Mechanism Of HTR7 Promoting Tumor Progression And Predicting Poor Prognosis Of LSCC Patients

Objective:Laryngeal cancer is one of the common malignancy of head and neck,which has a high incidence and high disability rate,seriously affecting the survival and living quality of patients.At present,some effective biomarkers are still in great need to explore to improve early diagnosis.Although there are more and more researches on the role of targeted drugs in the treatment of tumors and other diseases in recent years,there is still no effective targeted drug for the treatment of laryngeal cancer.G protein-coupled receptors(GPCRs)are a group of drug targets recently.There is no literature reporting about the expression level of any GPCR in laryngeal cancer tissues,or wether GPCRs can regulate the biological behavior of laryngeal cancer cells and the underlying mechanism.Based on the analysis of laryngeal cancer data files in TCGA database,we found that the expression of 5-hydroxytryptamine receptor 7(HTR7),a member of GPCR family,was significantly increased in laryngeal squamous cell carcinoma.Through a series of laboratory experiments,the research aimed to detect the expression of HTR7 in laryngeal squamous cell carcinoma(LSCC)specimens,to analyze the relationship between the expression of HTR7 in the LSCC specimens and the prognosis of LSCC patients,and to explore whether HTR7 plays a role in the biological behavior of laryngeal squamous cell carcinoma cells and its regulatory mechanism,so as to provide laboratory basis for early diagnosis and drug-targeted therapy of laryngeal cancer.Methods:1.Eight pairs of fresh laryngeal cancer tissues and adjacent laryngeal normal tissues were randomly selected from our specimens bank of laryngeal squamous cell carcinoma in our department.The expression of HTR7 was analyzed by q-PCR and Western blot.Immunohistochemical staining was performed to test the expression of HTR7 in laryngeal specimens of 113 patients with LSCC,and the correlation between HTR7 expression and the prognosis of LSCC patients was analyzed.2.The cells which overexpressed HTR7 and knocked-down HTR7 were constructed.MTT,Colony formation assay,BrdU incorporation assay,Soft agar growth assay and Xenograft tumor in nude mice were performed to detect the changes of biological behavior of laryngeal cancer cells under different expression levels of HTR7.3.Based on GSEA analysis,we found that HTR7 expression levels were positively correlated with PI3K/AKT pathway targets.FOXO luciferase reporter assay was conducted to explore whether overexpressed HTR7 can activate PI3K/AKT pathway.The laryngeal cancer cell lines were overexpressed or knocked down HTR7 expression,and the expression of PI3K/AKT pathway downstream genes were studied by q-PCR and Western blot.Inhibiting the PI3K/AKT pathway in HTR7 overexpressed cell lines,the LSCC cell lines were observed by Colony formation assay and Soft agar growth assay.Results:1.The mRNA and protein expression of HTR7 were found increased in laryngeal cancer tissues,using q-PCR and Western blot performed in eight pairs of fresh laryngeal cancer tissues and normal adjacent tissues.Immunohistochemical analysis of laryngeal cancer specimens and normal tissues adjacent to cancer showed that the expression of HTR7 in laryngeal cancer tissues was significantly higher than that in normal tissues adjacent to cancer.Survival analysis showed that the overall survival of LSCC patients with higher expression of HTR7 was significantly shortened.Univariate and multivariate COX regression analysis suggested that the expression of HTR7 was an independent factor for the prognosis of LSCC patients.2.MTT,Colony formation assay,BrdU incorporation assay,Soft agar growth assay showed that overexpressed HTR7 could promote the proliferation of laryngeal cancer cell lines,while knocking down the expression of HTR7 could inhibit the growth of LSCC cell lines.Xenograft tumor which overexpressed HTR7 LSCC cell lines growed more quickly and showed larger tumor volume,while xenograft tumor which knocked-down HTR7 expression growed slowly and showed smaller tumor volume.3.FOXO luciferase reporter gene experiment shows that HTR7 overexpression can activate PI3K/Akt pathway.Western blot assay showed HTR7 overexpression increased the phosphorylation of Akt,while HTR7 knockdown reduced the phosphorylation of AKT.Q-PCR analysis showed HTR7 overexpression promoted the expression of BCL2L1,BCL2A1,BIRC5,BCL2,XIAP,CCNE2,CCND2,CDK2,CDK4 and BAD,which are the targets of Akt of PI3K/Akt signaling pathway.We inhibited PI3K/Akt pathway activation using either small interference for Akt or Akt pathway inhibitor perifosine in HTR7 overexpressed laryngeal cancer cell,and Colony formation assay showed inhibition of PI3K/Akt pathway significantly inhibited cell proliferation,and soft agar growth assay also showed inhibition of PI3K/Akt pathway significantly inhibited cell proliferation.Conclusions:In this research,we found that HTR7 was significantly highly expressed in laryngeal cancer tissues,which was correlated to poor prognosis of laryngeal cancer patients.HTR7 overexpression promoted the proliferation and growth of LSCC cells by activating PI3K/Akt pathway.Herein,HTR7 may be perfomed as a marker to predict the prognosis of laryngeal cancer patients,and could be a drug target for the treatment of laryngeal cancer.