| Cancer is a kind of disease with complex pathogenesis.At present,there is no effective cure for cancer in the medical and academic community.Lung cancer is the one of the leading causes of cancer deaths in the world.In the treatment of lung cancer,radiotherapy and chemotherapy are the main treatment methods.Although radiotherapy and chemotherapy have certain effects on tumors,they are less effective in the treatment of lung squamous cell carcinoma(LUSC)and lung adenocarcinoma(LUAD)alone.In the clinical treatment of lung cancer patients,the combination of chemotherapy and radiotherapy is often used.However,radiotherapy and chemotherapy not only kill tumor cells,but also kill healthy cells of patients.Therefore,the application of targeted therapy is particularly important.In recent years,targeted therapy has become a hot topic in cancer treatment,and some targeted drugs have achieved good therapeutic effects.In different types of lung cancer,LUSC and LUAD have more mutation points than other tumors,but the abnormal molecules in the two tumors are not the same.More importantly,the genetic and epigenetic profiles in the process of tumorigenesis and development vary strikingly between LUAD and LUSC.There is a wide range of pivotal molecules verified for LUAD,which leads to great therapeutic improvement for recurrent or unresectable LUAD.Instead,there have been few therapeutic alternatives for recurrent LUSC due to the lack of specific driver molecules or mutations.Hence,accurate indicators in the tumorigenesis and development of LUSC are urgently required.Long non coding RNA(lncRNA,>200nt)is a class of non coding protein RNA.LncRNAs are emerging key factors in the regulation of various cellular processes,including genomic imprinting,X-chromosome silencing,chromatin modification,intranuclear transportation,transcriptional activation and transcriptional interference.In recent years,expression of a large number of lncRNAs have been reported to dysregulate in human cancer samples,which regulate the biology function of cancer as oncogenic or tumor suppressor.With the advent of the age of big data,on the basis of individualized medicine,a new model,precision medicine,has been developed by combining gene sequencing technology,biological information and the cross combination of big data.The essence of precision medicine is analyzing and treating patients through surgery firstly,and finding the cause of their disease accurately,and finding the feasible therapeutic targets.It is also possible to analyze the same disease at different stages,so as to achieve the goal of personalized and precise treatment,and improve the diagnosis and treatment efficiency of patients.In this study,we collected 3 fresh LUSC tissue samples and the corresponding adjacent tissue samples,and prepared lncRNA chips.A total of 12 down-regulated expressed lncRNAs(CYP2B7P,CHIAP2,SFTA1P,MIR99AHG,HLA-F-AS1,SIGLEC17P,PGM5-AS1,ECEL1P2,LINC00968,FENDRR,SIGLEC16 and LHFPL3-AS2)and 1 up-regulated lncRNA(BBOX1-AS 1)were achieved through the intersection of chip-sequencing data,LUSC microarray data and lncRNA chip data.Through the analysis of the research status of the 13 differentially expressed IncRNAs,we found that CHIAP2,SFTA1P,MIR99AHG,LINC00968 and FENDRR had been studied in LUSC,and abundant studies were published on LINC00968 and FENDRR.However,PGM5-AS1,HLA-F-AS1,LHFPL3-AS2,SIGLEC17P,SIGLEC16,ECEL1P2,CYP2B7P and BBOX1-AS1 have not been studied in LUSC,and few studies have been reported in other tumors.Therefore,3 lncRNAs,PGM5-AS1,HLA-F-AS1,and LHFPL3-AS2,were randomly selected,and were first selected to be researched in our further study.Our previous work has confirmed that PGM5-AS1,HLA-F-AS1 and LHFPL3-AS2 were obviously lowly expressed in LUSC tissiues than in normal lung.And we also confirmed the lowly expression of PGM5-AS1,HLA-F-AS1 and LHFPL3-AS2 in LUSC based on LUSC microarray data.But here has been no report on the expression,functions and potential mechanism of these 3 lncRNAs.In this study,the expression levels of PGM5-AS1,HLA-F-AS1 and LHFPL3-AS2 in 3 LUSC cell lines(NCI-H1703,NCI-H226 and SK-MES-1)and human normal lung epithelial cells BEAS-2B were determined by real time RT-qPCR.Lentivirus-mediated overexpressed PGM5-AS1 were constructed.LUSC cell lines,including NCI-H1703,NCI-H226 and SK-MES-1,were infected with Lentivirus-mediated overexpressed PGM5-AS1,HLA-F-AS1 and LHFPL3-AS2 to obtain the stable overexpressed cell lines to further detect the potential functions of PGM5-AS1,HLA-F-AS1 and LHFPL3-AS2 in LUSC.Then,we researched the effect of overexpressed PGM5-AS1、HLA-F-AS1 and LHFPL3-AS2 on cell proliferation,migration,cell cycle and apoptosis in LUSC cell lines via CCK-8 assay,Celigo wound healing assay,PI-FACS cell cycle assay,Annexin V-APC flow cytometry assay,Caspase 3/7 assay,migration and matrigel invasion assays.To screen the co-expressed differentially mRNAs ofPGM5-AS1,HLA-F-AS1 and LHFPL3-AS2 via MEM,Cbioportal and our in-house microarray.Also,we analysis the potential functions and mechanism of PGM5-AS1,HLA-F-AS1 and LHFPL3-AS2 via GO,KEGG and PPI analysis,and further to investigate the prospective possibility for these 3 lncRNAs to act as a molecular target for future lung cancer treatment strategies.We believe that with the further study of the mechanism of LUSC and the discovery of new therapeutic targets,patients with LUSC will benefit from it.The first part The screen of long non-coding RNA and mRNA in LUSC chip.Materials and Methods1.The collection of 3 pairs LUSC and normal lung tissues2.The preparation of lncRNA and mRNA chip in LUSC3.The screen of long non-coding RNA and mRNA in LUSC chip.4.The GO,KEGG and PPI analysis of mRNAs in mRNA chipResults1.A total of 1,564 obviously differentially lncRNAs were screened based on the lncRNAs in-house microarray data,containing 533 up-regulated lncRNAs and 1,031 down-regulated lncRNAs.And a total of 907 obviously differentially mRNAs were screened based on the mRNAs in-house microarray data,containing 412 up-regulated mRNAs and 495 down-regulated mRNAs.2.Based on GO and KEGG analysis,we found that the obviously differentially mRNAs were related to intracellular signal transduction,regulation of protein metabolic process,protein phosphorylation,cell adhesion,G-protein coupled receptor binding,calmodulin binding,and Adrenergic signaling in cardiomyocytes,ECM-receptor interaction and Protein digestion and absorption pathways.Based on PPI analysis,we found that ACTB,ALB,ACTL7B,HDAC1,CPS1,MIB2,UBD,LM07,ADCY5,EIF4E and PAICS were hub genes.The second part The computational biology analysis of lncRNAs in LUSC based on Sequencing dataMaterials and Methods1.The processing of Sequencing data and the screen of LUSC datasets2.To take the intersection of the differentially expressed lncRNAs3.The expression of differentially expressed IncRNAs based on Sequencing data4.The research status of the differentially expressed IncRNAsResults1.A total of 5,964 obviously differentially lncRNAs were screened based on LUSC microarray data,containing 1,924 up-regulated lncRNAs and 4,040 down-regulated lncRNAs.A total of 8,397 obviously differentially IncRNAs were screened based on lncRNA datasets,containing 4,057 up-regulated lncRNAS and4,340 down-regulated lncRNAs.2.A total of 12 down-regulated IncRNAs(CYP2B7P,CHIAP2,SFTA1P,MIR99AHG,HLA-F-AS1,SIGLEC17P,PGM5-AS1,ECEL1P2,LINC00968,FENDRR,SIGLEC16 and LHFPL3-AS2)and 1 down-regulated lncRNA(BBOX1-AS1)were screened.3.The expression of 13 differentially expressed lncRNAs in TCGA was similar with the in-house microarray data.CYP2B7P,CHIAP2,SFTA1P,MIR99AHG,HLA-F-AS1,SIGLEC17P,PGM5-AS1,ECEL1P2,LINC00968,FENDRR,SIGLEC16 and LHFPL3-AS2 were down-regulated in LUSC,while BBOX1-AS1 was up-regulated in LUSC.4.According to the research status of the differentially expressed IncRNAs,we found that several studies have reported the potential role of CYP2B7P、SFTA1P、MIR99AHG、LINC00968 and FENDRR.But no study related to PGM5-AS1,HL A-F-AS1,LHFPL3-AS2,SIGLEC16,ECEL1P2,CYP2B7P and BBOX1-AS1 was published.And we will further analysis the potential role of PGM5-AS1,HLA-F-AS1,LHFPL3-AS2,SIGLEC16,ECEL1P2,CYP2B7P and BBOX1-AS1 in LUSC.The third part The clinical significance,function and potential mechanism of PGM5-AS1 in LUSCMaterials and Methods1.The expression and clinical significance of PGM5-AS1.2.The SMD and diagnostic analysis of PGM5-AS1 in LUSC3.The expression of PGM5-AS1 in NCI-H1703,NCI-H226,SK-MES-1 and BEAS-2B based on real time RT-qPCR.4.Lentivirus-mediated overexpressed PGM5-ASlwere constructed.LUSC cell lines,including NCI-H1703 and SK-MES-1,were infected with Lentivirus-mediated overexpressed PGM5-AS1 or lenti-control virus to obtain the stable overexpressed cell lines to further detect the potential functions of PGM5-AS1 in LUSC.5.To determine the effect of overexpressed PGM5-AS1 on cell proliferation.,migration,cell cycle and apoptosis in LUSC cell lines,we performed the CCK-8 assay,Celigo wound healing assay,PI-FACS cell cycle assay and Annexin V-APC flow cytometry assay.6.To screen the co-expressed differentially mRNAs of PGM5-AS1 via MEM、Cbioportal and our in-house microarray.Also,to analysis the potential functions and mechanism of PGM5-AS1 via GO,KEGG and PPI analysis.Results1.Expression of PGM5-AS1 was obviously down-regulated in LUSC tissiues.But the verification with a large sample was still needed.2.Via SMD analysis,we found that the SMD of PGM5-AS1 is-3.67(-4.02,-3.32),indicating PGM5-AS1 was down-regulated in LUSC.Via diagnostic analysis,we found that the SROC of PGM5-AS1 is 0.97(0.95-0.98),indicating PGM5-AS1 has high diagnostic value in LUSC.3.Based on cell RT-qPCR,expression of PGM5-AS1 was obviously down-regulated in LUSC cells than in normal lung cells4.We successfully constructed lenti virus-mediated overexpressed PGM5-ASI,and infected into NCI-1703 and SK-MES-1 cells.5.In vitro experiments showed that overexpression of PGM5-AS1 in LUSC cells could inhibit the cell migration and promote apoptosis.And the cell cycle was blocked in S or G2/M phase.6.Via GO and KEGG analysis,PGM5-AS1 was confirmed to effected the extracellular matrix,focal adhesion,cytoskeletal protein binding,growth factor binding and protein complex binding of LUSC cells,and cGMP-PKG signaling pathway,PI3K-Akt signaling pathway,Rapl signaling pathway,Hippo signaling pathway,Wnt signaling pathway or MAPK signaling pathway.However,the specific molecular mechanism still needs to be verified by a large number of experiments.The fourth part The clinical significance,function and potential mechanism of HLA-F-AS1 in LUSC Mraterials and Methods1.The expression and clinical significance of HLA-F-AS12.The SMD and diagnostic analysis of HLA-F-AS1 in LUSC3.The expression of HLA-F-AS1 in NCI-H1703,NCI-H226,SK-MES-1 and BEAS-2B based on real time RT-qPCR.4.Lentivirus-mediated overexpressed HLA-F-AS 1 were constructed.LUSC cell lines,including NCI-H1703 and NCI-H226,were infected with Lentivirus-mediated overexpressed HLA-F-AS1 or lenti-control virus to obtain the stable overexpressed cell lines to further detect the potential functions of HLA-F-AS1 in LUSC.5.To determine the effect of overexpressed HLA-F-AS1 on cell proliferation and migration in LUSC cell lines,we performed the CCK-8 assay and Celigo wound healing assay.6.To screen the co-expressed differentially mRNAs of HLA-F-AS1 via MEM,Cbioportal and our in-house microarray.Also,to analysis the potential functions and mechanism of HLA-F-AS1 via GO,KEGG and PPI analysis.Results1.Expression of HL,A-F-AS1 was obviously down-regulated in LUSC tissiues.But the verification with a large sample was still needed.2.Via SMD analysis,we found that the SMD of HLA-F-AS1 is-0.42(-0.67,-0.78),indicating HLA-F-AS1 was down-regulated in LUSC.Via diagnostic analysis,we found that the SROC of HLA-F-AS1 is 0.89(0.86-0.91),indicating HLA-F-AS1 has high diagnostic value in LUSC.3.Based on cell RT-qPCR,expression of HLA-F-AS 1 was obviously down-regulated in LUSC cells than in normal lung cells4.We successfully constructed lentivirus-mediated overexpressed HLA-F-AS1,and infected into NCI-1703 and NCI-H226 cells.5.In vitro experiments showed that overexpression of HLA-F-AS1 in LUSC cells could inhibit the cell migration.6.Via GO and KEGG analysis,HLA-F-AS1 was confirmed to effected the regulation of cell activation,cell projection part,cytoskeletal protein bindin or phospholipase C activity of LUSC cells,and Calcium signaling pathway.However,the specific molecular mechanism still needs to be verified by a large number of experiments.The fifth part The clinical significance,function and potential mechanism of LHFPL3-AS2 in LUSCMaterials and Methods1.The expression and clinical significance of LHFPL3-AS2.2.The SMD and diagnostic analysis of LHFPL3-AS2 in LUSC3.The expression of LHFPL3-AS2 in NCI-H1703,NCI-H226,SK-MES-1 and BEAS-2B based on real time RT-qPCR.4.Lentivirus-mediated overexpressed LHFPL3-AS2were constructed.LUSC cell lines,including NCI-H1703 and SK-MES-1,were infected with Lentivirus-mediated overexpressed LHFPL3-AS2 or lenti-control virus to obtain the stable overexpressed cell lines to further detect the potential functions of LHFPL3-AS2 in LUSC.5.To determine the effect of overexpressed LHFPL3-AS2 on cell proliferation and migration in LUSC cell lines,we performed the CCK-8 assay and Celigo wound healing assay.6.To screen the co-expressed differentially mRNAs of LHFPL3-AS2 via MEM,Cbioportal and our in-house microarray.Also,to analysis the potential functions and mechanism of LHFPL3-AS2 via GO,KEGG and PPI analysis.Results1.Expression of LHFPL3-AS2 was obviously down-regulated in LUSC tissiues.But the verification with a large sample was still needed.2.Via SMD analysis,we found that the SMD of LHFPL3-AS2 is-4.68(-5.08,-4.28),indicating LHFPL3-AS2 was down-regulated in LUSC.Via diagnostic analysis,we found that the SROC of LHFPL3-AS2 is 0.94(0.91-0.95),indicating LHFPL3-AS2 has high diagnostic value in LUSC.3.Based on cell RT-qPCR,expression of LHFPL3-AS2 was obviously up-regulated in LUSC cells than in normal lung cells4.We successfully constructed lentivirus-mediated overexpressed LHFPL3-AS2,and infected into NCI-1703 and SK-MES-1 cells.5.In vitro experiments showed that overexpression of LHFPL3-AS2 in LUSC cells could promote the cell growth of NCI-1703 cells.Also,overexpression of LHFPL3-AS2 could inhibit migration of LUSC cells.6.Via GO and KEGG analysis,LHFPL3-AS2 was confirmed to effected the transmembrane transport,plasma membrane part,igated channel activity or oltage-gated cation channel activity,and Calcium signaling pathway,Jak-STAT signaling pathway or GnRH signaling pathway.However,the specific molecular mechanism still needs to be verified by a large number of experiments.Conclusion1.Together with our previous observation,PGM5-AS 1,HLA-F-AS 1 and LHFPL3-AS2 were all the differentially expressed.2.PGM5-AS1 was a suppressor gene,and PGM5-AS1 was down-regulated in LUSC tissues and cells.Also,overexpression PGM5-AS1 could inhibit the cell migration and inhibit apoptosis.And the cell cycle was blocked in S or G2/M phase.3.HLA-F-AS1 was a suppressor gene,and HLA-F-AS1 was down-regulated in LUSC tissues and cells.Also,overexpression HLA-F-AS1 could inhibit the cell migration.4.LHFPL3-AS2 was differentially expressed in LUSC tissues and cells.And the functions were also differentially in different LUSC cells.5.The possible functions and pathways of PGM5-AS1,HLA-F-AS1 and LHFPL3-AS2 were discussed via GO,KEGG and PPI analysis;However,the specific molecular mechanism still needs to be verified by a large number of experiments. |
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