expression of P-gp,MRP and LRP decreased,being consistent with the results in vitro experiments.Innovation:This part further explored the role of gene silencing TGM2 in inhibiting the growth of xenograft in nude mice of MCF-7/ADR from animal levels.Part 4 The research on tTG transferred by exosomes extracted from MCF-7/ADR mediating the drug resistance of MCF-7 to adriamycinObjective:To explore that tTG transferred by exosomes extracted from MCF-7/ADR mediate drug resistance of MCF-7 to adriamycinMethods:(1)The low temperature gradient centrifugation was used to extract and purify exosomes.(2)Observe the size and morphology of exosomes by transmission electron microscope.(3)The expression of exosome-specific markers CD63,TSG 101 and the negative control Calnexin as well as the expression of tTG in MCF-7,MCF-7/ADR,MCF-7-EXO/ADR was detected through western-blot.(4)The absorption of EXO/ADR labeled by PKH67 was observed by inverted fluorescence microscopy.(5)The expression of tTG was detected in EXO/S,EXO/ADR by flow cytometer;(6)the IC50 of MCF-7,MCF-7/ADR,MCF-7+EXO/ADR was detected by CCK8.Results:(1)The exosome was observed in a typical circular or cup-shaped form,with the exosome diameter between 30-150nm by transmission electron microscope.(2)The identification of exosome:the western-blot results showed that exosome-specific markers CD63,TSG101 expressed,and the negative control Calnexin did not expressed.(3)tTG expression in EXO/S,EXO/ADR:the flow cytometry results show that tTG was both included in EXO/S,EXO/ADR,and the expression of tTG in EXO/ADR increased significantly compared to EXO/S(p<0.05);(4)It is observed that the exosomes marked PKH67 was absorbed by MCF-7 through an inverted fluorescent microscope.(5)The western-blot results show that the expression in MCF-7+EXO/ADR increased compared to MCF-7.(8)The CCK8 results show that The IC50 of mcF-7+ exO/ADR is 6.16± 1.15,the IC50 of MCF-7 is 1.91 ±0.18,and the IC50 of MCF-7 cultured with EXO/ADR together is 3.23 times that of MCF-7.Conclusions:(1)The exosomes were secreted both by MCF-7 and MCF-7/ADR.(2)tTG expressed in both exosomes extracted by MCF-7 and MCF-7/ADR,and compare to EXO/S,the expression of tTG was increased in EXO/ADR.(3)The exosomes from MCF-7/ADR transferred tTG,and they can be absorbed by MCF-7.(4)tTG transferred by exosomes from MCF-7/ADR could mediate the drug resistance of MCF-7 to adriamycin.Innovation:After reviewing the literature and retrieval,there is no relevant research on tTG transferrd by exosomes extracted from MCF-7/ADR mediating the drug resistance of MCF-7 to adriamycin.In this part,it is firstly found that tTG transferrd by exosomes extracted from MCF-7/ADR mediating the drug resistance of MCF-7 to adriamycin.