LncRNA TPTEP1 Inhibits The Proliferation And Invasion Of Non-small Cell Lung Cancer Cells By Targeting MiR-761/LATS2

Objective:Lung cancer is one of the most rapidly growing morbidity and mortality rates and one of the most life-threatening malignant tumors.Non-small cell lung cancer(NSCLC)accounts for more than 80% of all lung cancers.The current treatment of NSCLC including chemotherapy,radiotherapy and surgery.However,NSCLC is a type of tumor with aggressive metastasis.The most prominent feature of malignant tumors is their invasive and metastatic capacities.Even the tumor was completely removed by surgery when it was first discovered,adjuvant chemotherapy and other treatments were also given to consolidate the treatment after surgery,some patients still experience recurrence or metastasis.Therefore,it is necessary to explore the invasion and metastasis mechanisms and regulatory factors in NSCLC.Recently,long non-coding RNAs(Lnc RNAs)have been shown to exert specific functions in many biology processes(such as epigenetic regulation,transcriptional and post-transcriptional regulation)as the form of RNA.Lnc RNAs have the ability to regulate expression of genes,and affect many disease processes,including cancer.It has reported that many Lnc RNAs regulated the tumor progression in NSCLC.Lnc RNA PTAR promotes cell proliferation,migration and invasion in NSCLC,while Lnc RNA NBR2 suppressed the progression of epithelial to mesenchymal transition(EMT).Previous studies revealed that Lnc RNA TPTEP1(herein referred to TPTEP1),played an antitumor role in many kinds of cancers,such as hepatocellular carcinoma,glioma.In NSCLC cells,TPTEP1 was reported to inhibit cell proliferation.However,the underlying mechanism of TPTEP1 in cancer progression of NSCLC remains unclear.It has been proved that Lnc RNAs can bind with micro RNA(miRNAs)sites as competitive endogenous RNAs(ce RNAs),thus influencing the expression of the target genes.The contribution of miRNAs to disease and tumor biology was widely described.In our study,we predicted that TPTEP1 targeted miR-761,which has been illustrated the oncogenic role on various cancers.For example,in triple-negative breast cancer,miR-761 induces aggressive phenotypes,and enhances the drug resistance in synovial sarcoma.In NSCLC cells,miR-761 is also reported to promote tumor progression and metastasis.The interaction between miR-761 and LATS2 was also predicted.LATS2,a Dbf2-related kinase,acts as a tumor suppressor.It is indicated that LATS2 serves as a central regulator of cell fates by modulating the function of the downstream effectors.LATS2 shows an inhibitory role in tumor metastasis,and is regulated by miRNAs,such as miR-302 and miR-372-3p.The antitumor effects of LATS2 are also implicated.The decreased expression of LATS2 indicates poor prognosis of NSCLC,and LATS2 can promotes cell apoptosis,inhibits cell growth,suppressed migration and motility.In NSCLC,although the respective roles of TPTEP1,miR-761 and LATS2 have been discovered,their interactions have never been studied.In this study,we investigated whether TPTEP1 inhibited the migration and invasion ability by regulating miR-761/LATS2 axis,in order to provide theoretical basis for the therapy of NSCLC.Methods:1.Cell culture: the cell lines used in the experiment were purchased from Shanghai cell bank,and the cell culture environment was:the medium containing 10%qualified fetal bovine serum and 100IU/ml double antibody(penicillin-streptomycin).Lung cancer A549 H1299 cell lines were cultured in RPMI(Roswell Park Memorial Institute)1640 medium.All cell lines and related cytological experiments were cultured in a 5% carbon dioxide incubator at 37?.2.Quantitative real-time polymerase chain reaction(PCR): RNA extraction(TRIzol reagent,Takara,Otsu,Japan),c DNA reversion(Revert Aid c DNA Synthesis Kit,Thermo Fisher Scientific)and amplified(ABI Prism 7900 HT platform,Applied Biosystems,Foster City,CA,USA)were performed according to previous study.m RNA expression data were calculated relative to U6 and GAPDH,respectively,using the2-??Cq method.3.MTT assay: The 5,000 cells were diluted in a 100 ?L medium,then were seeded and incubated in 96 well plate.20 ?L 5 mg/m L MTT solution were added in each well for reaction and standing at culture condition for 3?4 h.100 ?L DMSO were added after the supernatant was removed.The optical density(OD)value was determined by a Microtitre plate reader(Bio Tek,Winooski,VT,USA)at 570 nm.4.Colony formation assays: the monolayer cultured cells in logarithmic growth phase were digested with 0.25% trypsin and blown into a single cell.The cells were suspended in the culture medium of 10% fetal bovine serum.The cell suspension was diluted by gradient multiple and the cells were counted.The cells were cultured at a density of 500 cells per well in a 6-well plate containing the culture medium for about 8-10 days.When visible clone formation occurs in the petri dish,the culture is terminated.Discard the supernatant and rinse carefully with PBS for 2 times.Add pure methanol or [1:3 acetic acid/methanol] 5ml and fix for 15 minutes.Then go to the fixed solution,add appropriate amount of Giemsa to dye solution for 10 minutes and 30 minutes,then wash off the dye solution slowly with running water and dry the air.After air drying,quantitative analysis was carried out according to the number of counted colonies.5.Dual-Luciferase Reporter Assay: dilute 5*PLB(Passive Lysis Buffer)with distilled water to 1ml PLB,add 100 ml to each well in 96-well plate,blow the cells with liquid transfer gun,place in shaker at room temperature for 15 minutes,transfer the cell lysate to 1.5ml centrifuge tube,centrifuge at 4 degrees 12000 rpm for 10 minutes,take the supernatant and transfer it into a new tube;add LAR II(Luciferase Assay Reagent II)working liquid 100 ml to 96-well plate.20 ml cell lysate was added and mixed with liquid transfer gun for 2-3 times.The Firefly luciferase value was measured and recorded,which was the internal reference value.Add 100 ml Stop & Glo ?Reagent(Luciferase Assay Reagent,Progema),liquid transfer gun to blow and mix for 2-3 times,then measure and record the Renilla luciferase value,which is the reporter gene luminescence value.6.Transwell(invasion experiment): the cell invasion experiment was carried out in a24-well Transwell chamber with a diameter of 8 microns.After 24 hours of transfection,the cells in the experimental group and the control group were digested with trypsin and counted.600 ? l medium containing 10% fetal bovine serum was added to the 24-hole subplate chamber,which was careful not to produce bubbles and affect the chemotaxis.200 ? l medium containing 2% fetal bovine serum was added to the upper chamber.6 ×104 cells were inoculated into the upper chamber of transwell chamber.After 24 hours of culture,the cells on the surface of the superior ependyma were wiped with cotton swabs,fixed with 4% paraformaldehyde and stained with hematoxylin.Ten regions were randomly selected under the high-power field of view of the microscope to calculate the number of invasive cells.The experiment was repeated 3 times under the same conditions.7.Scratch assay :Cells(6 × 105)were added in 6 well plates for 12 h.Then,we used a sterile pipette tip to make a scratch in the surface of these wells,and each well was washed with PBS to remove debris.Pictures of these cells were taken immediately after the scratch was made and 48 h later.The relative migration rate of cells was obtained by counting the percentage of wound healing area.8.Statistical analysis:SPSS 18.0 software was used for statistical analyses,and data are presented as the mean ± SD from at least three independent experiments.The differences between groups were analyzed by Student's t-test or one-way ANOV A followed by Bonferroni's post hoc test.Significant differences are indicated by *p < 0.05.9.Biological information analysis: in the initial stage of practical experience,we consulted the GEPIA data base(http://gepia.cancer-pku.cn/)to analyze the differential expression analysis between tumor tissues and normal tissues.GEPIA database is an online analysis tool that can analyze the RNA sequencing expression data of 9736 tumors and 8587 normal samples from TCGA and GTEx databases.Results: 1.the expression of TPTEP1 in NSCLC tumor tissue was detected by q PCR.Compared with normal tissues adjacent to cancer,the expression level of TPTEP1 in 32 pairs of non-small cell lung cancer tissues was significantly decreased.Through bioinformatics analysis,it is predicted that TPTEP1 can combine with miR-761.In addition,there are binding sites between miR-761 and LATS2.Therefore,we also detected the transcriptional expression levels of miR-761 and LATS2.The results showed that compared with the matched normal paracancerous tissues,the expression of miR-761 was up-regulated in non-small cell lung cancer,while the expression of LATS2 was inhibited.We also analyzed the correlation among TPTEP1,miR-761 and LATS2.It was found that there was a negative correlation between the expression of Mi R-761 and TPTEP1,a negative correlation between the expression of miR-761 and LATS2,and a positive correlation between the expression of TPTEP1 and LATS2.2.A549 and H1299 cells(belong to NSCLC cell lines)were used for in vitro experiments.The cells were transfected with TPTEP1 pc DNA3.1 to overexpress TPTEP1.The cell viability was decreased significantly after overexpression of TPTEP1.The colony formation assay also indicated that TPTEP1 inhibited the cell proliferation significantly.the metastasis abilities of NSCLC cells were detected by scratch assay and transwell assay respectively.It was indicated that overexpression of TPTEP1 inhibited the migratory and invasive abilities of NSCLC cells.These results suggested that TPTEP1 played inhibitory roles on cell proliferation and metastasis.3.DIANA was used to predicted the binding site of TPTEP1 and miR-761.As shown in,after transfected with TPTEP1 pc DNA3.1,overexpression of TPTEP1 reduced expression of miR-761 significantly.In order to assess the interaction between miR-761 and TPTEP1,the RIP assay and dual luciferase reporter assay were used.miR-761 mimics enhanced the expression of miR-76.In contrast,miR-761 inhibitor reduced the expression of miR-761.Both in A549 and H1299 cells,RIP assay confirmed that there was an interaction between TPTEP1 and miR-761.In addition,miR-761 suppressed the luciferase activity notably in the cells transfected with wide type TPTEP1,while miR-761 inhibitor enhanced the luciferase activity.However,it was unchanged in the cells transfected with mutated TPTEP1.Therefore,miR-761 was targeted by TPTEP1.4.the effects of miR-761 on metastatic abilities of TPTEP1 was investigated.Overexpression of TPTEP1 inhibited the cell viability notably,which could be blocked by miR-761 mimics.This result was also verified by colony formation assay.As shown in,after co-overexpression of miR-761 and TPTEP1,miR-761 blocked the inhibitory effects of TPTEP1 on cell proliferation significantly.In addition,overexpression of TPTEP1 suppressed the ability of metastasis,which were also blocked by the overexpression of miR-761.Hence,the inhibitory effects of TPTEP1,including cell proliferation and metastasis suppression,were achieved by suppression of miR-761.5.The downstream mechanism of TPTEP1/miR-761 was also studied in A549 and H1299 cells.It was predicted by starbase 2.0 software that LATS2 might be a target of miR-761,and we verified that overexpression of TPTEP1 enhanced the expression of LATS2.In addition,the expression of LATS2 was decreased in the cells with overexpression of miR-761,while it was enhanced significantly in the cells with miR-761 knockdown.By western blot,expression of LATS2 protein was increased with overexpression or decreased in cells with knockdown of miR-761.To identified the interaction between miR-761 and LATS2,we performed dual luciferase reporter assay and found that LATS2 targeted by miR-761.These results showed that LATS2 was a downstream effector of TPTEP1/miR-761 and targeted by miR-761.6.LATS2 sh RNA reduced the expression of LATS2 significantly.After transfected with miR-761 inhibitor and LATS2 sh RNA,it was shown that knockdown of miR-761 suppressed the cell viability,while knockdown of LATS2 increased the cell viability,and LATS2 sh RNA reversed the inhibitory effects of miR-761 inhibitor on cell proliferation efficiency.The similar effects also verified in Fig.6.C by colony-formation assay.The inhibitory role of miR-761 inhibitor on cell proliferation could be blocked by LATS2 sh RNA.As for cell metastasis,we showed that miR-761 inhibitor suppressed the ability of cell migration and invasion,respectively.However,after knockdown of LATS2,the migratory and invasive abilities were increased significantly.Therefore,miR-761 played the antitumor role on NSCLC cells via regulating LATS2 negatively.7.The NSCLC cell lines were incubated with TPTEP1 pc DNA3.1,miR-761 mimics,miR-761 inhibitor or LA TS2 sh RNA.Then the EMT-related protein expression,such as N-cadherin,Twist,Vimentin,E-cadherin were detected.Overexpression of TPTEP1 promoted the expression of E-cadherin,while reduced the expression of N-cadherin,Twist,and Vimentin.The effects of TPTEP1 could be reversed by overexpression of miR-761 Furthermore,miR-761 knockdown increased the expression of E-cadherin,while decreased the expression of N-cadherin,Twist,and Vimentin.Again,these changes were reversed by the knockdown of LA TS2.In summary,in NSCLC cells,TPTEP1/miR-761/LA TS2 axis participated in the regulation of EMT.Conclusion: 1.TPTEP1,miR-761 and LATS2 have a coordinated control relationship.2.TPTEP1 inhibits the invasion of non-small cell lung cancer cells by miR-761/lats2.3.in NSCLC cells,TPTEP1/miR-761/LATS2 axis participated in the regulation of EMT.