The Function And Mechanism Of FAL1 On The Malignant Progression Of Non-small Cell Lung Cancer

Backgrounds:Lung cancer is one of the most common malignancies in the world.Its high mortality seriously threatens human health.Non-small cell lung cancer(NSCLC)accounts for the majority of lung cancer.Although with the development of technology,the diagnosis and treatment of lung cancer developed rapidly,the overall prognosis of NSCLC is still not optimistic.On the one hand,because many patients with NSCLC have already on the advanced stage when they are diagnosised,they miss the optimal treatment time window;on the other hand,resistance to drugs and radiation therapy also limits the effectiveness of treatment.These are related to the current unclear pathogenesis and development mechanism of NSCLC.The lack of early diagnostic markers and prognostic indicators coupled with unclear mechanisms of resistance has limited the efficacy of NSCLC.Therefore,it is extremely necessary to explore the mechanism under the progression of NSCLC and find novel targets for diagnose and treatment of NSCLC.With the development of high-throughput sequencing technology and genomics research,it has been found that about 98% of genes in the human genome do not have protein-coding functions,calling non-coding RNAs(nc RNAs).Of these,nc RNAs longer than 200 nt are called long non-coding RNAs(lnc RNAs).The gene-encoding RNAs and non-coding regulatory roles of nc RNAs play important roles in the development of NSCLC.In recent years,the role of lnc RNAs as a diverse nc RNAs involved in the regulation of epigenetics,pre-transcriptional and post-transcriptional modifications has been gradually discovered.Lnc RNAs can also participate in a variety of biological processes including tumorigenesis,metastasis and drug resistance,and have the potential to become tumor diagnosis and prognostic markers.To date,the characteristics and functions of several lnc RNAs in NSCLC have been tentatively explained.For example,MALAT1 could promote the growth and metastasis of NSCLC and predicted poor prognosis;high expression of HOTAIR predicted short-term survival of NSCLC patients,and down-regulation of HOTAIR levels could inhibit NSCLC drug resistance;MEG3 was low expressed in NSCLC tissues and could inhibits the proliferation and invasion of NSCLC cells via p53.Lnc RNA FAL1(focally amplified lnc RNA on chromosome 1,FAL1)was first reported in ovarian cancer and had the effect on promoting the proliferation of ovarian cancer cells and inhibiting cell apoptosis.However,the function of FAL1 in NSCLC has not yet appeared.Therefore,this study focuses on the role and mechanism of FAL1 in the malignant development of NSCLC,and explores how it could regulate the proliferation,migration,invasion and drug resistance of NSCLC cells,whether it can be used as a marker for malignant progression and prognosis of NSCLC and be a new molecular target for NSCLC treatment.This study has a certain clinical significance for a better understanding of the progress of NSCLC,and it could also provide good theoretical basis for the diagnosis,treatment and prognosis of NSCLC.Objective:This study aims to explore the role and molecular mechanism of FAL1 in the malignant development of NSCLC such as proliferation and metastasis,and to explore the influence and mechanism of FAL1 in the formation of cisplatin resistance in LAC.Method:Part 1: The expression of FAL1 in 100 NSCLC tissues and adjacent para-tumor tissues was detected by real-time quantitative RT-PCR(q RT-PCR).The correlation between FAL1 and clinical pathological data was analyzed.A FAL1 knockdown H1299 cell line was constructed using small interfering RNA(si RNA)and a SPCA1 cell line stably overexpressing FAL1 was constructed using a lentiviral vector.The abilities of proliferation,cell cycle,apoptosis,migration,and invasion of well-established cells were detected by CCK8,clone formation,flow cytometry,wound-healing,and Transwell assays.Western blot was used to detect the changes of epithelial cell-mesenchymal transition(EMT)markers and FAL1 downstream target genes.PTEN levels were knocked down by si RNA and the reliability of PTEN as a target gene was verified by several functional assays.Finally,models of xenograft tumor and in vivo metastasis analysis were constructed to verify the effect of FAL1 on the malignant progression of NSCLC in vivo.Part 2: The expression of FAL1 in parental A549 cells and cisplatin-resistant A549/DDP cells was detected by q RT-PCR.A549/DDP cells with low expression of FAL1 and A549 cells with FAL1 overexpression were constructed.The IC50 and proliferation of cells were detected by CCK8 method.Clone formation and Edu experiments were designed to observe cell growth.Cell cycle and apoptosis were analyzed by flow cytometry.Western blot was used to verify the expression of BTG2 and Cyclin D1 in cells.In vivo,the effect of FAL1 on cisplatin resistance in LAC was analyzed by mouse tumor-bearing experiments.Result:Part 1:1)The expression of FAL1 in NSCLC tissues was significantly higher than that in paired para-tumor tissues,and its high expression predicted a poorer degree of tumor differentiation,larger tumor size,higher TNM stage,and lymph node metastasis in NSCLC patients.2)FAL1 knockdown could inhibit the proliferation of NSCLC cells and induce cell G0/G1 arrest and overexpression of FAL1 could promote cell proliferation and promote cell transformation from G0/G1 phase to S phase.However,there was no effect on apoptosis.3)FAL1 knockdown significantly inhibited cell migration and invasion,while FAL1 overexpression promoted cell migration and invasion.4)FAL1 knockdown significantly inhibited the EMT process of cells while overexpression of FAL1 promoted the process.5)FAL1 inhibited the expression of PTEN via stabilizing BMI1,thereby regulating downstream AKT phosphorylation to function as an oncogene.6)Knockdown of PTEN expression partially reversed the effect of FAL1 knockdown on inhibition of NSCLC cell proliferation,invasion and metastasis.7)Overexpression of FAL1 can promote the growth of tumors in mice and cause the down-regulation of PTEN expression.Meanwhile,FAL1 can also promote lung metastasis of NSCLC in mice.Part 2:1)FAL1 was highly expressed in cisplatin-resistant A549/DDP cells than in parental A549 cells.And FAL1 level increased with the increasing DDP concentration.2)FAL1 knockdown could increase the sensitivity of A549/DDP cells to DDP and inhibit the proliferation of A549/DDP.3)FAL1 knockdown induced G0/G1 arrest of A549/DDP cells and promoted DDP-induced A549/DDP cell apoptosis via a DDP concentration-dependent way.4)Overexpression of FAL1 could promote the transformation of parental A549 from G0/G1 phase to S phase,and also inhibit DDP-induced apoptosis.These effects also had a concentration-dependent effect.5)FAL1 could induce the down-regulation of BTG2 expression through BMI1,thus regulating the expression of Cyclin D1 to exert its effect.6)Overexpression of FAL1 could increase the resistance of A549 cells to cisplatin in vivo.Conclusion:In summary,this study elaborated the role of FAL1 in the malignant progression of NSCLC and its mechanism to some extent:(1)FAL1 showed high expression in NSCLC,and it could promote the proliferation,invasion,migration and EMT process of NSCLC cells.;(2)FAL1 inhibited the expression of PTEN through BMI1,and then affected the downstream AKT pathway;(3)FAL1 could increase the cisplatin resistance of LAC cells through BTG2.These studies revealed the role of FAL1 in the epigenetic regulation of NSCLC,providing new insights into the relationship between lnc RNAs in NSCLC and providing a new target for the diagnosis and treatment of NSCLC.