Experimental Study On Invasion And Metastasis Of Non-small Cell Lung Cancer Through EMT Mediated By MiR-661/RB1/E2F1

Background and ObjectiveLung cancer is one of the most malignant tumour that is hazardious to people in the world.The non-small cell lung cancer（NSCLC）is a common subtype of lung cancer,consisting of 80-90% patiens.According to the data of National Cancer Institute published in 2015 that lung cancer is the most malignant tumour with highest mortality and morbidity in China.Due to the early diagnosis of lung cancer is difficult,There are about 30% of patients with distant metastasis at diagnosis,Distant metastasis occurre in50-60% in the course of treatment,Metastasis is responsible for approximately 80-90%of cancer-associated mortality.consequently,Metastasis of lung cancer is a major problem that has yet to conquer,To clarify the molecular mechanism of metastasis of lung cancer,To establish effective means of detection,to take measures to prevent and delay the occurrence of metastasis,can significantly improve patients’ quality of life and prolong the survival time of patients.Cascade reaction of tumor invasion-metastasis process is a complex process,Currently,it is believed that transformation of epithelial to mesenchymal cells（epithelial-to-mesenchymal transition,EMT）is a key part of tumor metastasis,In general,The epithelial cells have the feature with typical polarity of the top surface and the bottom surface,Close adhesion between epithelial cells limit the ability of migration of cells and free activities;But the cells will have the ability to invasion and metastasis,when the epithelial cells were transformed into cells with the morphological characteristics of stromal cells in the presence of certain elements,Studies have shown that the cell in the central region of tumor has the trait of epithelial cells phenotype,While those are phenotypic feature of mesenchymal cells in the infiltration area,When the infiltrating cells enter into the distance by the blood vessels or lymphatic metastasis,they can rebuild the cell junctions and cell skeleton to form metastases through the epithelium into interstitium（mesenchymal-epithelial transitions,MET）.So EMT is a multi-step process of reversible changes dynamically.MicroRNA is a class of non-coding small molecule RNA size of about 20-25 nucleotides which is widely present in the eukaryotic.The mature miRNA targets the3’untranslated region（3’UTR）of its target mRNA,and induces either translational repression、mRNA degradation or suppress other forms of regulation,Thus inhibits the expression of target gene.microRNA plays a key role in tumor formation,growth and metastasis,so they can be applied in the early diagnosis and treatment of tumors.Studies have shown that miR-661 is playing the effect of inhibition or facilitation proliferation,invasion and metastasis in breast cancer,ovarian cancer and glioma.However there is no study that have been reported about the role and molecular mechanism of miR-661 in the development of lung cancer.This project intends to detect the expression of miR-661 in non-small cell lung cancer,and to predict the target,to analyze the role and molecular mechanism associated with miR-661 in non-small cell lung cancer occurrence,development and metastasis.Methods1.MiR-661 expression in non-small cell lung cancer tissues and cell lines was detected using quantitative RT-PCRThe gene chip of miR-661 in lung cancer was found by mining of bioinformatic algorithms（gene code:GSE15008/GP/L8176/989）,Fluorescent quantitative RT-PCR method was applied for detection the expression of miR-661 in matched adjacent normal tissues in the non-smalll cell lung cancer tissue samples and miR-661 in normal lung epithelial cell EBAS-2B and seven lung cancer cell lines including A549,H460,GLC-82,SPC-A1,PC-9,H1299,H322.2.Screening and identification of miR-661 targets.1)Potential molecular targets of miR-661 were analysed by bioinformatic algorithms in the five commonly used databases including miRwalk 、 TargetScan 、microRNA.map、Pita、RNA22 was determined as the candidate target genes of miR-661 related with growth and metastasis in NSCLC by selecting the target gene with intersection high value of Prediction and through the literature.2)Western blot was used to detect RB1 expression in non-small cell lung cancer cells and tissues.3)The RB1 3’UTR was amplified by PCR and then cloned downstream of the Renilla luciferase open reading frame of the psi-CHECK2 vector（Promega）as Wt RB13’UTR;the predicted target site for miR-661 was mutated using the KOD Plus Mutagenesis Kit（Toyobo）,generating Mut RB1 3’UTR.A luciferase reporter assay was performed to test whether the RB1 was the target of miR-661.3.The effects of biological characters of miR-661 on NSCLC growth and metastasis in vitro and in vivo1)Transient transfection of miR-661 and anti-miR-661,The CCK-8 cell proliferation assays,Plate cloning experiments,cell cycle experiments,Transwell migration and scratch assay were carried out to detect cell proliferation,plate colony formation,migration and mobility ability in vitro after transient overexpression and interference of miR-661 in NSCLC cells lines A549 and SPC-A1.2)Xenograft tumours were generated by subcutaneous injection of tumor cells to assess the effect of stable overexpression of miR-661 on tumor growth in vivo.Tumor cells were injected into nude mice through the tail wein to evaluate the effect of stable overexpression of miR-661 on the metastasis of NSCLC cells in vivo.4.The mechanism involved in MiR-661 on non-small cell lung cancer was detected.Changes of EMT related marks were tested by Western blot in NSCLC cells after transient transfection of miR-661,anti-miR-661 and stable overexpression of miR-661;Changes of E2F1 and E-cadherin were checked by Western blot after transient transfection of RB1siRNA;the expression of Vimetin and Fibronectin was also examined by Western blot after transient transfection of E2F1cDNA;Localization of EMT related marks was detected by Confocal laser scanning after transient overexpression of miR-661.Chromatin immunoprecipitation technology（ChIP）was verified the interaction of RB1 and its downstream transcription factor E2F1.5.Statistical analysisSPSS 22.0 software was used for statistical analysis.Quantitative values of all experiments are expressed as the mean ± standard deviation（SD）.Relative quantification value(2^{-（35）（35）Ct})of qRT-PCR in cells were analysed by One-way ANOVA,with the SNK,LSD or Dunnett’s T3 tests for multiple comparisons.It also analysed through two-tailed independent-samples t-test.The data of colony formation assay,Transwell migration and invasion assay and relative luciferase activities were analysed through One-way ANOVA.The data of CCK8 assay was analysed by Factorial design analysis of variance.Results1.MiR-661 upregulation in NSCLC（1）miR-661 was upregulated in NSCLC tissues.The gene chip results showed: The average expression level of miR-661 was significantly increased in 60.9%（67 of 110）NSCLC specimens compared to their para-carcinoma tissues（t=-2.931,P = 0.004）.Comparison of miR-661 abundance in 50 paired primary NSCLC tissues,miR-661 upregulated in 60%（34/50）of non small cell lung cancer as compared to adjacent tissue（P < 0.001）,compared to their normal counterparts,with a 7.26-fold increase in the lungl cancer tissue samples.（2）miR-661 expression was detected in NSCLC lines.Compared to normal lung epithelial cell EBAS-2B,miR-661 expression in NSLCL lines was upregulated.Fluorescent quantitative RT-PCR results showed: The Dunnett ’s T3 multiple comparison indicated the expression of miR-661 in NSCLC cells were higher than that of the EBAS-2B（P < 0.001）,we selected A549 and SPC-A1 which have the higher erpression in the NSCLC cells for the follow-up experiment.2.Prediction and validation of miR-661 targets1)Five bioinformatic algorithms（Target Scan、microRNA.map、Pita、RNA22、miRwalk）were used to predict the targets with the search term of miR-661.We picked those have the intersect of the five databases and which were confirmed as tumor suppressor genes through the literature.we determined RB1 as the target gene of miR-661.2)RB1 was tested in NSCLC cells and tissues by RT-PCR and Western Blot.RB1 was downregulated in most of lung cancer cells related to the lung epithelial cell line;Compared to the para-carcinoma tissues,RB1 expression in NSLCL tissues was also downregulated.Spearman’s correlation analysis showed a negative relationship between the miR-661 expression level and the RB1 mRNA in 50 cases of NSCLC tissue samples.3)Dual luciferase reporter vectors of Wt RB1 3’UTR and Mut RB1 3’UTR was successfully constructed.There was scarcely any expression of miR-661 in the 293 T cell.First,we co-transfected the luciferase plasmids Wt RB1 3’UTR and Mut RB1 3’UTR 、pre-miR-661 and negative control pre-miRNA、miR-661 inhibitor and negative control pre-miRNA into the 293 T cells.We observed that the exogenous expression of miR-661 significantly decreased the luciferase activity of the Wt RB1 3′UTR but not the Mut RB13’UTR（P < 0.001）compared to the blank and control,in 293 T cell,and in the case of miR-661,when cloned 3’UTR RB1 gene plasmid was transfected.We also observed inhibitition of miR-661 significantly increased the luciferase activity of the Wt RB13′UTR but not the Mut Sox2 3’UTR（P < 0.001）compared to the blank and control（P<0.05）.It showed the fluorescence activity was changed by combination of hsa-miR-661 and RB1 3’UTR.Therefore,RB1 is a direct target of miR-661 and the negative regulation of RB1 by miR-661 is due to the miR-661-binding sites in the RB13’UTR.4)Immunofluorescence results demonstrated that RB1 located in the Cell Membranes of A549 and SPC-A1 cell with miR-661 overexpression group by transient tranfection,it also can support RB1 was the direct target of miR-661.3.MiR-661 represses RB1 to accelerate NSCLC growth and invasion in vitro and in vivo1)Stable transfection A549 and SPC-A1 cell packaged using the pPACKH1 lentivector packaging kit（System Biosciences）,A549 and SPC-A1 cell lines stably expressing miR-661 were generated.2)The expression of RB1 was obviously decreased after Transient transfection of miR-661 in A549 and SPC-A1 cells by Western blot.3)After transient transfection has-miR-661 and miR-661 inhibitor,CCK-8 cell proliferation experiment showed that: upregulation of has-miR-661 promotes proliferation of A549 cell and SPC-A1 cell compared to control cell（P < 0.001;P <0.001）,While interference of has-miR-661 supresse proliferation of A549 cell and SPC-A1 cell compared to control cell（P < 0.001;P < 0.001）,Transwell migration and scarification experiment indicate that miR-661 over-expression markedly promoted migration and mobility of A549 and SPC-A1 cells（P<0.001,P<0.001）compared to control cells,Howerver the knockdown of miR-661 significantly restained the migration and mobility of A549 and SPC-A1 cells（P<0.001,P<0.001）compared toscramble-transfected cells.4)Stable overpression miR-661 can increase the proliferation ability P < 0.001;P <0.001),Colony-forming ability（P < 0.001;P < 0.001）,Migration（P < 0.001;P < 0.001）and mobility（P < 0.001;P < 0.001）ability of A549 and SPC-A1 cells compared to vector-transfected cells.5)Rescued experimental result showed overpression miR-661 can increase the migration（P < 0.001;P < 0.001）ability of A549 and SPC-A1 cells compared to control cells,While co-expression miR-661/RB1 cDNA,the migration ability of them was weakened（P < 0.001;P < 0.001）.6)Subcutaneous tumour growth in the A549/LV-miR-661 group was faster than that in the A549/LV-NC（P<0.001）.Immunostaining confirmed that expression of RB1E-cadherin in miR-661 group was downregulated,while the expression of E2F1 and Vimentin was upregulated;the positive expression of cell proliferation index Ki-67 was very strong in A549/LV-miR-661 group compared to A549/LV-control group.7)Nude mouse tail in vivo transfer experiment results showed that A549/miR-661 group had an increase in the number of nodule number developed into lung metastasis in nude mice compared with A549/LV-NC group（T=-6.686 P=0.001;）;there was no metastasis nodule in other organ.4.Preliminary study on the mechanism of miR-661 regulation of NSCLC.1)Quantity One software was used to strip gradation analysis.Changes of EMT related marks were tested by Western blot in NSCLC cells after transient transfection of miR-661,anti-miR-661,The results showed the expression of epithelial markers（E-cadherin、β-catenin）and RB1 was lower in transient transfection miR-661 of A549 and SPC-A1 cells compared to control;While the expression of mesenchymal markers（Vimetin,Fibronectin）was higher in transient transfection miR-661 of A549 and SPC-A1 cells compared to control;Howerver the transient transfection of anti-miR-661 had the opposite result.These results indicate that miR-661 can induce the conversion of EMT（epithelial to mesenchymal）in NSCLC cells.miR-661 can promote EMT by its target RB1.meanwhile We observed the role of RB1 CDNA 、 RB1 siRNA and E2F1 CDNA in EMT,The results showed the expression of epithelial marker（E-cadherin、β-catenin）was higher in transient transfection RB1 CDNA of A549 and SPC-A1 cells compared to control;the expression of epithelial marker（E-cadherin）was lowerer while E2F1 was increased in transient transfection RB1 siRNA of A549 and SPC-A1 cells compared to control,While the expression of mesenchymal markers（Vimetin,Fibronectin）was high in transient transfection E2F1 cDNA of A549 and SPC-A1 cells compared to control;these also can demonstrate RB1 inactived and E2F1 actived were involved in the process of EMT in NSCLC cells.miR-661 can suppress RB1 and suppressed RB1 can promote E2F1 express,thus can develop a miR-661/ RB1/ E2F1 axis.2)Confocal laser scanning detect the Localization of EMT related marks after transient overexpression of miR-661 and control in A549 and SPC-A1 cells.The results shows epithelial marker E-cadherin locates in the cell membrane,while the β-catenin anchor in the cell nucleus compared to the control;mesenchymal markers（Vimetin,Fibronectin）locate in the cytoplasm;The location of epithelial marker（E-cadherin and β-catenin）was not obvious in the overexpression of miR-661 compared to the control,while there was a clear location of the mesenchymal markers（Vimetin,Fibronectin）in the overexpression of miR-661 compared to the control,These results showed EMT was the important process of the regulation of miR-661 on NSCLC.At the same time,we can observe that E2F1 was located obviously in the cell cytoplasm of the miR-661 group.It maybe conclude E2F1 also partcipte in the biological processes of miR-661.3)Chromatin immunoprecipitation technology（ChIP）verified the direct interaction of RB1 and its downstream transcription factor E2F1.this demonstrated that E2F1 participated in the course of growth and metastasis of NSCLC by combination miR-661 with RB1.Conclusion1.MiR-661 was up-regulated in NSCLC cells and tumors.We reasoned that expression of miR-661 is positively correlated with the metastatic potential of NSCLC and that miR-661 may promote NSCLC growth、tumor and metastatic ability.2.RB1 was validated as a target for miR-661.MiR-661 promote tumour growth and metastasis by down-regulating RB1 in vitro and in vivo.3.EMT has a key effect on the course of development and metastatis of NSCLC caused by mir-661.4.The RB1 and its downstream transcription factors E2F1 can participate in the course of metastatis of NSCLC cells by miR-661 through regulating of EMT.