HTR7 is upregulated in laryngeal carcinoma tissues and cells
We found that HTR7 was significantly upregulated in laryngeal cancer tissues compared with that in normal laryngeal tissues in data from The Cancer Genome Atlas (TCGA) database (Figure 1a). We also confirmed this result using eight pairs of laryngeal cancer tissues and their adjacent normal tissues: Q-PCR and western blotting showed that HTR7 was also upregulated in laryngeal tissues (Figure 1b and 1c). Q-PCR and western blotting further demonstrated that HTR7 was upregulated in laryngeal cells compared with that in normal laryngeal epithelia cells (Figure 1d). Gene Set Enrichment Analysis (GSEA) showed the high HTR7 expression correlated positively with strong tumorigenesis ability (Figure 1e). Finally, further analysis of TCGA data revealed that patients with high HTR7 expression had a shorter survival time than those with low HTR7 expression (Figure 1f). Taken together, these findings suggested HTR7 was upregulated in laryngeal cancer tissues and cells, and high HTR7 expression was associated with poor outcome.
HTR7 is an independent predictive factor for prognosis of patients with laryngeal cancer
According to above results, we determined the association between HTR7 expression and overall survival of patients with laryngeal cancer using long-rank test and multivariable Cox proportional hazard regression analysis. IHC was used to determine HTR7 expression in cohort of 113 patients with laryngeal cancer (Figure 2a). Kaplan–Meier survival analysis showed that patients with low HTR7 expression had longer survival than those with high HTR7 expression (Figure 2b). To identify independent prognostic factors for the survival of patients with laryngeal cancer, univariate and multivariate Cox regression models were used, which showed that relapse, distant metastasis, and HTR7 expression were significant prognostic predictors for overall survival of patients with laryngeal cancer. Relapse and HTR7 expression were identified as independent predictive factors for the prognosis of patients with laryngeal cancer (Figure 2c). Taken together, these results showed high HTR7 expression was a significant independent prognostic factor for poor prognosis in laryngeal cancer and could be used as a biomarker for prognosis in patients with laryngeal cancer.
HTR7 promotes laryngeal cancer proliferation in vitro
To determine the function of HTR7 in laryngeal cancer progression, we used GSEA to analysis the relationship between HTR7 expression and the expression of key genes associated with tumor proliferation and found that HTR7 expression correlated positively with CCND1 expression (Figure 3a). CCND1 is a key factor for G1/S transition, and accelerates cell cycle progression [11, 12]. Cyclin E promotes the cell cycle, and p21 and p16 inhibit the cell cycle [13-15]. We overexpressed HTR7 in the FaDu cell line, which has low HTR7 expression, and knocked down HTR7 expression in TU212, which has high HTR7 expression. Western blotting analysis showed HTR7 overexpression increased cyclin D1 and cyclin E levels and reduced p16 and p21 levels (Figure 3b), while HTR7 knockdown reduced cyclin D and cyclin E levels and increased p21 and p16 levels (Figure 4a). The MTT assay showed that HTR7 overexpression promoted cell proliferation significantly (Figure 3c), while HTR7 knockdown inhibited cell proliferation significantly (Figure 4b). Colony formation assays showed that HTR7 overexpression increased cell proliferation (Figure 3d), while HTR7 knockdown inhibited cell proliferation (Figure 4c). BrdU incorporation assays revealed that HTR7 overexpression promoted cell proliferation (Figure 3e), while HTR7 knockdown inhibited cell proliferation (Figure 4d). These results suggested that HTR7 promoted the proliferation of laryngeal cancer.
HTR7 promotes laryngeal cancer growth in vivo
To confirm above results, we used soft agar growth assays and a mouse model to determine the role of HTR7 in laryngeal cancer growth. Soft agar growth assays demonstrated that HTR7 overexpression promoted tumor anchorage-independent growth significantly, while HTR7 knockdown inhibited tumor anchorage-independent growth significantly (Figure 5a). The mouse model revealed that HTR7 overexpression increased the tumor volume significantly, while HTR7 knockdown reduced the tumor volume significantly (Figure 5b). These results suggested that HTR7 promoted laryngeal cancer growth.
HTR7 promotes laryngeal cancer growth by activating the PI3K/AKT pathway
To determine the regulatory mechanism of HTR7 in laryngeal cancer growth, we used GSEA to analyze the signaling pathways regulated by HTR7 and found that HTR7 expression levels correlated positively with those of PI3K/AKT pathway targets (Figure 6a). Luciferase reporter assays showed that HTR7 overexpression significantly increased the luciferase activity, while HTR7 knockdown significantly inhibited the luciferase activity, suggesting that HTR7 increased PI3K/AKT pathway activity (Figure 6b). Western blotting assays showed that HTR7 overexpression increased the level of phosphorylated AKT, while HTR7 knockdown reduced the level of phosphorylated AKT (Figure 6c), suggesting that HTR7 activated the AKT pathway. BCL2L1, BCL2A1, BIRC5, BCL2, XIAP, CCNE2, CCND2, CDK2, CDK4, and BAD are targets of AKT, and are associated with tumor proliferation and inhibition of apoptosis [16, 17]. Q-PCR analysis showed that HTR7 overexpression promoted their expression, while HTR7 knockdown inhibited their expression (Figure 6d), which supported the view that HTR7 activated the PI3K/AKT pathway.
To understand whether HTR7 promoted laryngeal cancer growth by activating the AKT pathway, we inhibited PI3K/AKT pathway activation using either small interfering RNAs targeting AKT or the AKT pathway inhibitor perifosine [18] in HTR7-overexpressing laryngeal cancer cells. Colony formation assays showed that inhibition of the PI3K/AKT pathway significantly inhibited cell proliferation (Figure 7a), which was confirmed using soft agar growth assays (Figure 7b). These results suggested HTR7 promoted laryngeal cancer growth by activating the PI3K/AKT pathway.
We confirmed these results using clinical samples. Western blotting assays showed that laryngeal cancer tissues with high HTR7 expression had high p-AKT levels, while laryngeal cancer tissues with low HTR7 expression had low p-AKT levels, and the expression of HTR7 and p-AKT levels correlated positively (Figure 7c), suggesting that HTR7 activated the PI3K/AKT pathway in clinical samples.