The Role Of TC-1(c8orf4) In Cell Proliferation, Migration And Invasion Of Non Small Cell Lung Cancer

AimsLung cancer is the leading cancer-related cause of morbidity and mortality worldwide.Cancer metastasis accounts for the lower survival of patients with lung cancer. TC-1, alsonamed as c8orf4, was found to be overexpressed in various solid tumors compared with itssurrounding normal tissues, and to be correlated with the tumour metastasis either.However, the relevance of TC-1in lung cancer has not yet been elucidated, and the role ofTC-1in lung cancer is still unclear. The aim of this study was to explore the role of TC-1in the proliferation, migration and invasion of non small cell lung cancer (NSCLC), and itspossible mechanism, which may provide a new target and open up a novel way for theprevention and treatment of lung cancer metastasis.Methods1. Smaples of147patients with NSCLC from cancer tissues and adjacent normal tissues, and clinical findings were collected. The expressions of TC-1in NSCLC andnornal lung tissues were detected by using immunohistochemical staining. The experssionof TC-1in NSCLC relatied with age, gender, histological type, TNM staging and regionallymph node metastasis was analyzed.2. Lentiviral expression constructs containing the TC-1gene (pLenti-TC-1), TC-1siRNA sequences (pLenti-TC-1si) and control empty vector (pLenti-E) were constructedand transfected into A549cells. Stable clones were isolated using limiting dilutionanalysis. A549-TC-1(+) cell clone with TC-1over-expression and A549-TC-1(-) withTC-1lower expression and empty vector (pLenti-E) transfected A549cells as control wereobtained respectively. MTT cell proliferation analyze, wound healing test and matrigelcoated invasion transwell test were performed to test the influence of TC-1on A549cells.3. To study the possible mechanism of TC-1in the proliferation, migration andinvasion in NSCLC, the expression of Wnt/β-catenin pathway geneβ-catenin anddownstream target genes VEGF, Cyclin D1, MMP-7, c-Myc, survivin on both mRNA andprotein levels were detected by using realtime PCR and western blot on A549-TC-1(+),A549-TC-1(-) and A549-control cell lines.Results1. TC-1protein exhibited and located in the cytoplasm of positive cells diffusely byimmunostaining with a rate of65.99％(97/147）in NSCLC. There was no significantdifference between gender, age and histological subtypes for the expression of TC-1(P＞0.05). However, the expresssion of TC-1was significantly correlated with TNM stagingand regional lymph node metastasis(P＜0.01). The rates of TC-1expression of NSCLCwere58.00%（29/50）in stage Ia/b,63.16%(24/38) in stage IIa/b and74.58%(44/59) instage IIIa/b; and were59.38%(38/64) in N0,60.60%(20/33）in N1and78.00%(39/50) inN2. These results indicated that the expression of TC-1in NSCLC was closely relatedwith TNM staging and regional lymph node metastasis,but not with gender, age andhistological subtypes.2. Lentiviral expression of pLenti-TC-1，pLenti-TC-1si，pLenti-E were constructed and transfected into A549cells. A stable clones of over-expression and silencing TC-1A549cell clones named as A549-TC-1(+) and A549-TC-1(-)were obtained respectively.And the A549cells were transfected with pLenti-E as control. A549-TC-1(+)demonstrated the highest expression of TC-1, on the contrary, A549-TC-1(-) showed thelowest expression of TC-1and the A549-control did as same as normal. In order to ensurereliability and repeatability, these three clones were used in following exprements. Effectof the cell proliferation, migration and invasion were tested by using MTT, Wound scratchassay and Matrigel coated invasion transwell test respectively. The results showed thatA549cell proliferation with overexpressed of TC-1greatly was improved, and on thecontrary, the cell proliferation was greatly reduced in TC-1inhibited A549cells (P＜0.05).Wound healing test demonstrated that A549-TC-1(+) migrated quickly than A549-control,and A549-TC-1(-) has the slowest migratory ability in the three groups. Matrigel coatedinvasion transwell test shown that migrated A549-TC-1(+) cells was significantly morethan the number of A549-control, and A549-TC-1(-) has the lowest invasion ability (P＜0.05). Our data demonstrated that TC-1positively regulate A549lung cancer cellsproliferation, invasion and migration behaviors.3. Realtime PCR and western blot on A549-TC-1(+), A549-TC-1(-) and A549-controlcell of Wnt/β-catenin pathway geneβ-catenin and downstream target genes VEGF,Cyclin D1, MMP-7, c-Myc, survivin shown that: compared with the control groupA549-TC-1(+) shown increase in β-catenin, VEGF, Cyclin D1, MMP-7, c-Myc andsurvivin mRNA and protein expression respectively. On the contrary, A549-TC-1(-)caused in reduction in β-catenin, VEGF, Cyclin D1, MMP-7, c-Myc and survivin mRNAand protein expression respectively. That means TC-1can regulate Wnt/β-cateninpathway.Conclusions1. In NSCLC tissures, the expression of TC-1was upregulated. The expression ofTC-1was significantly associated with TNM staging and regional lymoh nodes metastasis,but not with age, gender and histological subtyoes. 2. The TC-1over expressed, less expressed and control A549cell line wassuccessfully cloned. MTT cell proliferation analyze, Wound healing test and Matrigelcoated invasion transwell test demonstrated that TC-1enhance A549lung cancer cellsproliferation, invasion and migration behaviors.3. In A549lung cancer cells, overexpression of TC-1could promote the express of β-catenin, VEGF, Cyclin D1, MMP-7, c-Myc and survivin at mRNA and protein levels. Onthe contrary, TC-1less expressed A549shown lower express of β-catenin, VEGF, CyclinD1, MMP-7, c-Myc and survivin. These experiments indicated that TC-1may regulate theWnt/β-catenin signaling pathway.In this study, the expression of TC-1in lung cancer tissures and the role of TC-1in theproliferation, migration and invasion of A549lung cancer cells were systematicallyinvastigated. We also inestgate the relationship between TC-1and Wnt/β-cateninsignaling pathway. It was proved that TC-1was significantly associated with TNM stagingand regional lymoh nodes metastasis in NSCLC, could promote lung cancer cellsproliferation, invasion and migration as TC-1act as a upstream regulator of Wnt/β-catenin signaling pathway, all of which provided a new target and opened up a new wayfor the prevention and treatment of lung cancer metastasis.