Biological Effect And Mechanism Of 5-HT₇ Receptor In Non-small Cell Lung Cancer

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death.A significant proportion of lung cancer patients have a group of histological subtypes jointly known as NSCLC(non-small-cell lung cancer),of which lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC)are the most common subtypes.Along with the development of immune-checkpoint inhibitors(ICIs)and targeted therapy,the NSCLC treatment prospects have notably progressed.Because only a small portion of patients can benefit from molecular targeted therapy or immunotherapy and do not develop therapeutic resistance,continued research on new targets is warranted.Serotonin has recently emerged as a growth factor for tumor cells,and its receptors may be potential therapeutic targets.The 5-HT₇receptor is a serotonin receptor.Since it was discovered in1993,there has been extensive research into its role in the central nervous system.Recently,it has been suggested to be involved in several kinds of cancer.In our study,the first time,over-expression of 5-HT₇receptor in NSCLC tumor tissues compared to adjacent normal lung tissues was reported.Further evaluation of its role in NSCLC progression and the possible mechanism was conducted.Part? The expression of HTR7 in NSCLC and the correlation of HTR7 expression with clinicopathologic characteristics.Objective:Using gene expression data and immunohistochemical analysis to explore the expression of HTR7 in NSCLC and to detect the correlation between HTR7 expression and clinicopathologic characteristics.Methods:(1)Gene profiles(Illumina Hi Seq,log2(x+1)-transformed RSEM normalized data)of the HTR7 in tumor and correlating adjacent normal lung tissues from male smokers in the LUSC(TCGA lung squamous cell carcinoma,n=28)and LUAD(TCGA lung adenocarcinoma,n=11)datasets were retrieved from the UCSC Xena browser.The m RNA expression of HTR7 was compared between the paired tissues.(2)HTR7 expression data for tumors in male smokers with NSCLC of different pathological stages were retrieved from the UCSC Xena browser.The m RNA expression of HTR7 among different pathological stages was explored.(3)The expression of HTR7 in paired NSCLC tissues was tested using immunohistochemistry.(4)The average optical density(AOD)value was used to present expression level of HTR7 in tumor tissues.The clinical data analysis was conducted to explore the correlation of HTR7 expression level and clinicopathologic characteristics.Results:(1)Tumor tissues expressed HTR7 m RNA at a higher level than normal tissues in both LUSC(P<0.01)and LUAD(P<0.05)data.(2)The m RNA expression of HTR7 in LUSC patients showed a trend of being increased in stage?,while the data of LUAD patients did not show the same trend.(3)The result of immunohistochemistry indicated higher expression of HTR7 in NSCLC tumor tissues compared with adjacent normal tissues.(4)The correlation between HTR7 expression and clinicopathological features revealed that HTR7 expression levels were not associated with age,sex,smoking status or tumor size.But higher HTR7 expression levels were correlated with lymph node metastasis(P=0.007)and advanced TNM stage(P=0.000)in NSCLC patients.Conclusion:Both m RNA and protein expression of HTR7 were higher in tumor than in paired normal tissue in NSCLC patients,and it might be an important risk factor for the development of NSCLC;Higher expression of HTR7 might be correlated with lymph node metastasis and advanced TNM stage,which indicated that HTR7 might be an important predictor for prognosis of NSCLC patients.Part? Exploring the functional significance of HTR7 using bioinformatic analysisObjective:Using gene expression analysis to find out whether HTR7 was one of different expressed genes(DEGs)of paired NSCLC tissues.GO(Gene Ontology)enrichment and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analyses to find out the involvement of HTR7 in important biological process(BP),cellular component(CC),molecular function(MF)and signaling pathways.Methods:(1)The m RNA expression(raw read counts),clinical,meta-and manifest data of the aforementioned paired tissues were downloaded from The Cancer Genome Atlas(TCGA)using Genomic Data Commons Data Portal(GDC Data Portal).Then,m RNA expression data were converted using the R language and Perl software.Differentially expressed genes were identified by the R package“edge R”in Bioconductor.A false discovery rate(FDR)of 0.05 and a log-fold change of 2 were considered as cutoffs for a significant difference.Volcano plots and heat maps were generated by using the packages“gplot”and the“TBtools”software.(2)GO enrichment was performed with the package“cluster Profile”and package“org.Hs.eg.db”.(3)KEGG pathway analyses were performed with the packages“enrichplot”and package“org.Hs.eg.db”.Results:(1)The HTR7 was one of the significantly elevated genes in LUSC data;Although HTR7 expression was not markedly increased in LUAD tumor samples,its expression still showed an increasing trend.(2)A substantial proportion of LUSC DEGs were enriched in the“biological process”term“G-protein-coupled peptide receptor”,while a significant number of LUAD DEGs were enriched in the“cellular component”term“G-protein coupled peptide receptor”;HTR7 belongs to the GPCR(G-protein coupled receptor)super family.(3)The majority of the DEGs of both LUSC and LUAD data were enriched in the“neuroactive ligand receptor interaction”pathway;HTR7 can serve as a neurotransmitter receptor.Conclusion:The expression of HTR7 increased in both LUSC and LUAD tumor;The GO enrichment and KEGG pathway analyses all indicated functional significance of HTR7 in the differences between NSCLC from normal samples.Part? Knockdown HTR7 to explore its regulation in NSCLC cell proliferation,migration and invasion.Objective:Knockdown expression of HTR7 in NSCLC cells to find out its effects on proliferation,migration and invasion,and to explore the mechanism for its involvement.Methods:(1)The gene expression of HTR7 was knocked down using small interfering RNA(si RNA)in two NSCLC cell lines:A549 and H1299;q RT-PCR and Western blot were used to detected the m RNA level and protein level respectively.(2)Colony formation assay and CCK-8 assay were performed to detect role of HTR7 in cell proliferation;Transwell assay was used to determine the effect of HTR7 on cell migration and invasion.(3)Western blot was used to detect the protein level of PCNA,Survivin and MMP9;Western blot was used to detect the phosphorylation of Src,P38,JNK,ERK,m TOR,Akt.Results:(1)Knockdown HTR7 notably reduced the colony formation of the two NSCLC cell lines,and significantly attenuated migration and invasion capacity in NSCLC cells.(2)Knockdown HTR7 significantly decreased the expression of PCNA(P<0.05),survivin(P<0.01)and MMP9(P<0.05)in A549 cells;Knockdown HTR7 notably reduced expression of PCNA(P<0.05),survivin(P<0.05)and MMP9(P<0.05).(3)Knockdown HTR7 exppression suppressed phosphorylation of Src(P<0.05),P38(P<0.05),Akt(P<0.05)in A549 cells;Downregulation HTR7 expression attenuated activation of Src(P<0.05),JNK(P<0.01)in H1299 cells.Conclusion:Knockdown HTR7 expression significantly suppressed proliferation,migration and invasion in A549 and H1299 cells;Downregulation of HTR7 decreased expression of PCNA,survivin and MMP9 in both NSCLC cells;HTR7 might regulate Src,P38 and Akt signaling pathways in A549 cells;HTR7 might act upstream of Src and JNK in H1299 cells.Part? Stimulation of HTR7 with selective agonist LP-211 to validation its involvement of proliferation,migration and invasion in NSCLC cells and the modulatory mechanismObjective:NSCLC cells were incubated with LP-211 and appropriate pathway inhibitor to determine its involvement in proliferation,migration and invasion,and to validate its modulatory mechanism.Methods:(1)A549 cells were incubated with LP-211 at varied concentration,and phosphorylation of Src,P38,Akt were detected using Western blot;Further validation was performed using colony formation assay in both NSCLC cells.(2)A549 and H1299 cell were treated with appropriate concentration of LP-211,and cells were harvested at various times to explore the downstream molecular.(3)Both NSCLC cells were incubated with appropriate concentration of LP-211 at appropriate time,then were tested migration and invasion capacity using transwell assay.(4)Both NSCLC cells were pretreated with corresponding pathway inhibitors and then incubated with appropriate concentration LP-211 at appropriate time;Proteins were extracted to explore the expression of PCNA,survivin and MMP9.(5)Further validation of pathways involved in HTR7-regulated migration and invasion in NSCLC cells was carried out by Transwell assay.Results:(1)10nM LP-211 was chosed according to its activation of pathways that were down-regulated in knockdown experiments and its promotion of colony formation.(2)The highest phosphorylation of P38 was obtained at 2 h,and the highest p-Akt level was observed at 6 h in A549 cells;The highest level of MMP9 and survivin was observed at 12h in A549 cells;As with H1299 cells,p-JNK and p-Src both reached their maximum levels at 2 h;The highest expression of MMP9,PCNA and survivin was observed at 24 h in H1299 cells.(3)LP-211 notably enhanced the migration and invasion capacities in both NSCLC cells.(4)The P38 inhibitor BMS582949 significantly reversed the effect of LP-211on MMP9 expression,and the Akt inhibitor MK2206 suppressed the expression of survivin,in A549 cells;The Src inhibitor AZD0530 could reverse the influence of LP-211 on MMP9,PCNA and survivin in H1299 cells,and the JNK inhibitor SP600125 partly reversed the effect of LP-211 on survivin expression.(5)LP-211-induced migration and invasion were significantly suppressed by BMS582949 in A549 cells.In H1299 cells,the enhanced migration and invasion was reversed by AZD0530.Conclusion:Activation of 5-HT₇receptor promoted proliferation,migration and invasion in A549 cells and H1299 cells;HTR7 might be an upstream regulator of P38 and Akt,and also mediates the proliferation or metastasis in A549 cells by activating these pathways respectively.While in H1299 cells,pathways that might be regulated by 5-HT₇receptor to influence proliferation and metastasis were JNK and Src signaling pathways.