The Biological Function And Mechanism Of MiR-1-3p In Lung Adenocarcinoma

BackgroundLung cancer is the cancer with the highest incidence and one of the main causes of tumor-related death worldwide.It has become a malignant disease that seriously threatens people's life and health.Lung cancer is mainly divided into non-small cell lung cancer(NSCLC)and small cell Iung cancer(SCLC),accounting for 80-85%and 15-20%of all lung cancers,respectively.The most common type of NSCLC is adenocarcinoma,which accounts for about 55 percent of NSCLC and is even higher in non-smoking Asian patients.Because early symptoms are insidious and nonspecific,many patients are diagnosed in tumor advanced-stage.With lung cancer treatment concept updating,new targeted therapeutic drugs have emerged,new targeted therapeutic drugs are emerging one after another,but unfortunately,the 5-year survival rate of patients with adanced lung cancer still is only 10-15%,its main reason is that no matter what kind of drugs in the late stage of lung adenocarcinoma will develop resistance,lead to the lack of long-term effective clinical treatment.Therefore,it is of great significance to find new markers for early diagnosis and therapeutic targets of lung adenocarcinoma.MicroRNA(miRNA)is a small class of endogenous RNA discovered in recent years,consisting of 18-24 nucleotides and belonging to the non-coding RNA family.To date,about 2,000 species of niRNAs have been identified in the human genome(http://www.mirbase.org).As a major regulator of the post-transcriptional level of coding genes,miRNAs leads to the dysfunction of the target gene through the binding complementary paired of 3'-non-translation region(3'-utr)of target gene nRNA.Abnormal miRNAs expression results in multiple dysfunction of tumor cells,including proliferation,migration,invasive drug resistance and recurrence.Therefore,miRNA is considered as a potential biomarker for a variety of tumors.For example,miR-122 is down-regulated in liver cancer,while miR-221,miR-222 and miR-21 are overexpressed in hepatocellular carcinoma(HCC).As tumor suppressors,miR-125b and miR-145 are down-regulated in tumors,and miR-21,miR-155,miR-24,miR-125b,miR-21,miR-19 and miR-155 are important cancer-promoting miRNAs,which are overexpressed in tumors.As the first numbered miRNAs,miR-1-3p is associated with a variety of human diseases and is known primarily for its regulatory role in skeletal muscle and cardiac tissue,including myocytogenesis,muscle proliferation and differentiation.Current research has shown that miR-l-3p plays a role in a variety of tumor types,including rhabdomyosarcoma and tumors such as thyroid,prostate,bladder,colorectal,and hepatocellular carcinoma.However,how miR-1-3p affects the biological functions of lung adenocarcinoma,such as proliferation,migration and apoptosis,and which target genes interact with it and related mechanisms have not been systematically studied.To explore the pathways of action between miR-1-3p and downstream target genes can deepen the understanding of the molecular mechanism of the development of lung adenocarcinoma,thus promoting the discovery of improved therapy for lung adenocarcinoma.Therefore,this topic is mainly devoted to the study of the role and specific mechanism of miR-1-3p in the occurrence and development of lung adenocarcinoma.The research content is divided into the following two parts.?.The expression,biological function and clinical significance of miR-1-3p in lung adenocarcinoma?.Screening and identification key target gene of miR-1-3p,exploring the target gene function and clinical significance.?.Screening and validation of the molecular regulatory mechanism of PRC1 in downstream lung adenocarcinomaPart ? The expression,biological function and clinical significance of miR-1-3p in lung adenocarcinomaObjectMir-1-3p is one of the most important miRNAs related to tumorigenesis and development,and its abnormal expression has been detected in many types of tumors(such as ovarian cancer,prostate cancer,etc.),and is widely involved in tumor proliferation,migration,invasion,angiogenesis and other processes.In lung adenocarcinoma,however,its function and clinical significance have not been clarified.This part aims to study the expression,clinical significance and biological function of miR-1-3p in lung adenocarcinoma.Contents and methodsSurgical specimens of fresh lung adenocarcinoma and adjacent normal tissues were collected and qRT-PCR was applied to detect the expression of miR-1-3p in the above tissues.Meanwhile,the differential expression of miR-1-3p in lung adenocarcinoma cells(A549.H1299 and H1975 cells)and normal alveolar epithelial ceils(HPAEPIC cells)were also detected by qRT-PCR.MiR-1-3p mimics was used to transfection lung adenocarcinoma cells(A549,H1299 and H1975 cells)instantaneously,and the transfection efficiency was confirmed by qRT-PCR to establish miR-1-3p overexpressed cell lines.Lung adenocarcinoma cell lines with overexpression of miR-1-3p(A549,H1299 and H1975 cells)were used to detect the effect of miR-1-3p on proliferation of lung adenocarcinoma cells by MTT,invasion and metastasis by transwell,and expression changes of related proteins by Western blot.Overexpression plasmids of miR-1-3p were constructed and stably transfected into lung adenocarcinoma cell lines(A549,H1299 and H1975 cells).Subcutaneous tumor formation test was conducted in nude mice to investigate the effect of miR-1-3p on tumor growth.According to the patient information collected from the pathological tissues of lung adenocarcinoma,relevant clinical datas(including tumor size,TNM stage,peripheral lymph nodes and distant metastasis,etc.)were tracked,and the clinical significance of miR-1-3p was analyzed based on the expression level of miR-1-3p.ResultsThe expression levels of miR-1-3p in lung adenocarcinoma tissues and cells and the normal control group were detected by qRT-PCR.The results showed that the expression level of miR-1-3p in lung adenocarcinoma tissues was significantly lower than that in normal lung tissues.Similarly,significant downregulation of miR-1-3p expression was also observed in human lung adenocarcinoma cell lines A549,H1299 and H1975,compared with normal alveolar epithelial cell line(HPAEPIC).The above results suggest that the abnormal expression of miR-1-3p may be involved in the physiological process of lung adenocarcinomaTransient transfection of miR-1-3p mimic caused a significant increase in miR-1-3p expression in lung adenocarcinoma cells(A549,H1299 and H1975 cells).MTT assay was used to detect the difference in the proliferation rate between the above cells and normal alveolar epithelial cell(HPAEPIC cells),and it was found that the overexpressed miR-1-3p significantly inhibited the proliferation of these three lung adenocarcinoma cell lines in a time-dependent manner.These results suggest that miR-1-3p 1s sufficient to inhibit the growth of lung adenocarcinoma cells in vitro.To further investigate the effect of miR-1-3p on the invasion and metastasis of lung adenocarcinoma,we performed transwell assay on miR-1-3p overexpressed cell lines(A549,H1299 and H1975 cells)and normal alveolar epithelial cell(HPAEPIC cells).The results showed that the ability of invasion and metastasis of lung adenocarcinoma cells with miR-1-3p overexpressed were significantly decreased compared with that in normal alveolar epithelial cell.Moreover,we use western blot to detect the mark proteins(fibronectin,N-cadherin and vimentin)in the process of epithelial-interstitial transformation(epithelial-mesenchymal transition,EMT)the results showed that in the miR-1-3p overexpressed cell lines,the expression levels of above three kinds of proteins are reduced.These data demonstrated that miR-1-3p overexpression weakened the invasion and metastasis abilities of lung adenocarcinoma cells by inhibiting the expressions of fibronectin,N-cadherin and vimentin.In addition to vitro experiments,we inoculated miR-1-3p overexpressed and control lung adenocarcinoma A549 cells in nude mice subcutaneously.After 3 weeks of inoculation,the tumor size and weight of miR-1-3p overexpressed groups were significantly decreased compared with the control group.The above results showed:?that overexpression of miR-1-3p inhibited tumor growth in mice.By analyzing the correlation between the expression of miR-1-3p in the pathological specimens of lung adenocarcinoma and the patient's tumor size,staging,peripheral lymph nodes and distant metastasis,it was found that low-level expression of miR-1-3p was closely related to tumor TNM staging,lymph node metastasis and distant metastasis.These data suggests that the development of lung adenocarcinoma may be related to the low expression of miR-1-3p.ConelutionsMiR-1-3p is low expressed in lung adenocarcinoma tissues and cell lines,and the overexpression of miR-1-3p significantly inhibits the proliferation of lung adenocarcinoma cell lines,and reduces the invasion and metastasis ability of lung adenocarcinoma cells by inhibiting the expressions of fibronectin,N-cadherin and vimentin.In vivo,overexpression of miR-1-3p in lung adenocarcinoma cells significantly inhibited tumor growth in mice.Clinically,patients with low expression of miR-1-3p have rapid tumor progression and early metastasis.Part ? Screening and identification key target gene of miR-1-3p,exploring the target gene function and clinical significance.ObjectCurrently,miR-1-3p is known to be involved in biological processes such as proliferation and metastasis of lung adenocarcinoma,and its high expression significantly inhibits the occurrence and development of lung adenocarcinoma.The main way of its function is to bind to the 3'-non-translation region(3'-UTR)of the downstream target gene mRNA,and achieve post-transcriptional regulation of the downstream genes by inhibiting its translation,leading to the dysfunction of the downstream target genes.However,it is not clear which target gene acted by miR-1-3p.This part of the study aims to predict the potential downstream target genes of miR-1-3p through relevant databases,and to screen out the genes with strong correlation for identification,discuss the function of this target gene in lung adenocarcinoma,and analyze the relationships between its expression level and relevant clinical infornation of patients.Contents and methodsUsing starbase website(http://starbase.sysu.edu.cn/)to predict potential miR-1-3p downstream target genes,combined with TCGA data and lung adenocarcinoma expression profile chip,find candidate target genes PRC1.Western blot analysis of PRC1 expression in lung adenocarcinoma cells(A549,H1299 and H1975 cells)and nornal alveolar epithelial cells(HPAEPIC cells)were performed.At the same time,the changes of PRC 1 expressions after miR-1-3p overexpression in three types of lung adenocarcinoma cells were further detected.The basic expression levels of miR-1-3p and PRC1 genes in lung adenocarcinoma tissues and cells and those in normal lung tissues and cells were detected by qRT-PCR,and the correlation was analyzed.Luciferase reporter assay was used to confirm whether PRC1 is the direct target gene of miR-1-3p.To determine whether PRC1 can compensate for the regulatory effects of upstream miR-1-3p in lung adenocarcinoma,rescue experiment was used to detect whether overexpression of PRC1 can reverse the effects of miR-1-3p on lung adenocarcinoma cells.lmmunohistochemistry detected the differential expressions of PRC1 in lung adenocarcinoma tissues and normal adjacent tissues of patients with follow-up data.TCGA data were used to analyze the expression differences of PRC1 in lung adenocarcinoma and normal tissues.Patient information and tumor-related clinical data(including tumor size,TNM stage,peripheral lymph nodes and distant metastasis,etc.)were collected during follow-up and the clinical significance of PRC I was analyzed combination with the expression level of PRC1.TCGA data were also used to analyze the effect of PRC1 on the prognosis of patients with lung adenocarcinoma.ResultsIn the PITA database of stabrase website,complementary base pairing were predicted between miR-1-3p and PRC 1's 3'-UTR.Combined with the expression profile chip and TCGA data of lung adenocarcinoma,PRC1 might be the target gene of miR-1-3p,and there was a strong negative correlation between them(R=-0.415,p?9.58E-23).To verify this finding,we used western blot to detect the differential expression of PRC1 in lung adenocarcinoma cells(A549,H1299 and H1975 cells)and normal alveolar epithelial cells(HPAEPIC cells),and the results showed that PRC1 expression in the three types of lung adenocarcinoma was higher than that in normal alveolar epithelial cells.In addition,PRC1 expression level decreased after miR-1-3p was overexpressed in three types of lung adenocarcinoma cells.qRT-PCR detection also showed that the expression of PRC1 in lung adenocarcinoma tissues and cells was also higher than that in normal lung tissues and cells,which was negatively correlated with the expression of miR-1-3p.In luciferase reporter gene assay,miR-1-3p reduced the fluorescence activity of the constructed PRC1 gene 3'utr wild vector rather than the mutant vector,indicating that miR-1-3p plays a role by binding to PRC1 gene 3'utr.Rescue experiment results showed that the reduced invasion and metastasis ability were restored after the addition of PRC1 in miR-1-3p overexpressed cells.Immunohistochemical results showed that PRC1 expression in lung adenocarcinoma tissues was higher than that in normal lung tissues.TCGA data also showed that PRC1 expression level in lung adenocarcinoma tissue were notably higher than that in normal lung,and the higher PRC1 expression level suggested larger earlier metastasis(later TNM stage)and worse prognosis.ConclutionsPRC1 is a key downstream target gene of miR-1-3p in lung adenocarcinoma,and their expression is negatively correlated.PRC1 is highly expressed in lung adenocarcinoma tissues and cells.The overexpression of PRC1 significantly reversed the effect of miR-1-3p overexpression on metastasis and invasion of lung adenocarcinoma.High expression of PRC1 is associated with early metastasis and poor prognosis.Part ? Screening and validation of the molecular regulatory mechanism of PRC1 in downstream lung adenocarcinomaObjectIt has been determined that miR-1-3p plays a role through PRC1 gene,but it is unclear which pathway and mechanism downstream of PRC1 regulates the progression of lung adenocarcinoma.This part aims to screen the potential mechanism of PRC 1 downstream through transcriptome chip technology and verify it.Contents and methodsA549 cells were transfected with siRNA to interfere PRCI gene expression,and PRC1 silenced cell lines were constructed.Transcriptome sequencing analysis was used to evaluate the gene expression profiles of PRC1 interfered A549 cells and control cells.Differential genes[log2(Fold change)]?1 and q<0.05 were selected as significant differential genes.In order to further study the PRC1-mediated functional changes and related mechanisms,we conducted a KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment analysis on all the different Genes.Qrt-pcr was used to determine the transcription levels of several genes in A549 cells after PRC1 interference.In addition,Spearman rank correlation analysis was used to analyze the correlation between PRC1 and transcription level expression of representative genes in lung adenocarcinoma specimens.ResultsThe results showed that siRNA had a high efficiency in interfering with PRCI expression,and the siRNA-3 with the highest interference efficiency was selected to transfect A549 cells for transcriptome sequencing analysis.Transcriptome sequencing analysis was used to evaluate the gene expression profiles of A549 cells and control cells after PRC1 interference,and 10,936 differentially expressed genes in A549 cells after PRC1 interference were identified,including 8,887 down-regulated DEGS and 2,049 up-regulated DEGS.At the same time,the clustering of all differentially expressed genes was plotted.The results of KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment analysis showed that Wnt,VEGF,toll-like receptor,Toll and LMD and TNF pathways were significantly enriched,and the Wnt pathway was most down-regulated in the cells after PRC1 interference.Qrt-per was used to determine the decreased transcription levels of several representative genes in the Wnt pathway in A549 cells after PRC1 interference.such as Myc,CCND1,CTNNB1,Jun and RHOA.In addition,Spearman rank correlation analysis in 10 lung adenocarcinoma specimens showed that PRC1 transcription level was positively correlated with beta-catenin,Myc,Jun and RHOA.ConclutionsBy transcriptome sequencing analysis,it was found that after PRC1 expression silenced,the differentially expressed genes were mainly enriched in the Wnt signaling pathway.Further RT-PCR was applied in cell lines and human tissues to verify the positive correlation between PRC1 gene and important genes in Wnt signaling pathway.Innovations of this studyIn this paper,the biological functions and related mechanisms of miR-1-3p in the occurrence and development of lung adenocarcinoma were mainly studied.The innovations are as follows:1.The expression and function of miR-1-3p in lung adenocarcinoma were systematically analyzed,and the role of its tumor suppressor miRNA was fully confirmed.2.It has been proved for the first time that PRC1 is a direct downstream target gene of miR-1-3p,and its role in promoting the occurrence and development of lung adenocarcinoma is negatively regulated by miR-1-3p.3.The clinical significance of miR-1-3p and PRC1 in lung adenocarcinoma were elucidated for the first time.4.It has been proved that PRC1 palyed roles through Wnt singal pathway.