DRAM1 Regulates EMT Of Lung Cancer A549 Cells And Its Mechanism Study

Backgroud: Lung cancer is the most common cause of cancer-related morbidity and mortality worldwide. The invasion and metastasis of lung cancer is the major cause of patients’ treatment failure. How to suppress the invasion and metastasis of lung cancer is still an urgent challenge in lung cancer therapy. Many studies showed that epithelial mesenchymal transition(EMT) occurs frequently in epithelium originated tumors, which characterizes with lower level or missing of E-adherin and β-catenin, the hallmark of epithelium, and obtaining of mesenchymal phenotype, such as higher expression of vimentin, results in loss of cell adhesion and enhancement of invasion ability. Therefore, the EMT signaling pathways and its molecular mechanism involved in lung cancer need to be further explored to discover the potential novel therapeutic strategy.Autophagy is an evolutionary conservative to maintain the cellular homeostasis while cells under stress. It is a multistep process, including wrapped the damaged organelles or protein, transferred to the lysosome for degradation and biosynthesis, and organelles updates. Increasing evidence firmly demonstrates that autophagy functions are associated with several human diseases including cancer. Regulation of autophagy could mediate the cell proliferation, apoptosis, invasion and metastasis, which has become a hotspot in the cancer research field currently.Damage-Regulated Autophagy Modulator(DRAM) is a highly evolutionary conservative gene, has been showed that it could induce autophagic lysosome accumulation, resulting in autophagy activation in several studies. There are five members in the DRAM family, and DRAM1 was the most researched among them. Previous data showed that DRAM1 is a downstream target of p53, inducing autophagy and p53-dependent apoptosis, also was found declined level of DRAM1 in many malignancies, including lung cancer. Current studies showed that autophagy related genes p62 / SQSTM1 could regulate EMT in the tumor cells. In addition, TGF-beta 1 initiates EMT in tumor cells, affects the cellular autophagy flux simultaneously, suggesting that autophagy may relate with EMT somehow. As an autophagy regulation gene DRAM1, whether could mediate EMT in lung cancer cells through regulating p62 / SQSTM1, which remains unknown.In this study, DRAM1 is employed to investigate the autophagy and EMT in lung cancer cells A549, and in TGF-beta 1 inducing EMT as well for further unveiling the potential mechanism involved in autophagy and EMT, to provide some new ideas and potential therapeutic targets on prevention and cure of lung cancer.Objectives:1. To clarify the role of DRAM in EMT of A549 cells2. To explore the involved mechanism of DRAM regulating EMT in A549 cellsMethods:1. The expression of DRAM1 was detected by real-time PCR when the A549 cells was treated with 1ng/mL, 2ng/mL, 5ng/mL and 10ng/mL TGF-β1 for 24 h and with 5ng/m L TGF-β1 for 12 h, 24 h and 48 h, respectively.2. pEGFP-N1-DRAM1 和 pEGFP-N1 was isolated and transfected into A549 cells for 48 h, and then the transfection efficiency was detected by immunofluorescent microscope, and the expression of DRAM1, E-cadherin, Vimentin, Atg5, p62 and Twist was evaluated by real-time PCR and western blot, respectively.3. The migration of A549 cells were measured by scratch-wound healing test and transwell migration assay.4. The mRNA and protein expression of Atg5, p62 and Twist were detected by real-time PCR and western blot when cells were treated by 5ng/mL TGF-β1 for 24 h.5. A549 cells was tranfected with DRAM1 siRNA and NC, and then the mRNA and protein expression of Atg5, p62 and Twist were ananlyzed by real-time PCR and western blot upon 5ng/mL TGF-β1 exposure for 24 h.Results:1. Compared to the control group, the DRAM1 was significant higher expressed after 1ng/mL, 2ng/mL, 5ng/mL and 10ng/mL TGF-β1 for 24 h treatment(p <0.05), and the peak of DRAM1 expression was at 5ng/mL TGF-β1 group. There was no difference of DRAM between 5ng/mL and 10ng/mL TGF-β1 group(p >0.05). Additionally, the DRAM1 showed a time-dependent manner of increase with 5ng/mL TGF-β1 treatment, compared with the control group(p <0.05).2. The plasmid DNA of pEGFP-N1(vector) and pEGFP- N1-DRAM had been isolated and transfected into A549 cells. The expression of DRAM1, Vimentin, Atg5, p62, Twist and LC3 B II/I were much higher than those in the control and the vector group(p <0.05), respectively. On the contrary, the E-cadherin level was decreased in DRAM1 transfected cells compared with the control and the vector group(p <0.05).3. The migration ability of A549 cells was significant enhanced in pEGFP-N1-DRAM1 transfected cells, compared to the control and the vector.4. The expression of Twist, p62 and vimentin were higher in TGF-β1+NC and TGF-β1+DRAM1 siRNA than those in the control group(p <0.05) respectively, meanwhile the results in TGF-β1+NC group showed elevated compared with that in the control group(p <0.05). However, E-cadherin showed lower expression in TGF-β1+NC and TGF-β1+DRAM1 siRNA than that in the control group(p <0.05), also lower E-cadherin in TGF-β1+NC comparing with the control group(p <0.05)Conclusions:1. TGF-β1 elevated the expression of DRAM1, also induced DRAM1 related autophagy and promoted the EMT in A549 cells.2. Up-regualtion of DRAM1 promoted the EMT and migration abilities of A549 cells, while knock-down of DRAM1 could inhibit the EMT process in cells.3. DRAM1 might regulate the EMT process of A549 cells via p62-twist pathway.