Long Non-coding RNA HHIP-AS1 Inhibits Invasion And Metastasis Of Gastric Cancer Cells Via Hedgehog Signal Pathway By Targeting Regulation Of HHIP

Background and objective:Because of high degree of malignancy,strong abilities of invasion and metastasis,and high peritoneal and distant metastasis rates,gastric cancer(GC)usually leads to poor prognosis for patients.New breakthroughs for the treatment of GC might be found by exploring the molecular mechanism of invasion and metastasis and new therapeutic targets.Long non-coding RNAs(lnc RNAs)participate in the regulation of tumor cell proliferation,metastasis,invasion,drug resistance,etc.through various pathways,and are closely related to the prognosis of tumor patients.A large number of abnormally expressed Lnc RNAs in GC tissues may be involved in the occurrence and development of GC,but the specific functions and molecular regulatory mechanisms of most Lnc RNAs are still unclear.In this study,the differentially expressed Lnc RNA HHIP-AS1 in GC tissues was screened by gene chip,and its correlations with the clinical expressions and pathological characteristics of GC patients,as well as its regulation on the invasion and metastasis of GC cells were explored.Method:1.According to the results of gene chip,The differentially expressed Lnc RNA HHIP-AS1 was screened,and the expression level of HHIP-AS1 in GC tissues and paracancerous tissues was further analyzed and verified by the TCGA database.Then,expression differences of HHIP-AS1 in GC cell lines and normal gastric mucosal cells was verified by real-time quantitative PCR(RT-q PCR).2.Summarizing and analyzing the correlation between the expression level of HHIPAS1 in GC tissues and the clinicopathological characteristics of patients,and speculating that HHIP-AS1 might play a functional role in the occurrence and development of GC.3.The co-expressed gene of HHIP-AS1 were predicted by online databases,and its expression was detected by RT-q PCR and Western blotting(Western Blot,WB)in GC tissues and cell lines.The interaction between HHIP-AS1 and its co-expressed gene was analyzed and clarified.4.HHIP-AS1 was overexpressed in GC cells to observe its effect on the invasion and metastasis of GC cells in vitro cell functional experiments.At the same time,the expression levels of target genes HHIP and Hedgehog signaling pathway were detected to deeply explore the role of HHIP-AS1 in the process of invasion and metastasis of GC cells and the corresponding molecular regulatory mechanisms.Result:1.Compared with the para-cancerous tissues and gastric mucosa epithelial cells,the expression levels of HHIP-AS1 in GC tissues and GC cell lines was significantly decreased.2.Low expression of HHIP-AS1 was associated with lymph node metastasis and TNM stage in GC patients.Moreover,multivariate analysis indicated that the expression level of HHIP-AS1 could be an independent risk factor for GC invasion and metastasis.3.The survival analysis results of the TCGA database and 76 cases of GC patients collected by us showed that the survival time of GC patients with low expression of HHIP-AS1 was shorter and the prognosis was worse than that of patients with high expression of HHIP-AS1.4.The expression of HHIP in GC tissues and GC cell lines was significantly lower than para-cancerous tissues and normal gastric mucosal cells,and the expression level of HHIP was positively correlated with HHIP-AS1.Combined with the prediction of the online databases,the results suggested that HHIP might be a regulatory target gene of HHIP-AS1.5.The overexpression of HHIP-AS1 by plasmid transfection in AGS and SNU-1 GC cell lines inhibited the invasion and metastasis of GC cells significantly,which suggested that HHIP-AS1 was involved in the regulation of the biological behavior of GC cells invasion and metastasis.6.After overexpressing HHIP-AS1 through plasmid transfection in AGS and SNU-1 cell lines,not only the expression level of HHIP in the cells was significantly upregulated,but also the expression levels of key proteins SHH and GLI1 in the Hedgehog signaling pathway were significantly down-regulated and the Hedgehog signaling pathway was inhibited,which suggested that the Hedgehog signaling pathway was activated in GC cells,and contributes to the invasion and metastasis of GC cells Conclusion:1.Lnc RNA HHIP-AS1 showed a low expression level in GC tissues and cell lines,which was negatively correlated with lymph node metastasis and TNM stage of GC.At the same time,survival analysis showed that GC patients with low expression of HHIPAS1 had lower overall survival rate and worse prognosis,suggesting that HHIP-AS1 may be involved in the regulation of the biological behavior of GC cells invasion and metastasis.2.The expressions of HHIP-AS1 and HHIP were significantly down-regulated in GC tissues and cell lines,showing a positive correlation.After overexpression of HHIPAS1 in GC cells,the expression of HHIP was also up-regulated,suggesting that HHIP is most likely regulatory target gene of HHIP-AS1.3.The overexpression of HHIP-AS1 by plasmid transfection could significantly inhibit the invasion and metastasis of GC cells,and the Hedgehog signaling pathway was correspondingly inhibited,which suggested that HHIP-AS1 might be a tumor suppressor gene and affect the Hedgehog signaling pathway by targeting the HHIP gene,and then participated in the regulation of the biological behavior of GC invasion and metastasis.