Study On NEU4 Hydrolyzes Sialic Acids On CD44 To Inhibit Motility Of Liver Cancer Cells

Hepatocellular Carcinoma（HCC）is one of the most common types of malignant tumors with high metastasis and high recurrence,which resulted in a high mortality rate for patients.The sialic acid（SA）is a common nine carbon sugar,SAs level on cell surface is significantly increased in many types of tumors.This change could then impact on cell migration ability and the ability to binding with vascular endothelium,which lead to metastases formation and worse prognosis.In mammals,the level of SAs is mainly regulated by sialic transferase and sialidase,sialidase is mainly responsible for the removal of SAs from glycoconjugates,the expression and function of sialidase have received wide attention.NEU4 is an important member of sialidase,the expression of NEU4 is significantly down-regulated in HCC.However,the function of NEU4 in HCC has rarely been reported.TCGA and GTEx database showed NEU4 expression in clinical HCC tissues was significantly decreased compaired with normal liver tissues（p<0.05）.Kaplan-Meier Plotter also showed that HCC patients with low NEU4 expression had poor prognosis.On this basis,the NEU4 analyzed by q RT-PCR and western blot showed NEU4experission was down-regulated in HCC（p<0.05）.What’s more,tissue array slides containing 165 paired HCC tissues also showed the NEU4 expression was decreased in HCC（p<0.0001）,and NEU4 expression was related to the clinical stage,overall survival and disease-free survival.These results all indicate that NEU4 expression is down-regulated in HCC and is related to the occurrence and development of HCC.In order to explore the regulation mechanism of NEU4 in HCC,SK-Hep1 and MHCC-97H cell lines with low NEU4 expression were treated with histone-deacetylase inhibitor TSA,results showed TSA increased the expression of NEU4（p<0.05）.Treatment of SK-Hep1 and MHCC-97H cells with specific histone deacetylase inhibitors and HDAC1/HDAC2 si RNA also suggested that HDAC1 and HDAC2inhibits NEU4 expression（p<0.05）.Ch IP assay further demonstrated that NEU4promoter could specifically bound with H3K9ac and H3K27ac,suggested that the low expression of NEU4 in HCC could be resulted by the deacetylation of H3K9ac and H3K27ac induced by HDAC1/HDAC2.On this basis,the function of NEU4 in HCC cells was then discussed.The SK-Hep1,MHCC-97H and HUH7 with stable NEU4 over-expression were used in cell migration,invasion and adhesion analysis.It was found that the exogenous expression of NEU4 could significantly inhibit the migration of HCC cells and promote its adhesion ability to matrigel（p<0.05）.Meanwhile,the Tet-on system showed the inhibition of NEU4 on cell migration could be recovered.Then the nude mice were further used to detect the metastasis of HCC cells with NEU4 over-expression,and results showed NEU4 over-expression could inhibit the experimental pulmonary metastasis in nude mice（p<0.05）.These results indicate that NEU4 could inhibit the motility of HCC cells both in vitro and in vivo.In order to clarify the influence of NEU4 enzyme activity on migration-related phenotypes in HCC cells,the content of SAs in NEU4 over-expressed SK-Hep1 and MHCC-97H cells were detected,results showed cell membranes SAs were significantly down-regulated（p<0.05）.Then the NEU4^E235was found to be a potential active center of NEU4,and overexpression of NEU4^E235Acould no longer inhibit HCC cell migration ability.Meanwhile,NEU4 activator TQ significantly inhibited the migration of SK-Hep1 and MHCC-97H cells（p<0.05）.SS-NEU4 was then constructed,the extracellular incubation with SS-NEU4 could reduce the SAs on cell membrane,and inhibit the mobility of HCC cells（p<0.05）.These results suggested that NEU4 could hydrolyze the SAs on HCC cells’membrane through their enzyme activity,and could inhibit the motility of HCC cells.To further explore the mechanism of NEU4 on HCC cell migration ability,Co-IP was used to enrich NEU4 and its potential substrates in SK-Hep1,then the CD44 was identified through mass spectrum.Further experiments showed theα2,3-linked SAs on CD44 was decreased（p<0.05）and the hyaluronic acid binding ability was increased（p<0.01）when NEU4 over-expressed or activated.Additionally,the mutation of N-glycosylation sites on CD44 caused sensibility decreasement to NEU4 activation or SS-NEU4 extracellular incubation in SK-Hep1 and MHCC-97H,for their cell migration changed little compared with wild-type CD44.These results suggest that NEU4promotes the binding of HCC cells with extracellular matrix component hyaluronic acid by removing SAs from CD44,thus inhibiting the mmotility of HCC cells.In summary,our results revealed the mechanism of low expression of NEU4 in HCC and its inhibitory effect on cell migration by removal of SAs on CD44,which may provide new treatment strategies to control the motility and metastasis of HCC.