miR-19a mediates the mechanism by which SPHK2 regulates hypopharyngeal squamous cell carcinoma progression through the PI3K/AKT axis

Abstract

This study explored the expression of sphingosine kinase 2 (SPHK2) and microRNA miR-19a-3p (miR-19a-3p) in patients with Hypopharyngeal squamous cell carcinoma (HSCC) together with pathways affecting HSCC invasion and metastasis. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) were performed to assess the differential expression of SPHK2 and miR-19a-3p in patients with HSCC lymph node metastasis (LNM). Immunohistochemical (IHC) results were analyzed together with clinical information to evaluate their clinical significance. Subsequently, the functional effects of SPHK2 overexpression and knockdown on FaDu cells were evaluated in in vitro experiments. We performed in vivo experiments using nude mouse to assess the effects of SPHK2 knockdown on tumor formation, growth and LNM. Finally, we explored upstream and downstream signaling pathways associated with SPHK2 in HSCC. SPHK2 was significantly elevated in HSCC patients with LNM and survival was lower in patients with enhanced SPHK2 expression (P < 0.05). We also demonstrated that SPHK2 overexpression accelerated the proliferation, migration, and invasion. Using animal models, we further verified that SPHK2 deletion abrogated tumor growth and LNM. In terms of mechanism, we found that miR-19a-3p was significantly reduced in HSCC patients with LNM and was negatively associated with SPHK2. MiR-19a-3p and SPHK2 could regulate tumor proliferation and invasion through the PI3K/AKT axis. SPHK2 was found to contribute significantly to both LNM and HSCC patient prognosis and was shown to be an independent risk factor for LNM and staging in HSCC patients. The miR-19a-3p/SPHK2/PI3K/AKT axis was found to contribute to the development and outcome of HSCC.

Keywords: PI3P/AKT pathway; SPHK2; hypopharyngeal squamous cell carcinoma; lymph node metastasis; miRNA-19a-3p.

Figure 1

The expression of SPHK2 in HSCC patients and its associated prognosis. A. mRNA expression levels of SPHK2 were detected by qRT-PCR in HSCC tissues (10 samples from HSCC patients with LN metastasis patients and another 10 from patients without LN metastasis) and 10 corresponding adjacent normal tissues; B. SPHK2 protein expression was detected by WB in HSCC tissues (6 samples from HSCC patients with LN metastasis patients and another 6 from patients without LN metastasis) and 6 corresponding adjacent normal tissues; C. The immunohistochemical results of SPHK2 staining were as follows: a. Positive immunohistochemistry in tumor tissues (200× magnification). b. Positive immunohistochemistry in tumor tissues (200× magnification). c. Phosphate buffer saline (PBS) instead of primary antibody incubation was selected as negative control (200× magnification). d. Phosphate buffer saline (PBS) instead of primary antibody incubation was selected as negative control (200× magnification). D. Correlation between SPHK2 expression and overall survival in patients with HSCC. The red line represent the survival rate of patients in the positive expression group, the blue line represents the survival rate of patients in the negative expression group. Abbreviations: N, normal tissues; NL, tumor tissue without LN metastasis; L, tumor tissue with LN metastasis.

Figure 2

Changes in FaDu cell function after SPHK2 over-expression and silencing. A. SPHK2 mRNA and protein levels in SPHK2-over-expression FaDu cells; B. SPHK2 mRNA and protein levels in SPHK2-silenced FaDu cells; C. The CCK8 staining assay was used to measure the cell viability of FaDu cells at 24 h, 48 h, 72 h and 96 h, after transfection, respectively; D, E. The proliferation of FaDu cells was determined by colony formation assay; F, G. Transwell assay was used to determine the migration and invasion abilities of FaDu cells (200× magnification after 24 h). Abbreviations: Notes: ns P > 0.05, P < 0.05, *P < 0.001. Abbreviations: Fadu-WT, Wild-type Fadu cell lines; lv-SPHK2 NC, Overexpression of negative control lentiviral plasmids; lv-SPHK2, Overexpression of SPHK2 cell line transfected with lentivirus; sh-SPHK2 NC, silencing of negative control lentiviral plasmids; sh-SPHK2, silencing of SPHK2 cell line transfected with lentivirus.

Figure 3

The tumor model of foot pad was established in nude mice. A. The tumor model of foot pad was established in nude mice, using a Fadu cell line stably transfected with sh-sphk2-NC and sh-sphk2, grossly after 35 days; B. Naked eye view of isolated tumour; C. Naked eye view of isolated metastatic inguinal lymph nodes; D. The weight of tumer; E. The volume of tumer; F. The weight of lymph node.

Figure 4

In vivo and vitro experiments to validate the effect of SPHK2 on the PI3K/AKT pathway. A. KEGG pathway enrichment analysis; B, C. Expression of each target protein of Pl3K/AKT pathway in different cell lines; D. Pl3k/akt pathway was investigated in animal experimental tumors.

Figure 5

Changes in FaDu cell function after miR-19a silencing. A. Venn diagram of SPHK2 targeting miRNAs based on 3 databases: miRTarBase, TarBase and targetsacn; B. Binding site between miR-19a-3p and SPHK2; C. Immunofluorescence test results; D. mRNA expression levels of miR-19a were detected by gRT-PCR in HSCC tissues (10 samples from HSCC patients with LN metastasis patients and another 10 from patients without LN metastasis) and 10 corresponding adjacent normal tissues; E. miR-19a mRNA and SPK2 protein levels in mrR-19a-silenced FaDu cells; F. The CCK8 staining assay was used to measure the cell viability of FaDu cells at 24 h, 48 h, 72 h and 96 h, after transfection, respectively; G. The proliferation of FaDu cells was determined by colony formation assay; H. Transwell assay was used to determine the migration and invasion abilities of FaDu cells (200× maanification after 24 h).

Figure 6

The silent SPHK2 rescued to changes in cell function induced by silencing mir-19a. A. The CCk8 staining assay was used to measure the cell viability of FaDu cells at 24 h, 48 h, 72 h and 96 h, after transfection, respectively; B. The proliferation of FaDu cells was determined by colony formation assay; C. Transwell assay was used to determine the migration and invasion abilities of FaDu cells (200× magnification after 24 h); D. Expression of each target protein of PI3K/AKT pathway in different cell lines.

Figure 7

Altered functional changes in cells induced by silencing miR-19a with the addition of PI3K/AKT inhibitors. A. The CCK8 staining assay was used to measure the cell viability of FaDu cells at 24 h, 48 h, 72 h and 96 h, after transfection, respectively; B. The proliferation of FaDu cells was determined by colony formation assay; C. Transwell assay was used to determine the migration and invasion abilities of FaDu cells (200× magnification after 24 h); D. Expression of each target protein of PI3K/AKT pathway in different cellines.