Long Non-coding RNA ZFAS1 Interacts With MiR-150 That Targets On ZEB1 And Regulates The Oncogenic And Metastatic Process Of Lung Cancer

Lung cancer is considered as the leading reason of cancer death worldwide that threatens human health.Long non-coding RNAs(lncRNAs)and micro RNAs(miRNAs)were identified as important biomarkers in the diagnosis and treatment of lung cancer.We determined expression of lncRNAs in patients with lung cancer by RNA-seq and identified lnc RNA ZFAS1 to be significantly up-regulated in lung cancer tissue.Further assays by RT-PCR presented that increased expression of lnc RNA ZFAS1 induced decreased level of miR-150 indicating lnc RNA ZFAS1 might inhibit miR-150 in expression.miR-150 was proven to target on ZEB1.LncRNA ZFAS1 was found high-expressed in patients with lung cancerRNA-seq was performed to screen the significant differential expressed lncRNAs of lung cancer.According to the results,lnc RNA ZFAS1 was targeted and further validated in 40 patients by RT-PCR assay.The results of RT-PCR were in correspondence with the results of RNA-seq.The expression level of lnc RNA ZFAS1 was 4-fold increased in the lung cancerous tissue compared with the adjacent normal tissue.The difference was statistical significant(P < 0.05).On the other hand,the expression of miR-150 was significantly decreased at 60% in the cancerous tissue compared with the adjacent normal tissue(P < 0.05).The results suggested that lnc RNA ZFAS1 might be taken part in the regulation during lung cancer pathogenic process.It is negatively correlated with miR-150 indicating the functions of lnc RNA ZFAS1 might be performed relying on miR-150.miR-150 targets on ZEB1 and regulates lung cancerous cellsSince ZEB1 was predicted to be the target of miR-150 by the complementary principle,we conducted luciferase assay to validate the interaction between miR-150 and ZEB1.The result was shown in the experimental report.The luciferase activity was detected obviously decreased in the wild-type vector that contains 3’UTR sequences of ZEB1.However,it was almost no change to the luciferase activity in the mutant-type vector.It suggested that the miR-150 mimics,normally raises the level of miR-150,subsequently induced inhibition to the expression of ZEB1.Therefore,miR-150 was proven to be inhibitor targeting on ZEB1.On the other hand,we conducted transfection of miR-150 mimics and inhibitors to NCI-H460 cells.According to the results,miR-150 mimics obviously induced increased amounts of miR-150 while miR-150 inhibitors decreased the level of miR-150.At this moment,the protein level of ZEB1 was determined by western blotting.The results suggested that the treatment of miR-150 mimics transfection led to decline of ZEB1 level at 30% compared with control group.However,when the cells were transfected with miR-150 inhibitors,the protein level of ZEB1 was increased almost 3-fold compared with the inhibitor negative control group.The cell viability was thereby evaluated by the CCK-8 assay.For both negative control groups of miR-150 mimic and inhibitor,it is almost no difference throughout 5 days.However,miR-150 mimic led to lower cell viability since 2nd day indicating the cell proliferation process was suppressed.Oppositely,the miR-150 inhibitor induced higher cell viability compared with the negative control group indicating that the cells could reproduce more robustly than the normal cells.Apart from cell viability,the cell apoptosis and cell cycle were determined by flow cytometry.By raising the level of miR-150 using miR-150 mimic,the apoptotic cells were obviously increased.Meanwhile,the cells in S phase were decreased significantly but most of the cells were retained at G1 and G2 phases.However,inhibiting the expression of miR-150 using miR-150 inhibitor led to opposite results that the cell apoptosis was obviously suppressed and cells were mostly retained at S phase indicating the induction of more robust cell proliferation compared with the cells in negative control group.Because of cancerous cells,capacity of robust cell proliferation can be expected to induce the potential cell invasion and cell migration.Therefore,we conducted transwell assay and wound healing assay to estimate of the capacity of cell invasion and cell migration.Corresponding to the hypothesis,the results proved that the invasive cells were greatly increased and cells migration was accelerated when it was inhibition to the expression of miR-150.In opposite,the invasive cells became fewer and the cells migrated slower as long as the expression of miR-150 increased.According to the results so far,miR-150 was proven to be one of the key factors in regulating the pathogenic process of lung cancer: it suppressed the cell proliferation and enhances cell apoptosis and thereby inhibits the cancerous cells from invasion and migration.ZEB1 as a target of miR-150 is important in regulating lung cancerous cellsSince miR-150 targets on ZEB1,we attempted to further validate the relationship between ZEB1 and the functions of miR-150 in regulating the NCI-H460 cells in various processes.Therefore,overexpression of miR-150 and/or ZEB1 was induced to NCI-H460.According to the results,the transfection was done successfully that the level of miR-150 and ZEB1 was increased when miR-150 mimic and lenti-ZEB1 were transfected.The overexpression of miR-150 obviously lowered down the level of ZEB1,but overexpression of both miR-150 and ZEB1 eventually induced higher expression of ZEB1 compared with the control group.Meanwhile,CCK-8 assay was performed to detect the cell viability in each group.Increased expression of miR-150 suppressed the cell viability,but the cell viability was increased when both miR-150 and ZEB1 were higher expressed compared with the control group.The results suggested that the ZEB1 should be a cytokine that enhances the cell viability and it is regulated by miR-150.Both cell apoptosis and cell cycle were further verified by flow cytometry.The results proved that miR-150 performs functions in inducing cell apoptosis and suppressing cell proliferation.When the level of miR-150 was increased,the amount of the apoptotic cells was increased and cells in S phase became fewer compared with the control group.However,when co-transfected miR-150 and lenti-ZEB1 to the cells that induced higher expression of both miR-150 and ZEB1,the apoptotic cells were dramatically decreased and more cells retained in S phase that is benefits for cell proliferation.Transwell assay and wound healing assay were conducted in verifying the capacity of both cell invasion and cell migration.Compared with control group,overexpression of miR-150 only suppressed the cell invasion and cell migration.However,overexpression of both miR-150 and ZEB1 improved the capacity of cell invasion and cell migration.Thus,it can be observed that the invasive cells were significantly increased and the migration of cells was accelerated.According to the results so far,it can be summarized that miR-150 targets and suppresses the expression of ZEB1 and thereby regulates cell activities including cell proliferation and cell apoptosis.Consequently,the capacity of the cancerous cell invasion and migration was suppressed.The interaction between miR-150 and ZEB1 affects tumor formationIn the cell assays,we proved the functions of both miR-150 and ZEB1.Since the in vivo environment is normally in more complicated regulation network,we further performed tumor formation assay in nude mice in the purpose of validating the functions of both miR-150 and ZEB1.The weight of the tumor was decreased,which formed by NCI-H460 cells transfected with miR-150 mimic.However,for the tumor formed by the cells with overexpression of miR-150 and ZEB1,the weight was significantly greater than the control group.Regarding to the shape of the tumor,the overexpression of miR-150 induced smaller tumor indicating potential weaker cell proliferation and enhanced cell apoptosis during tumor formation;overexpression of both miR-150 and ZEB1 led to larger shape of tumor suggesting that expression of ZEB1 is positively correlated to tumor formation.