Effect Of Transglutaminase Type 2 On Proliferation And Invasion/metastasis Of Breast Cancer

Breast cancer is the most common cancer in women globally and is one of the principal causes of death among women.This poor outcome is associated to the fact that most patients are present with locally advanced or metastatic disease.Hence,identification of valid and specific biomarkers for prediction of proliferation and invasion/metastasis in breast cancer and development of novel treatment approaches are imminently needed.Uncontrolled proliferation is a vital marker of malignant cancer,as high proliferative activity of cancer cells is the basis and precondition of cancer invasion and metastasis.Epithelial-to-mesenchymal transition(EMT),a process in which epithelial cells show changes in morphology,lose their epithelial characteristics,and acquire their mesenchymal phenotype and the potential of high motility,is thought to be a critical event during tumor invasion and metastasis.TGM2(gene:TGM2:protein:TG2)is a multifunctional gene which encodes a stress-responsive,structurally complex and functionally sophisticated protein known as tissue transglutaminase(TG2 or tTG).TG2 plays an essential role in the control of a variety of proteins involved in survival,proliferation,invasion,angiogenesis,and metastasis of tumor cells.Silencing of TGM2 in the xenografts significantly inhibited pancreatic cancer cell proliferation and growth of TG2-positive tumors.TG2 induces epithelial-to-mesenchymal transition(EMT)and promotes ovarian cancer cell invasion and metastasis.The focus of the present study is to explore the effects of TG2 on cell proliferation and invasion/metastasis in breast cancer cells.Part ⅠConstruction and Identification of the Lentiviral Vector of Human TGM2 GenePurpose To construct a lentiviral vector for RNAi(RNA interference)of human TGM2 gene;and to investigate the effect of gene silencing of TGM2 in breast cancer cell line MDA-MB-231.Methods GV115(hU6-MCS-CMV-EGFP)-TGM2 expression plasmid was constructed by DNA recombinant method.Double-stranded oligonucleotides(dsOligo)were subsequently confirmed by DNA sequencing analysis.GV115-TGM2 was constructed along with pHelper 1.0 and pHelper 2.0 into 293T to package lentiviral particles.According to the EGFP expression,the functional titer was determined after transduction into 293T cells.The silencing effect of TGM2-RNAi-LV in MDA-MB-231 cells was assessed by Real-Time PCR assay.Results An effective TGM2-RNAi-LV was constructed.After cotransfection,lentiviral vector can be packaged in 293 T cell and the titer of lentivirus was detected.The expression of TGM2 mRNA was down-regluated in MDA-MB-231 cells infected with TGM2-RNAi-LV.Conclusions Lentiviral vector of human TGM2 gene has been successfully constructed,and an effective TGM2-RNAi-LV could suppress the expression of TGM2 gene in MDA-MB-231 cells in vitro,which provides a tool for investigating the role of TGM2 gene in proliferation and invasion/metastasis of breast cancer.Part ⅡKnockdown of TGM2 Suppressed The Proliferation And Invasion/Metastasis of MDA-MB-231 Cells In VitroPurpose To determine the effect of TG2 on proliferation,invasion and migration of MDA-MB-231 cells.Methods The proliferation of MDA-MB-231 cells was determined by MTT assay and colony formation assay.Apoptosis rate and cell cycle of MDA-MB-231 cells was determined by flow cytometry assay.The migration ability of MDA-MJ3-231 cells was examined by means of a scratch assay,and transwell assay was performed to examine the invasion ability of MDA-MB-231 cells.Results The lentiviral vector containing the shRNA-TGM2-expressing cassette achieved the reduction of mRNA-TGM2 expression in MDA-MB-231 cells.Downregulation of TG2 promoted apoptosis and suppressed the cell viability,proliferation,invasion and migration abilities in MDA-MB-231 cells transfected with shRNA-TGM2.TG2 facilitated tumor growth and proliferation and migration of MDA-MB-231 cells.Compared with control groups,knockdown of TGM2 expression inhibited the expression of proliferation-related and EMT-related molecules such as p-AKT,Bcl-2,cyclinD1,Vimentin and Snail,while the expression of epithelial marker E-cadherin was elevated in MDA-MB-231 cells.Further analysis revealed that shRNA-TGM2 could induce apoptosis and G0/G1 phase arrest which mediated by down-regulation of cyclinD1 in MDA-MB-231 cells.Conclusions Knockdown of TGM2 expression might induce apoptosis and inhibit the cell viability,proliferation,invasion and migration abilities in MDA-MB-231 cells.TG2 might promote cell proliferation,invasiveness and migration ability in MDA-MB-231 cells.Part Ⅲ Suppression Of Proliferation And Invasion/Metastasis By shRNA-TGM2 In Nude Mice.Purpose To observe the shRNA-TGM2 inhibition on breast cancer growth and to further demonstrate suppression of proliferation and invasion/metastasis genes by shRNA-TGM2 in breast carcinoma model of nude mice.Methods The animal model of human breast cancer was set up by subcutaneous injection to explore the effect of TG2 on cell proliferation and invasion/metastasis.Breast cancer xenograft model was established in nude mice with the MDA-MB-231 cell line.Breast tumors were measured bidimensionally with calipers to observe the change of tumor volume.Tumor volume was calculated as L × W2/2,where L is length and W is width.Mice were sacrificed six weeks after tumor implantation and tumor tissues were collected and weighted.The percentage of tumor inhibition was calculated according to the formula[1-(R/N)]× 100 where R and N represent the mean tumor volumes of RNAi group and NC group,respectively.In implanted tumor,the expression levels of TG2,Ki67,MMP-9 and MVD(CD34)were detected by immunohistochemical technique.Results TGM2-shRNA markedly suppressed the growth of orthotopic breast cancer xenografts by approximately 36.9%,when compared with NC group(P<0.05).And the tumor weight was lighter than that of NC group(P<0.05).TGM2-shRNA down-regulated the expression of Ki67,MMP-9 and MVD(CD34).Silencing TGM2 with shRNA could decrease tumor angiogenesis in female nude mice.Conclusions Silencing TGM2 could downregulate levels of Ki67,MMP-9 and MVD(CD34).TGM2 knockdown reduced tumorigenesis and inhibited proliferation and invasion/metastasis of MDA-MB-231 cells in the human breast carcinoma xenograft model.Part ⅣTG2 Expression and its Correlation with Clinicopathological Factors in Breast Cancer PatientsPurpose To detect the expression of TG2 and its correlation with clinicopathological parameters in breast cancer patients.Methods The expression levels of TG2,Ki67 and MMP-9 were detected by immunochemistry staining method,and their relationship with clinicopathological variables were analyzed in 107 evaluable specimens for pathology examination.We searched microarray dataset for TGM2 as a differentially expressed gene in breast cancer.TGM2 cRNA were identified in the analysis of GSE8977 in ONCOMINE database.Results We found that TG2 was upregulated in breast cancerous tissue compared with pericarcinomatous tissue.Bioinformatics analysis revealed that TGM2 cRNA was over-expressioned in breast cancer(p<0.001)..Between the TG2 expressions,we found no significant differences in the distribution according to age,histological grade or menopause.However,we did observe significant correlations of TG2 with tumor size(χ2=4.82,P=0.028),lymph node metastasis(χ2=12.38,P<0.001)and TNM stage(χ2=11.259,P=0.001).Spearman correlation analysis showed that TG2 was correlated with Ki-67(r=0.34,p<0.001)and MMP-9(r=0.36,p<0.001).Conclusions TG2 might serve an important role in the regulation of proliferation,invasion and migration of breast cancer and act as a novel biomarker for clinical diagnosis and prognosis in patients with breast cancer.