MiR-19a-3p Mediates The Mechanism By Which SPHK2 Regulates Hypopharyngeal Squamous Cell Carcinoma Progression Through The PI3K/AKT Axis

Objective.To investigate the expression of sphingosine kinase 2(SPHK2)and micro RNA miR-19a-3p in patients with hypopharyngeal carcinoma and the related mechanisms affecting the invasive metastasis of hypopharyngeal carcinoma.Methods.(1)Quantitative polymerase chain reaction(q RT-PCR)and Western blot(WB)were used to detect differences in SPHK2 expression in normal hypopharyngeal tissues,hypopharyngeal carcinoma with lymph node metastasis and hypopharyngeal carcinoma without lymph node metastasis,followed by immunohistochemical analysis,and the semi-quantitative results were analysed with the clinical information collected.(2)SPHK2 overexpression and SPHK2 knockdown plasmids were transfected into hypopharyngeal carcinoma cell line(Fadu),and cell function assays were performed using CCK-8 assay for cell viability,colony formation assay for cell proliferation,and Transwell for cell invasion and migration ability.(3)A tumour-forming model of nude mouse footpad was constructed and after 35 days,the formation of primary tumour foci was observed,the size of tumours,tumour and lymph node weights were assessed,and the role of SPHK2 in tumourigenesis,growth and lymph node metastasis was confirmed in in vivo experiments.(4)The signalling pathways associated with SPHK2 were analysed by bioinformatics tools,and the expression of key target proteins of the downstream pathways was verified in vivo and in vitro by WB.(5)The differences in the expression of the upstream target miR-19-3p were verified by RT-PCR in normal hypopharyngeal tissues,hypopharyngeal carcinoma with lymph node metastasis and hypopharyngeal carcinoma without lymph node metastasis,respectively,and the changes in the functions of survival,proliferation and invasion of Fadu cell lines after knockdown of miR-19-3p were verified by cell function assays.AKT pathway reverted to miR-19-3p action.Results.(1)SPHK2 was significantly elevated in hypopharyngeal carcinoma with lymph node metastasis.The survival rate of patients with high SPHK2 expression was lower than that of patients with low SPHK2 expression(p<0.05).(2)In cellular assays,the overexpression of SPHK2 group promoted the proliferation,migration and invasion of Fa Du cells compared with the blank group(all p<0.01),while knockdown of SPHK2 inhibited the proliferation,migration and invasion of Fa Du cells(all p<0.01).(3)The tumor size and lymph node metastasis rate of nude mice injected with SPHK2 Fadu cells were significantly lower than those of the corresponding empty vector group(p<0.05).(4)Further mechanistic studies revealed that the PI3K/AKT pathway is one of the downstream pathways of SPHK2 in hypopharyngeal carcinoma,and studies on cell function showed that the PI3K/AKT pathway was activated after overexpression of SPHK2 and inhibited after inhibition of SPHK2.A further WB assay on the primary foci of nude mouse foot pads revealed that SPHK2 expression was also positively correlated with PIK3 and AKT in vivo.(5)Upstream,miR-19a-3p was lowly expressed in hypopharyngeal carcinoma patients with lymph node metastasis(p<0.01),and there was a binding site between miR-19a-3p and SPHK2,and inhibition of miR-19a-3p increased SPHK2 expression,which was negatively regulated,and could affect tumor proliferation and invasion through PI3K/AKT pathway;knockdown of SPHK2 and inhibition of PI3 K /AKT pathway could reverse the effect of miR-19a-3p silencing on cancer cell progression.Conclusion.SPHK2 is highly expressed in hypopharyngeal carcinoma patients and significantly affects hypopharyngeal carcinoma lymph node metastasis,which is one of the risk factors for clinical patient prognosis.Inhibition of SPHK2 inhibited tumor growth and lymph node metastasis,and overexpression of SPHK2 promoted tumor growth,invasion and migration.miR-19a-3p was lowly expressed in hypopharyngeal carcinoma patients with lymph node metastasis and negatively regulated with SPHK2,inhibiting tumor proliferation and invasive migration.Mechanistically,miR-19a-3p/SPHK2/PI3K/AKT is a new signaling pathway affecting the development and invasion and migration of hypopharyngeal carcinoma.