Mechanism Study Of Long Noncoding RNA ENST00000434223 Suppressing Tumor Progression In Non-small Cell Lung Cancer

Background and AimsNon-small cell lung cancer(NSCLC), including adenocarcinoma and squamous cell carcinoma, is the most common type of cancer and a leading cause of cancer-related death worldwide. Most NSCLC patients are diagnosed at the advanced stages as they are usually asymptomatic at early stages. Despite the fact that great advances have been made in surgical techniques and chemoradiation therapy, the prognosis of lung cancer remains to be dismal, the 5-year survival rate is about 15 %. Therefore, it is important to reveal the molecular mechanisms of the progression of NSCLC.One recent study described the expression profile of lnc RNAs in human lung adenocarcinoma at an early stage and the corresponding adjacent non-tumorous tissues(NT) by microarray. From the microarray analysis, we found that ENST00000 434223 was significantly down regulated in cancerous tissues. However, the role of ENST00000434223 and the underlying molecular mechanism in the development of NSCLC remains to be unexplored. This research is divided into three parts, to reveal the correlation between ENST00000 434223 expression level and clinic pathologic characteristics of the NSCLC patients and the mechanism by which it regulates NSCLC progression, to investigate the regulation of ENST00000434223 to the proliferation, metastasis and invasion of NSCLC cells using the technologies of over expression and si RNA knockdown, in addition, to check the effect of ENST00000434223 on cell cycle, apoptosis and epithelial-mesenchymal transition(EMT), and to explore another new target for the early diagnosis and treatment of patients with NSCLC.Part One: The investigation of lnc RNA ENST00000434223 expression spectrum and the correlation between the expression level and clinic pathological characteristics Objective:To determine whether ENST00000434223 is down regulated in NSCLC cell lines and 56 pairs of NSCLC tissues and matched adjacent noncancerous tissues, and to explore the correlation between ENST00000434223 expression level and clinic pathological factors and determine the possibility of ENST00000434223 as a new target for the early diagnosis and treatment of patients with NSCLC. Methods:To find out whether ENST00000434223 is downregulated in non-small cell lung cancer, we first determined its expression level in NSCLC cell lines including adenocarcinoma and squamous carcinoma subtypes, and 56 pairs of NSCLC tissues and matched adjacent noncancerous tissues utilizing Real time-PCR,we also explored the correlation between ENST00000434223 expression level and clinic pathological factors to determine the possibility of ENST00000434223 as a new target for the early diagnosis and treatment of patients with NSCLC. Results:When normalized to normal bronchial epithelial cell line(16HBE), the expression levels of ENST00000434223 were down regulated in A549, SKMES-1, SPC-A1, and NCI-H1975 cell lines,and A549 cells express the highest level of ENST00000434223, while NCI-H1975 cells have the lowest ENST00000434223 expression level.The expression levels of ENST00000434223 were significantly down regulated in cancerous tissues compared with paired adjacent normal tissues(p< 0.01). Low ENST00000434223 expression level was correlated with tumor diameter(p<0.05),lymph node metastasis(p <0.05), and advanced TNM stage(p < 0.05). However, ENST00000434223 expression level was not correlated with other parameters, such as age, gender, and tumor differentiation. Conclusions:The expression levels of ENST00000434223 were down regulated in NSCLC cell lines. ENST00000434223 expression levels were also significantly down regulated in cancerous tissues compared with paired adjacent normal tissues and low ENST00000434223 expression level was correlated with tumor diameter, lymph node metastasis, and advanced TNM stage, but was not correlated with other parameters, such as age, gender, and tumor differentiation.Part Two: Mechanism investigation of lnc RNA ENST00000434223 inhibiting proliferation and growth of NSCLC cells Objective:To explore the role of the long non-coding RNA ENST00000434223 on the proliferation and growth of NSCLC cells in vitro and in vivo and explore the underlying mechanism by which lnc RNA ENST00000434223 affects the proliferation and growth of NSCLC cells. Methods:As illustrated in the last part of study, ENST00000434223 was expressed at the lowest level in NCI-H1975 cells. In order to explore the role of ENST00000434223 in NSCLC development, we selected H1975 cells for ENST00000434223 over expression and A549 for ENST00000434223 knockdown. Forty-eight hours after transfection, the expression level of ENST00000434223 was detected with Real time-PCR analysis. We demonstrated the cell viability of H1975 cells with ectopic expression of ENST00000434223 and A549 cells with ENST00000434223 knockdown utilizing cell-counting kit-8 assays. In addition, we also determined the PCNA protein level of the H1975 cells with ectopic expression of ENST00000434 223 and the A549 cells after transfection with si-ENST00000434223 by immunostaining. Then, we examined whether over expression of ENST00000434223 would have an effect of ENST00000434223 over expression in H1975 cells and ENST00000434223 knockdown in A549 cells by flow cytometric assay. To investigate the underlying molecular mechanism of the effect of ENST00000434223 on cell cycle regulation, we determined the G0/G1 arrest marker p21 with immunostaining analysis in the above two cell lines. Flow cytometric analysis was also performed to examine whether the anti-proliferative effects of ENST00000434223 could possibly be through inducing cancer cell apoptosis. To validate the effect of ENST00000434223 on NSCLC cell tumorigenesis in vivo, H1975-pc DNA3.1- ENST00000434223 or H1975-pc DNA3.1- control cells were injected into flanks of nude mice. Palpable tumors formed within 3 days. Tumor volume was measured on a weekly basis. Furthermore, immunostaining was used to check the positive rate of Ki67 of the tumors from the pc DNA3.1-ENST00000434223 group. Results:Cell viability was decreased in H1975 cells with ectopic expression of ENST00000434223, while cell viability was increased in A549 cells with ENST00000434223 knock down. Consistent with the reduced cell viability, H1975 cells had obviously decreased protein levels of proliferating cell nuclear antigen(PCNA) compared to control cells with ectopic expression of ENST00000434223. On the other hand, the expression of PCNA was increased in A549 cells after transfected with si-ENST00000434223. These data indicate that ENST00000434223 plays a role in the regulation of cell proliferation.ENST00000434223 over expression induced a significant increase in the percentage of cells in G0/G1 phase as determined by flow cytometric assay. Consistently, the percentage of cells at G0/G1 phase was decreased in si-ENST00000434223 transfected A549 cells. These data suggest that ectopic expression of ENST00000434223 may inhibit cell proliferation through inducing G0/G1 arrest. The results of western blot analysis revealed that the G0/G1 arrest marker p21 protein levels were increased with ENST00000434223 over expression. In the meantime, the protein level of p21 was decreased with ENST00000434223 knockdown in A549 cells.ENST00000434223 over expression induced cell apoptosis in H1975 cells. These results demonstrated that ENST00000434223 may inhibit the proliferation of NSCLC cancer cells through inducing cell G0/G1 arrest and apoptosis.Consistent with the previous in vitro results, tumor growth in the pc DNA3.1-ENST00000434223 group was significantly decreased relative to the pc DNA3.1-control group. Furthermore, immunostaining revealed that tumors from the pc DNA3.1-ENST00000434223 group exhibited a decreased positive rate of Ki67. Taken together, our data demonstrate that ENST00000434223 suppresses NSCLC tumor growth both in vitro and in vivo. Conclusions:ENST00000434223 inhibits proliferation of NSCLC cells in vitro. ENST00000434223 decreased protein levels of proliferating cell nuclear antigen(PCNA). ENST00000434223 induces G0/G1 arrest of NSCLC cells. ENST00000434 223 increases G0/G1 protein levels of arrest marker p21. ENST00000434223 induces apoptosis of NSCLC cells. ENST00000434223 inhibits growth of NSCLC cells in vivo and the positive rate of Ki67 of the tumor. Taken together, these results demonstrate that ENST00000434223 suppresses NSCLC tumor growth both in vitro and in vivo by inducing G0/G1 arrest and apoptosis. Part Three: Mechanism investigation of lnc RNA ENST00000434223 inhibiting invasion of NSCLC cells Objective:To determine the function of the long non-coding RNA ENST00000434223 on the invasion of NSCLC cells and explore the underlying mechanism by which lnc RNA ENST00000434223 affects the invasion of NSCLC cells. Methods:To investigate the effect of ENST00000434223 on cell migration, we over expressed long non-coding RNA ENST00000434223 in H1975 cells and knock down ENST00000434223 in A549 cells, and then determined the function of the lnc RNA ENST00000434223 on the invasion of NSCLC cells by the transwell assays. Epithelial-mesenchymal-transition(EMT) has been demonstrated to promote cancer invasion. We then explored whether ectopic expression of ENST00000434223 could reverse EMT in NSCLC cell lines by checking the protein levels of EMT E-cadherin, N-cadherin and vimentin by immunofluorescence analysis and immunostaining. Results:We performed cell invasion assay to determine the effect of ENST00000434223 on cell migration and found that ectopic expression of ENST00000434223 significantly decreased cancer cell migratory capacity in H1975 cells, while ENST00000434223 knockdown promoted tumor cell migration in A549 cells.We then explored whether ectopic expression of ENST00000434223 could reverse EMT to inhibit invasion in NSCLC cell lines by immunofluorescence analysis and immunostaining analysis. Immunofluorescence results demonstrated that ENST00000434223- overexpressing cells acquired a more rounded, epithelial morphology. ENST00000434223 over expressing resulted in a marked increase in the E-cadherin protein level and a significantly decrease in expression of N-cadherin and vimentin. Moreover, immunostaining analysis confirmed the results of immunofluorescence analysis. Conclusions:ENST00000434223 inhibits invasion of NSCLC cells. ENST00000434223 could reverse EMT in NSCLC cell lines. Taken together, long non-coding RNA ENST00000434223 inhibits invasion of NSCLC cells by reducing EMT.