A Peptide Encoded By LINC00998 Promotes Liver Cancer Progression

Background & Aims:As one of the most advanced tumor,human primary liver cancer represents the third leading cancer causing death worldwide.Herein,hepatocellular carcinoma(HCC)accounts for 85%-90% of human primary liver cancer and is treatment-resistant to almost all currently available therapies.It is well known that the carcinogenesis process of normal liver cells develop by the accumulation of numerous driver mutations and many such kinds of mutations often occur in the so-called desert regions of human genome.The massive non-coding RNAs(ncRNAs)accounted for 98% of the whole transcriptome and have long been linked to cancer as essential regulators.All these ncRNAs act through different mechanisms to influence diverse cancer cell properties as cell proliferation,cell-cycle progression,cell apoptosis,or even cell invasion and migration.Among them,lncRNAs are associated with kinds of malignant phenotypes frequently and attract much attention.The dysregulation expression of lncRNAs has been observed in various human cancers and the molecular mechanisms by which lncRNAs exhibit their important biological functions are intricacy.However,despite numerous existing research results,the important role of lncRNAs or other ncRNAs in normal development and cancer progression remains an area of active investigation and the subject of controversy.Growing evidence suggests that ncRNAs with small open reading frames(smORFs)possess special structure including 5’cap,3’ polyA tail and some hairpin structure.For instance,lncRNAs with smORFs are present in cytoplasm where they can be translated by ribosomes.Therefore,some lncRNAs with smORFs are translated to be functional peptides or proteins,which is proved to be greatly underestimated.These putative peptides and proteins may mediate oncogenic or tumor suppressing effects and may be a new class of cancer therapeutic targets.Nevertheless,the precise mechanisms of peptides or proteins generated from ncRNAs in the development and progression of HCC remains unclear.Thus,given the tissue-specific nature of lncRNAs expression in various cancer types,we hypothesized that some hidden peptides might be used to regulate the biological process of HCC and meet the specific needs of liver cancer cells.We also aimed to identify novel ncRNA/peptide based strategies for the diagnosis and therapies of HCC.Methods:1.Rip-seq was performed to screen the ribosome-associated lncRNAs in cancer cells.Rip-seq analyses with RPS6 were performed to screen lncRNAs with coding potential.As for genes binding with RPS6,their properties were demonstrated by GO and KEGG analyses.ORF finder and Coding Potential Calculator(CPC)were used to find potential smORFs in indicated lncRNAs.Gene expression profiling interactive analysis(GEPIA)and quantitative polymerase chain reaction(qPCR)were used to evaluate the expression level of identified lncRNAs.RNA folding was used to predict the second structures of lncRNAs.2.Western blot(WB)was performed to detect the expression of peptide encoded by LINC00998 and its properties were further analyzed.Rapid amplification of cDNA ends(RACE)was performed to determine the transcriptional initiation sites of lncRNA.Fluorescence in situ hybridization(FISH)was performed to find the subcellular localization of lncRNAs.Mass spectrum(MS)was performed to validate the endogenously express of peptide.Related plasmids were performed to validate the express of peptide and.Online tools include ClustalX,TMHMM,ProtScale,and Signal IP 4.0 was used to describe the structure of SMIM30.3.Analysis of the molecule function of target lncRNA and its corresponding peptide in vivo and in vitro.Lentivirus-mediated shRNAs were used to knock down the expression of LINC00998/SMIM30.Cell counting kit-8(CCK8)assays,EdU staining assays,transwell assays,wound healing assays and flow cytometry assays were used to evaluate the functional role of LINC00998/SMIM30.Special constructs as expressed peptide or not were used to performed the rescue experiments.Nude mouse tumorigenicity assay and transplanted tumor assay were used to investigate the role of targeted peptide in vivo.4.Co-immunoprecipitation(Co-IP)and MS were used to seek proteins interacted with targeted peptide.Protein-protein interaction assay network analyses were also performed to demonstrate proteins interacting with targeted peptide.Immunoflourescence double-labeling was used to performed the colocalization of proteins and targeted peptide.5.Evaluating the molecule mechanisms of SMIM30 involved in HCC progression.JASPAR was used to predict the potential transcription factor.Chromatin immunoprecipitation(ChIP)and luciferase analyses were performed to verify the binding transcription factor.RNA-seq analysis was performed to analyze the gene expression profile affected by targeted peptide knocking down in Huh7 cells.WB analyses were performed to evaluate the expression changing in mitogen-activated protein kinase(MAPK)signaling pathway.Results:In this study,we identified ribosome-associated lncRNAs in cancer cell lines by RIP-seq combined with RPS6 antibody.These lncRNAs are uniform in human genome and have coding potential.Combined with qPCR analysis and related databases,we found that the expression of LINC00998 was high in HCC tissues compared with adjacent tissues.And the high expression of LINC00998 was related to the poor prognosis and different stages of HCC patients.What’s more,the expression of LINC00998 was also high in HCC cell lines.Results of FISH experiments showed that LINC00998 with smORFs was mainly localized in the cell cytoplasm.We further found that peptide SMIM30 was translated by the smORF sequence of LINC00998.The expression of SMIM30 was endogenous and increased in HCC tissues.SMIM30 is a membrane molecule and located in the cell membrane.It mediates the membrane anchoring of some huge molecules.The sequence of SMIM30 is conserve,and it has significant signal peptide cleavage sites,transmembrane domains and strong hydrophobicity.SMIM30 has an important role in promoting the growth,proliferation,invasion and migration of HCC cells,and alters the cell cycle.Therefore,SMIM30 is a key tumor enhancer.SMIM30 can interact with non-receptor tyrosine kinase SRC/YES1 and mediate their membrane anchoring.By mediating their membrane anchoring,peptide SMIM30 also activate SRC/YES1.Further,after knocking down the expression of SMIM30 in HCC cells,many important signaling pathways have been changed.And the MAPK pathway is very related to activated SRC/YES1.Through the expression validation of important proteins,we found that SMIM30-SRC/YES1 complex promotes the development and metastasis of HCC by regulating ERK/p38-MAPK signaling pathway.These findings will be helpful to develop novel diagnostic strategies and therapeutic targets based on ncRNAs/peptides.Conclusions:There are large numbers of lncRNAs binding to ribosomes and have coding potential in cancer cells.LINC00998 is highly expressed in HCC tissues and cell lines,and is related to the poor prognosis of HCC patients.In addition,SMIM30,the peptide encoded by LINC00998,was also significantly increased in HCC tissues.By analyzing the structure and properties of SMIM30,we found that the sequence of SMIM30 was conserve and it was a membrane molecule with strong membrane anchoring ability.SMIM30 has significant transmembrane region,signal peptide cleavage sites and strong hydrophobicity.Furthermore,it is peptide SMIM30,rather than LINC00998,promotes the proliferation,migration and invasion of HCC cells.SMIM30 activates SRC/YES1 by binding with SRC/YES1 and mediating their membrane anchoring.SMIM30-SRC/YES1 complex promotes the development of HCC by regulating ERK/p38-MAPK signaling pathway.