Defective Expression Of TGFBR3 Gene And Its Molecular Mechanisms In Non-small Cell Lung Cancer Cell Lines

Background and Objective: Lung cancer is one of malignant tumors, which is characterized by highly morbidity and mortality worldwide. The two major forms of lung cancer are non–small cell lung cancer (NSCLC, accounting for about 85% of all lung cancers) and small-cell lung cancer (SCLC, accounting for about 15%). Despite advances in early detection and standard treatment, NSCLC is often diagnosed at an advanced stage and has a poor prognosis. Better understanding for the molecular variation mechanisms and evolution of the disease is preferentially important. Transforming growth factor beta (TGF-β) signalling system is postulated to play an important role in lung carcinogenesis. Alterations in key components of TGF-βsuperfamily signaling pathways, including mutation or deletion of receptors and other signaling components, are frequent molecular events in human cancers, resulting in loss of function of tumor suppressor in these pathways. However, no mutation was commonly detected in NSCLC. On the contrary, TGF-βsuperfamily co-receptor, transforming growth factor beta receptor III (TGFBR3) (or betaglycan) is lost at the mRNA and protein levels in majority of NSCLC specimens. However, its molecular mechanisms remain unclear. In the present study, we investigate the expression of TGFBR3 in six NSCLC cell lines and tissues. Expression of TGFBR3 in Human Bronchial Epithelial Cell (HBEpiC) was used as a positive control. Then we explore potential molecular mechanisms underlying inactivation of TGFBR3 gene.Methods: Western blot was performed to determine the expression of TGFBR3 in cell lines and NSCLC tissues. Automatic image analysis was carried out to estimate relatively expression of TGFBR3 protein in cell lines. We screened for mutation of the promoter region of TGFBR3 gene in all seven cell lines using DNA direct sequencing. Bisulfite-sodium modification sequencing was used to detect the methylation status of TGFBR3 promoter in seven cell lines. Using methylation-specific PCR (MSP) method, we examined methylation status for TGFBR gene in 36 NSCLC tissues and paired normal tissues. Results: TGFBR3 protein level was abnormally reduced in NSCLC cell lines as compared with HBEpiC (P<0.001). There was significant difference in TGFBR3 expression between the highly metastatic cell line 95D and non-metastatic cell lines, including LTEP-α-2, A549 and NCI-H460 (P= 0.003; 0.017; 0.011). The expression of TGFBR3 is clearly down–regulated in NSCLC tissues compared with the control lung tissues. MethylPrimer software analysis showed that in the TGFBR3 gene proximal promoter region upstream sites -165 to -75 there were 13 CpG island sites which located in -82, -84, -88, -94, -103, -118, -120, -122, -134, -142, -145, -154 and -164. As for distal promoter,we detected 8 CpG sites located in -292, -290, -286, -277, -270, -218, -214 and -202. No mutation and methylation was found in upstream sites -165 to -75 of the proximal promoter of TGFBR3 in HBEpiC and NSCLC cell lines. Hypermethylation was shown in upstream sites -314 to -199 of the distal promoter of TGFBR3 in HBEpiC and NSCLC cell lines. No aberrant DNA methylation was found in TGFBR3 promoter of 36 NSCLC tissues.Conclusion: Reduced expression of TGFBR3 was observed in NSCLC cell lines and tiusses, especially in 95D cell line, suggesting that TGFBR3 might play a role in development and progression of NSCLC and correlate with NSCLC invasion and migration. The methylation event occurring at TGFBR3 promoter is not a major cause for reduction of TGFBR3 expression. Aberrant methylation of critical transcription elements in TGFBR3 promoter region may infrequently occur in NSCLC.