The Mechanism Of LncRNA AK126420 Promoting Gastric Cancer Metastasis Through Its Binding Protein HNRNPH1

Purposes:Gastric cancer(GC)is one of the most common human malignancies in the world,and it is characterized by the poor prognosis and high recurrence rate.The invasion and metastasis of cancer cells are the main causes of poor prognosis in patients with GC,especially in patients with advanced GC.Invasion and metastasis in GC are a complex process involving many biological morphological changes.However,the underlying molecular mechanisms of invasion and metastasis remain largely unclear.In addition to surgical resection,the therapeutic approaches for advanced GC are limited.All of these facts emphasize the urgency to identify the mechanism of invasion and metastasis in GC patients.Comprehensive understanding of the mechanism of invasion and metastasis would help us to find effective therapeutic targets for successful intervention in metastasis and improve the prognosis of patients with GC.Long noncoding RNAs(lncRNAs)are known as transcripts greater than 200 nucleotides in length with no protein coding potential.In cancer,lncRNAs have been demonstrated to take part in tumor formation and metastasis through activation/inactivation different pathway by interacting with DNA,RNA or protein.In GC,some lncRNAs have been reported to regulate carcinogenesis before.However,how lncRNAs regulate the invasion and metastasis of GC remains largely elusive.In this study,Arraystar Human 8x60K LncRNA Microarray v2.0 was used to screen the differential expression of lncRNAs between GC tissues with and without lymph node metastasis(LNM).And we found that AK126420 was a lncRNA closely related to GC metastasis.In addition,we detected the expression of AK126420 in GC tissues,clarified its basic gene information and analyzed the relationship between the expression of AK126420 and the clinical parameters of GC such as LNM and distant metastasis.The effect of AK126420 on invasion and metastasis of GC cells was investigated in vitro and in vivo.We identified a protein that interacts with AK126420 and elucidated the molecular mechanism of AK126420 in regulating GC metastasis at the lncRNA-binding protein complex level.In addition,we explained the upstream regulation mechanism of AK126420 abnormal expression in GC tissues at the transcription factor level.Methods:High-throughput lncRNA expression profiling chips were used to screen lncRNAs associated with GC metastasis.Real-time quantitative PCR was used to detect the expression of AK126420 in GC tissues and cell lines.Correlation between AK126420 expression level and clinicopathological parameters of GC patients was analyzed.To clarify its chromosomal location and ability to encode proteins,the genetics of AK126420 was determined by bioinformatics analysis.The cellular localization of AK126420 was determined using RNA Nuclear/Cytoplasmic Isolation assay and RNA Fluorescent in Situ Hybridization assay.The core promoter region of AK126420 was determined by luciferase assay.The transcription factors that may bind to the AK126420 core promoter region were predicted using software.The regulatory effect of transcription factors on AK126420 was verified by real-time quantitative PCR,luciferase assay and chromatin immunoprecipitation assay(ChIP assay).The effects of AK126420 on the biological behavior of gastric cancer cells were detected by cell migration,invasion,proliferation and apoptosis assays in vitro.In vivo,subcutaneous tumor formation and tail vein injection assays were used to study the effects of AK126420 on growth and metastasis in gastric cancer.AK126420 was identified to bind to HNRNPH1 by RNA pull down,mass spectrometry and RNA immunoprecipitation assay(RIP assay).Real-time quantitative PCR,Western blot and RIP assay were used to identify the target genes regulated by the AK126420/HNRNPH1 protein complex and the involved signaling pathways.Results:(1)lncRNA AK126420 is highly expressed in gastric cancer tissues with LNMHigh-throughput screening of lncRNA microarray showed that there were 1057 differentially expressed lncRNAs between primary GC tissues with and without LNM The lncRNA microarray was further analyzed and found that AK126420 is a lncRNA associated with GC metastasis.Real-time quantitative PCR results indicated that AK126420 was up-regulated in primary GC tissues with LNM.Correlation analysis of clinicopathological parameters showed that high expression of AK126420 was positively correlated with LNM,distant metastasis and poor prognosis,and its expression could distinguish GC tissues with LNM and without LNM.Trough UCSC and NCBI,we found that AK126420 is located on human chromosome 11,consisting of three exons and contains 3019 nucleotides in length.NCBI open reading frame analysis,CPAT bio-software prediction combined with RNA pull down confirmed that AK126420 does not have protein coding ability.RNA nuclear/cytoplasmic isolation assay and RNA fluorescence in situ hybridization(RNA FISH)showed that AK126420 was mainly localized in the nucleus of GC cells.(2)JUN upregulates the expression of AK126420 in GCTo clarify the mechanism of AK126420 up-regulation in GC,we first examined whether the genomic of AK126420 exists amplification,and the results indicated that there is no genomic amplification of AK126420 in GC cells.Subsequently,we found that DNA methyltransferase and histone deacetylase inhibitors had no effect on the expression of AK126420 in GC cells.Bioinformatics analysis showed that there are multiple transcription factor binding sites in the promoter region of AK126420,suggesting that transcription factors may be involved in the regulation of AK126420 abnormal expression.The promoter series truncation and dual luciferase experiments indicated that the AK126420 core promoter region was mainly located between-128bp and-75bp upstream of the transcription start site(TSS).The PROMO software predicts that multiple transcription factors such as JUN,AP-1,and XBP-1 can bind to the AK126420 core promoter region.Through real-time quantitative PCR,this study confirmed that JUN could up-regulate the expression of AK126420.Dual luciferase and ChIP experiments indicated that JUN interacts with the AK126420 core promoter region and activates transcription of AK126420.(3)AK126420 promotes GC cell migration and invasion in vitroAfter cell transfection with AK126420 overexpression or shRNA plasmid,real-time quantitative PCR was used to detect the efficiency.It was found that compared with the control group,AK126420 overexpression plasmid significantly increased the expression of AK126420,while its shRNA plasmid significantly inhibited the expression of AK126420.Transwell assays showed that AK126420 could promote the migration and invasion of GC cells.MTS,EdU and apoptosis assays showed that AK126420 had no significant effect on the proliferation and apoptosis of GC cells.(4)AK126420 promotes GC cell metastasis and growth in vivoWe next investigated the role of AK126420 in GC tumor development in vivo.First,we stably overexpressed AK126420 using lentivirus-mediated overexpression vector in MKN45 cells.MKN45 cells stably overexpressing AK126420 or control were injected into athymic nude mice via the tail vein to investigated the effects of AK126420 on lung metastatic ability.The results showed that overexpression of AK126420 promoted lung metastasis of GC cells.We further explored the role of AK126420 on growth and local invasion of GC cells through subcutaneous injection in mice.The results showed that,compared with the control group,cells overexpressing AK126420 developed larger tumors,more obvious local invasion,and less central necrosis in tumor.Tumor vascular density analysis showed that the number of blood vessels in AK126420 overexpression group was higher than that in the control group.In addition,vascular endothelial cell migration and tube formation assays showed that overexpression of AK126420 promoted vascular endothelial cell migration and tube formation,suggesting that AK126420 can promote angiogenesis.(5)AK126420 promotes GC metastasis through its binding protein HNRNPH1RNA pull-down combined with mass spectrometry analysis revealed that AK126420 may bind to many proteins.Combined with related research and cell localization,we selected the proteins HNRNPH1 and YBX1 for verification from the results of mass spectrometry.Only the protein HNRNPH1 was detected by Western blot after the RNA pull down of AK126420,indicating that AK126420 only binds to HNRNPH1.In addition,RIP assays confirmed the specific interaction between AK126420 and HNRNPH1 protein.Subsequently,we used a series of truncated AK126420 for RNA pull down assays to analyze specific regions of AK126420 interacting with HNRNPH1,and the results indicated that the nucleotides 2519-3019 at the 3' end of AK126420 are required for interaction with HNRNPH1.Real-time quantitative PCR and Western blot showed that HNRNPH1 had no significant effect on the expression of AK126420,while AK126420 up-regulated the protein level of HNRNPH1,suggesting that AK126420 may play a role through HNRNPH1.Through biological online software and real-time quantitative PCR,we found that HNRNPH1 was highly expressed in gastric cancer tissues.Transwell assays showed that HNRNPH1 could promote the migration and invasion of GC cells.MTS and EdU assays showed that HNRNPH1 had no significant effect on the proliferation of GC cells.In order to clarify whether AK126420 acts through HNRNPH1,Transwell assays were carried out by truncating the nucleotides 2519-3019 at the 3' end of AK126420.The results showed that AK126420 could not promote the migration and invasion of gastric cancer cells in this case.Subsequently,rescue experiments showed that inhibiting the expression of HRNRPH1 could reverse the migration and invasion which induced by AK126420 overexpression in GC cells.(6)AK126420/HNRNPH1 complex activates GRB2 signaling pathway in GCA number of studies have shown that HNRNPH1,a splicing factor,regulates the alternative splicing of RON in tumors,allowing them to produce activating mutants RON?165.Activated RON activates the RAS-ERK signaling pathway by interacting with GRB2.Therefore,we speculated that the AK126420/HNRNPH1 complex can activate GRB2 signaling pathway through RON and affect the development of GC.To verify this hypothesis,real-time quantitative PCR and RIP assays were performed and the results confirmed that HNRNPH1 binds to RON pre-mRNA and up-regulates the expression of RON?165,an activating mutant of RON.Real-time quantitative PCR and rescue experiments showed that AK126420 up-regulated the expression of RON?165 through HNRNPH1.Western blot was used to examine the effect of AK126420/HNRNPH1 complex on RON phosphorylation and GRB2 signaling pathway.The results indicated that AK126420 can up-regulate the phosphorylation of RON and activate the expression of GRB2 RAF,RAS,MEK,ERK,MYC,ELK,HIF-la and VEGF in GC cells.Subsequently,we performed rescue experiments through simultaneously transfecting AK126420 overexpression plasmid and HNRNPH1 siRNA in GC cells.The results showed that knockdown of HNRNPH1 reversed the effect of overexpression of AK126420 on RON protein phosphorylation level and GRB2 and its downstream target genes,indicating that AK126420 can regulate RON through HNRNPH1,thereby activating GRB2 signaling pathway.Conclusions:LncRNA AK126420 is up-regulated in GC tissues with LNM,and its high expression is associated with poor prognosis in patients with gastric cancer.The transcription factor JUN can up-regulate the expression of AK126420 in gastric cancer by binding to the AK126420 promoter region to activate transcription of AK126420.AK126420 can regulate the alternative splicing of RON exon 11 by binding to HNRNPH1 and produce an activating variant RON?165,increase the phosphorylation level of RON,thereby activating GRB2 signaling pathway and promoting the invasion and metastasis of GC cells.