Cancer stem cells(CSCs)are thought to drive uncontrolled tumor growth,and the existence of CSCs has recently been proven by direct experimental evidence,including tracing cell lineages within a growing tumor.However,C...Cancer stem cells(CSCs)are thought to drive uncontrolled tumor growth,and the existence of CSCs has recently been proven by direct experimental evidence,including tracing cell lineages within a growing tumor.However,CSCs must be analyzed in additional cancer types.Cancer stem cell-like cells(CSCLCs)are a good alternative system for the study of CSCs,which hold great promise for clinical applications.OCT4,NANOG,and SOX2 are three basic transcription factors that are expressed in both CSCLCs and embryonic stem cells(ESCs).These transcription factors play critical roles in maintaining the pluripotence and self-renewal characteristics of CSCLCs and ESCs.In this review,we discuss the aberrant expression,isoforms,and pseudogenes of OCT4,NANOG,and SOX2 in the CSCLC niche,which contribute to the major differences between CSCLCs and ESCs.We also highlight an anticancer therapy that involves killing specific cancer cells directly by repressing the expression of OCT4,NANOG,or SOX2.Importantly,OCT4,NANOG,and SOX2 provide great promise for clinical applications because reducing their expression or blocking the pathways in which they function may inhibit tumor growth and turn-off the cancer"switch."In the future,a clear understanding of transcription factor regulation will be essential for elucidating the roles of OCT4,NANOG,and SOX2 in tumorigenesis,as well as exploring their use for diagnostic and therapeutic purposes.展开更多
Objective: Multiple mechanisms underlying the development of portal vein tumor thrombus(PVTT) in hepatocellular carcinoma(HCC) have been reported recently. However, the origins of PVTT remain unknown. Increasing multi...Objective: Multiple mechanisms underlying the development of portal vein tumor thrombus(PVTT) in hepatocellular carcinoma(HCC) have been reported recently. However, the origins of PVTT remain unknown. Increasing multi-omics data on PVTTs in HCCs have made it possible to investigate whether PVTTs originate from the corresponding primary tumors(Ts).Methods: The clonal relationship between PVTTs and their corresponding primary Ts was investigated using datasets deposited in public databases. One DNA copy number variations dataset and three gene expression datasets were downloaded for the analyses.Clonality analysis was performed to investigate the clonal relationship between PVTTs and Ts from an individual patient.Differential gene expression analysis was applied to investigate the gene expression profiles of PVTTs and Ts.Results: One out of 19 PVTTs had no clonal relationship with its corresponding T, whereas the others did. The PVTTs with independent clonal origin showed different gene expression and enrichment in biological processes from the primary Ts. Based on the unique gene expression profiles, a gene signature including 24 genes was used to identify pairs of PVTTs and primary Ts without any clonal relationship. Validation in three datasets showed that these types of pairs of PVTTs and Ts can be identified by the 24-gene signature.Conclusions: Our findings show a direct evidence for PVTT origin and consolidate the heterogeneity of PVTTs observed in clinic.The results suggest that PVTT investigation at a molecular level is clinically necessary for diagnosis and treatment.展开更多
Objective:The dysregulation of ribosome biogenesis is associated with the progression of numerous tumors,including hepatocellular carcinoma(HCC).Small nucleolar RNAs(sno RNAs)regulate ribosome biogenesis by guiding th...Objective:The dysregulation of ribosome biogenesis is associated with the progression of numerous tumors,including hepatocellular carcinoma(HCC).Small nucleolar RNAs(sno RNAs)regulate ribosome biogenesis by guiding the modification of ribosomal RNAs(r RNAs).However,the underlying mechanism of this process in HCC remains elusive.Methods:RNA immunoprecipitation and sequencing were used to analyze RNAs targeted by ribosome proteins.The biological functions of SNORA23 were examined in HCC cells and a xenograft mouse model.To elucidate the underlying mechanisms,the 2′-O-ribose methylation level of r RNAs was evaluated by q PCR,and the key proteins in the PI3 K/Akt/m TOR pathway were detected using Western blot.Results:Twelve sno RNAs were found to co-exist in 4 cancer cell lines using RPS6 pull-down assays.SNORA23 was downregulated in HCC and correlated with the poor prognoses of HCC patients.SNORA23 inhibited the proliferation,migration,and invasion of HCC cells both in vitro and in vivo.We also found that SNORA23 regulated ribosome biogenesis by impairing 2′-O-ribose methylation of cytidine4506 of 28 S r RNA.Furthermore,SNORA23,which is regulated by the PI3 K/Akt/m TOR signaling pathway,significantly inhibited the phosphorylation of 4 E binding protein 1.SNORA23 and rapamycin blocked the PI3 K/AKT/m TOR signaling pathway and impaired HCC growth in vivo.Conclusions:SNORA23 exhibited antitumor effects in HCC and together with rapamycin,provided a promising therapeutic strategy for HCC treatment.展开更多
Metastases account for the overwhelming majority of cancer-associated deaths.The dissemination of cancer cells from the primary tumor to distant organs involves a complex process known as the invasion-metastasis casca...Metastases account for the overwhelming majority of cancer-associated deaths.The dissemination of cancer cells from the primary tumor to distant organs involves a complex process known as the invasion-metastasis cascade.The underlying biological mechanisms of metastasis,however,remain largely elusive.Recently,the discovery and characterization of non-coding RNAs(ncRNAs)have revealed the diversity of their regulatory roles,especially as key contributors throughout the metastatic cascade.Here,we review recent progress in how three major types of ncRNAs(microRNAs,long non-coding RNAs,and circular RNAs)are involved in the multistep procedure of metastasis.We further examine interactions among the three ncRNAs as well as current progress in their regulatory mechanisms.We also propose the prevention of metastasis in the early stages of cancer progression and discuss current translational studies using ncRNAs as targets for metastasis diagnosis and treatments.These studies provide insights into developing more effective strategies to target metastatic relapse.展开更多
In recent years,noncoding gene(NCG)translation events have been frequently discovered.The resultant peptides,as novel findings in the life sciences,perform unexpected functions of increasingly recognized importance in...In recent years,noncoding gene(NCG)translation events have been frequently discovered.The resultant peptides,as novel findings in the life sciences,perform unexpected functions of increasingly recognized importance in many fundamental biological and pathological processes.The emergence of these novel peptides,in turn,has advanced the field of genomics while indispensably aiding living organisms.The peptides from NCGs serve as important links between extracellular stimuli and intracellular adjustment mechanisms.These peptides are also important entry points for further exploration of the mysteries of life that may trigger a new round of revolutionary biotechnological discoveries.Insights into NCG-derived peptides will assist in understanding the secrets of life and the causes of diseases,and will also open up new paths to the treatment of diseases such as cancer.Here,a critical review is presented on the action modes and biological functions of the peptides encoded by NCGs.The challenges and future trends in searching for and studying NCG peptides are also critically discussed.展开更多
Objective:To identify potential early diagnostic markers for hepatitis B progression to primary liver carcinoma using routine immunological tests based on 6 cytokine combinations.Methods:Eight hundred and ninety-nine ...Objective:To identify potential early diagnostic markers for hepatitis B progression to primary liver carcinoma using routine immunological tests based on 6 cytokine combinations.Methods:Eight hundred and ninety-nine patients with hepatitis B progressing to early primary liver carcinoma admitted to and treated at Changhai Hospital,Naval Military Medical University,Shanghai,China between March 2015 and June 2017 were included in this observational study,including 666 patients with HBsAg^(+),HBeAb^(+),HBcAb^(+)liver carcinoma and 233 patients with HBsAg+,HBeAg+,HBcAb+liver carcinoma.Receiver operating characteristic(ROC)curves were used to evaluate the efficiency of the different cytokine in the diagnosis of hepatocellular carcinoma in patients with hepatitis B.This study was approved by the Institutional Review Board of Changhai Hospital,Naval Military Medical University,China(approval No.CHEC2020-080)on June 6,2020.Results:Changed levels of interleukin(IL)-1β,IL-2R,IL-8,and tumor necrosis factor(TNF)-a were statistically significant(P<0.05).The area under the ROC curve,sensitivity,specificity,positive predictive value,negative predictive value,and Youden index for the diagnosis of primary liver carcinoma using the combination of IL-1β,IL-2R,IL-8,and TNF-α were 0.938,79.2%,96.7%,96%,82.0%,0.759,respectively.The serum alpha-fetoprotein level in patients with primary liver carcinoma was positively correlated with IL-2R(r=0.3502,P<0.001),IL-8(r=0.1558,P=0.0273),and TNF-α(r=0.2544,P<0.001)levels.The equation fitted to the results was logit(P)=0.086+0.01IL-2R–0.001IL-8–0.033TNF-α–0.041IL-1β.Conclusion:Our study establishes a novel,potentially valuable diagnostic model based on four cytokines related to the early stages of liver carcinoma.展开更多
Objective: Emerging data have shown that non-coding RNAs (ncRNAs) can encode micro-peptides (≤100 amino acids) that play an important role in regulating physiological and pathological processes. Herein, we explored n...Objective: Emerging data have shown that non-coding RNAs (ncRNAs) can encode micro-peptides (≤100 amino acids) that play an important role in regulating physiological and pathological processes. Herein, we explored ncRNAs that may encode micro-peptides that are involved in the development of hepatocellular carcinoma (HCC).Methods: High-throughput sequencing of ribosomal protein S6 (RPS6) was performed in four cancer cell lines using RNA-immunoprecipitation (RIP). UCSC databases obtained the full length of the gene sequences and quantitative polymerase chain reaction (qPCR) was used to evaluate expression levels of ncRNAs of interest. The coding activity of ncRNA was assessedin vitro by co-immunoprecipitation, plasmid transfection, western blot, immunofluorescence and RNA fluorescencein situ hybridization. Mass spectrometry was performed to explore the potential functions of candidate micro-peptide in HCC. This study involving human tissue specimens was conducted in accordance withDeclaration of Helsinki and approved by the Institutional Review Board of Changhai Hospital, Naval Military Medical University, China (approval No. CHEC2020-081) on June 6, 2020.Results: We performed RIP assay using primary antibodies for RPS6 and high-throughput sequencing. A total of 223 overlapping genes were captured by RPS6-RIP. Venn diagram analysis revealed that 60 overlapping genes were detected in four cancer cell lines. QRT-PCR showed that six of the candidate genes (RP11-298J20.4, RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, HCP5, RP11-517B11.7) were expressed in Huh7 and Hep3B cells. Further analysis of these six candidate genes and found that five (RP11-298J20.4, RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, RP11-517B11.7) displayed higher expression levels in HCC cell lines (Huh7, Hep3B) and tumor tissues than in liver cell lines (L-02, QSG-7701) and non-tumor tissues, respectively. Performed additional RIP assays and confirmed that four of the genes (RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, RP11-517B11 .7) bound RPS6. We obtained the full length of the four gene sequences from the UCSC database and analyzed the open reading frames by ORF Finder;to determine the translation potential of the four candidate small open reading frames (smORFs), we subcloned a FLAG epitope tag into the C-terminal of the four selected smORFs before the stop codon, and the fusion sequences were then cloned into three different plasmid vectors (pSPT19, pcDNA3.1, and PEGFP-N1). We performed coupled transcription and translation reactions and found that the pSPT19 plasmids encoded small peptidesin vitro. After then transfected the pcDNA3.1 constructs into Huh7 cells, and a single 7.2 kDa micro-peptide was encoded from the candidate smORF of RP11.119F7.5. We transfected the recombinant pEGFP-N1 plasmids with smORFs in HCC cells, and western blot analysis revealed a band above GFP in the RP11.119F7.5 recombinant plasmid lane. The coding potential of the RP11-119F7.5 vector was also confirmed by immunofluorescence assay. Fluorescencein situ hybridization assay revealed that RP11-119F7.5 was localized in the cytoplasm and nucleoplasm of HCC cells. Gene ontology enrichment analysis showed that the micro-peptide–interacting proteins were mainly involved in extracellular exosomes. We also found the identified proteins were involved in several biological functions like protein binding, poly(A) RNA binding, translational initiation, and the nuclear-transcribed mRNA catabolic process. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed the peptide-interacting proteins might participate in several critical pathways including ribosome, biosynthesis of amino acids, carbon metabolism, biosynthesis of antibiotics, glycolysis and gluconeogenesis, pathogenicEscherichia coli infection and influenza A.Conclusion: Our study revealed a novel micro-peptide translated by ncRNA RP11-119F7.5, highlighting the coding ability and potential role of ncRNAs in HCC.展开更多
Objective:Liver cancer stem cells(CSCs)are the culprits of hepatocellular carcinoma metastasis and recurrence.Only by eliminating tumor stem cells can malignant tumors be fundamentally cured.This study aimed to identi...Objective:Liver cancer stem cells(CSCs)are the culprits of hepatocellular carcinoma metastasis and recurrence.Only by eliminating tumor stem cells can malignant tumors be fundamentally cured.This study aimed to identify the role and underlying mechanism of aberrant Collagen Type XIV Alpha 1 Chain(COL14A1)overexpression in liver CSCs,and improve understanding of the molecular basis of hepatocellular carcinoma metastasis and recurrence.Methods:First,quantitative real-time polymerase chain reaction was used to confirm aberrant high-expression of COL14A1 in liver CSCs.Next,interference experiments were performed to determine the key role of COL14A1.To explore the mechanism of COL14A1 overexpression in liver CSCs,putative microRNA(miRNAs)targeting COL14A1 were analyzed using the miRTarBase database.Next,quantitative real-time polymerase chain reaction,western blotting,and luciferase reporter assays were performed to verify the interaction between miR-7108-3p and COL14A1.Lastly,key target proteins of the COL14A1-extracellular-regulated signal kinase(ERK)signaling pathway were identified through western blotting analysis.This study was approved by the Ethics Committee of Shanghai Fourth People’s Hospital,Tongji University School of Medicine,China(approval No.2019tjdx17)on February 21,2019.Results:COL14A1 is abnormally highly expressed in liver CSCs,which is necessary for liver CSCs to maintain their self-renewal capability.Mechanistically,COL14A1 is post-transcriptionally regulated by miR-7108-3p in a negative manner.Low expression of miR-7108-3p increased translation of COL14A1,which subsequently activated ERK signaling,ultimately maintaining the self-renewal and stem cell-like properties of liver CSCs.Conclusion:COL14A1,which is negatively regulated by miR-7108-3p,was found to play a crucial role in maintaining the selfrenewal and stem cell-like properties of liver CSCs through activation of ERK signaling.展开更多
基金supported by grants from National Natural Science Foundation(No.81071750,81172472)Shanghai Science and Technology Committee(No.10JC1417600)Program for New Century Excellent Talents in University and specially-appointed Professor of Shanghai
文摘Cancer stem cells(CSCs)are thought to drive uncontrolled tumor growth,and the existence of CSCs has recently been proven by direct experimental evidence,including tracing cell lineages within a growing tumor.However,CSCs must be analyzed in additional cancer types.Cancer stem cell-like cells(CSCLCs)are a good alternative system for the study of CSCs,which hold great promise for clinical applications.OCT4,NANOG,and SOX2 are three basic transcription factors that are expressed in both CSCLCs and embryonic stem cells(ESCs).These transcription factors play critical roles in maintaining the pluripotence and self-renewal characteristics of CSCLCs and ESCs.In this review,we discuss the aberrant expression,isoforms,and pseudogenes of OCT4,NANOG,and SOX2 in the CSCLC niche,which contribute to the major differences between CSCLCs and ESCs.We also highlight an anticancer therapy that involves killing specific cancer cells directly by repressing the expression of OCT4,NANOG,or SOX2.Importantly,OCT4,NANOG,and SOX2 provide great promise for clinical applications because reducing their expression or blocking the pathways in which they function may inhibit tumor growth and turn-off the cancer"switch."In the future,a clear understanding of transcription factor regulation will be essential for elucidating the roles of OCT4,NANOG,and SOX2 in tumorigenesis,as well as exploring their use for diagnostic and therapeutic purposes.
基金supported by grants of China National Funds for Distinguished Young Scientists (Grant No. 81425019)National Natural Science Foundation of China (Grant No. 81672899)+2 种基金the State Key Program of National Natural Science Foundation of China (Grant No. 81730076)Shanghai Science and Technology Committee Program (Grant No. 18XD1405300)Specially-Appointed Professor Fund of Shanghai (Grant No. GZ2015009)
文摘Objective: Multiple mechanisms underlying the development of portal vein tumor thrombus(PVTT) in hepatocellular carcinoma(HCC) have been reported recently. However, the origins of PVTT remain unknown. Increasing multi-omics data on PVTTs in HCCs have made it possible to investigate whether PVTTs originate from the corresponding primary tumors(Ts).Methods: The clonal relationship between PVTTs and their corresponding primary Ts was investigated using datasets deposited in public databases. One DNA copy number variations dataset and three gene expression datasets were downloaded for the analyses.Clonality analysis was performed to investigate the clonal relationship between PVTTs and Ts from an individual patient.Differential gene expression analysis was applied to investigate the gene expression profiles of PVTTs and Ts.Results: One out of 19 PVTTs had no clonal relationship with its corresponding T, whereas the others did. The PVTTs with independent clonal origin showed different gene expression and enrichment in biological processes from the primary Ts. Based on the unique gene expression profiles, a gene signature including 24 genes was used to identify pairs of PVTTs and primary Ts without any clonal relationship. Validation in three datasets showed that these types of pairs of PVTTs and Ts can be identified by the 24-gene signature.Conclusions: Our findings show a direct evidence for PVTT origin and consolidate the heterogeneity of PVTTs observed in clinic.The results suggest that PVTT investigation at a molecular level is clinically necessary for diagnosis and treatment.
基金supported by the China National Funds for Distinguished Young Scientists(Grant No.81425019)the Shanghai Science and Technology Committee Program(Grant No.18XD1405300)the National Natural Science Foundation of China(Grant No.32001786)。
文摘Objective:The dysregulation of ribosome biogenesis is associated with the progression of numerous tumors,including hepatocellular carcinoma(HCC).Small nucleolar RNAs(sno RNAs)regulate ribosome biogenesis by guiding the modification of ribosomal RNAs(r RNAs).However,the underlying mechanism of this process in HCC remains elusive.Methods:RNA immunoprecipitation and sequencing were used to analyze RNAs targeted by ribosome proteins.The biological functions of SNORA23 were examined in HCC cells and a xenograft mouse model.To elucidate the underlying mechanisms,the 2′-O-ribose methylation level of r RNAs was evaluated by q PCR,and the key proteins in the PI3 K/Akt/m TOR pathway were detected using Western blot.Results:Twelve sno RNAs were found to co-exist in 4 cancer cell lines using RPS6 pull-down assays.SNORA23 was downregulated in HCC and correlated with the poor prognoses of HCC patients.SNORA23 inhibited the proliferation,migration,and invasion of HCC cells both in vitro and in vivo.We also found that SNORA23 regulated ribosome biogenesis by impairing 2′-O-ribose methylation of cytidine4506 of 28 S r RNA.Furthermore,SNORA23,which is regulated by the PI3 K/Akt/m TOR signaling pathway,significantly inhibited the phosphorylation of 4 E binding protein 1.SNORA23 and rapamycin blocked the PI3 K/AKT/m TOR signaling pathway and impaired HCC growth in vivo.Conclusions:SNORA23 exhibited antitumor effects in HCC and together with rapamycin,provided a promising therapeutic strategy for HCC treatment.
基金supported by grants from the State Key Program of the National Natural Science Foundation of China(No.81730076)the China National Funds for Distinguished Young Scientists(No.81425019)the Changhai Hospital Funds for Young Clinical Doctors(No.2018QNB001).
文摘Metastases account for the overwhelming majority of cancer-associated deaths.The dissemination of cancer cells from the primary tumor to distant organs involves a complex process known as the invasion-metastasis cascade.The underlying biological mechanisms of metastasis,however,remain largely elusive.Recently,the discovery and characterization of non-coding RNAs(ncRNAs)have revealed the diversity of their regulatory roles,especially as key contributors throughout the metastatic cascade.Here,we review recent progress in how three major types of ncRNAs(microRNAs,long non-coding RNAs,and circular RNAs)are involved in the multistep procedure of metastasis.We further examine interactions among the three ncRNAs as well as current progress in their regulatory mechanisms.We also propose the prevention of metastasis in the early stages of cancer progression and discuss current translational studies using ncRNAs as targets for metastasis diagnosis and treatments.These studies provide insights into developing more effective strategies to target metastatic relapse.
基金This work was supported by the China National Funds for Distinguished Young Scientists(81425019)the State Key Program of National Natural Science Foundation of China(81730076)+4 种基金Shanghai Science and Technology Committee Program no.18XD1405300the Specially Appointed Professor Fund of Shanghai(GZ2015009)We also thank the financial fund from Shanghai Key Laboratory of Medical Biodefense,Shanghai,ChinaS.R.L.also thank the State Key Laboratory of Oncogenes and Related Genes(90-17-04)for fundingC.B.M.would thanks the National Institutes of Health(EB021339)for financial support.
文摘In recent years,noncoding gene(NCG)translation events have been frequently discovered.The resultant peptides,as novel findings in the life sciences,perform unexpected functions of increasingly recognized importance in many fundamental biological and pathological processes.The emergence of these novel peptides,in turn,has advanced the field of genomics while indispensably aiding living organisms.The peptides from NCGs serve as important links between extracellular stimuli and intracellular adjustment mechanisms.These peptides are also important entry points for further exploration of the mysteries of life that may trigger a new round of revolutionary biotechnological discoveries.Insights into NCG-derived peptides will assist in understanding the secrets of life and the causes of diseases,and will also open up new paths to the treatment of diseases such as cancer.Here,a critical review is presented on the action modes and biological functions of the peptides encoded by NCGs.The challenges and future trends in searching for and studying NCG peptides are also critically discussed.
基金This work was supported by the State Key Program of National Natural Science Foundation of China(No.82030073,to SL)the National Natural Science Foundation of China(No.81501401,to CH)Shanghai Science and Technology Committee(No.18XD1405300,to SL).
文摘Objective:To identify potential early diagnostic markers for hepatitis B progression to primary liver carcinoma using routine immunological tests based on 6 cytokine combinations.Methods:Eight hundred and ninety-nine patients with hepatitis B progressing to early primary liver carcinoma admitted to and treated at Changhai Hospital,Naval Military Medical University,Shanghai,China between March 2015 and June 2017 were included in this observational study,including 666 patients with HBsAg^(+),HBeAb^(+),HBcAb^(+)liver carcinoma and 233 patients with HBsAg+,HBeAg+,HBcAb+liver carcinoma.Receiver operating characteristic(ROC)curves were used to evaluate the efficiency of the different cytokine in the diagnosis of hepatocellular carcinoma in patients with hepatitis B.This study was approved by the Institutional Review Board of Changhai Hospital,Naval Military Medical University,China(approval No.CHEC2020-080)on June 6,2020.Results:Changed levels of interleukin(IL)-1β,IL-2R,IL-8,and tumor necrosis factor(TNF)-a were statistically significant(P<0.05).The area under the ROC curve,sensitivity,specificity,positive predictive value,negative predictive value,and Youden index for the diagnosis of primary liver carcinoma using the combination of IL-1β,IL-2R,IL-8,and TNF-α were 0.938,79.2%,96.7%,96%,82.0%,0.759,respectively.The serum alpha-fetoprotein level in patients with primary liver carcinoma was positively correlated with IL-2R(r=0.3502,P<0.001),IL-8(r=0.1558,P=0.0273),and TNF-α(r=0.2544,P<0.001)levels.The equation fitted to the results was logit(P)=0.086+0.01IL-2R–0.001IL-8–0.033TNF-α–0.041IL-1β.Conclusion:Our study establishes a novel,potentially valuable diagnostic model based on four cytokines related to the early stages of liver carcinoma.
基金Key Program of National Natural Science Foundation of China(No.82030073).
文摘Objective: Emerging data have shown that non-coding RNAs (ncRNAs) can encode micro-peptides (≤100 amino acids) that play an important role in regulating physiological and pathological processes. Herein, we explored ncRNAs that may encode micro-peptides that are involved in the development of hepatocellular carcinoma (HCC).Methods: High-throughput sequencing of ribosomal protein S6 (RPS6) was performed in four cancer cell lines using RNA-immunoprecipitation (RIP). UCSC databases obtained the full length of the gene sequences and quantitative polymerase chain reaction (qPCR) was used to evaluate expression levels of ncRNAs of interest. The coding activity of ncRNA was assessedin vitro by co-immunoprecipitation, plasmid transfection, western blot, immunofluorescence and RNA fluorescencein situ hybridization. Mass spectrometry was performed to explore the potential functions of candidate micro-peptide in HCC. This study involving human tissue specimens was conducted in accordance withDeclaration of Helsinki and approved by the Institutional Review Board of Changhai Hospital, Naval Military Medical University, China (approval No. CHEC2020-081) on June 6, 2020.Results: We performed RIP assay using primary antibodies for RPS6 and high-throughput sequencing. A total of 223 overlapping genes were captured by RPS6-RIP. Venn diagram analysis revealed that 60 overlapping genes were detected in four cancer cell lines. QRT-PCR showed that six of the candidate genes (RP11-298J20.4, RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, HCP5, RP11-517B11.7) were expressed in Huh7 and Hep3B cells. Further analysis of these six candidate genes and found that five (RP11-298J20.4, RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, RP11-517B11.7) displayed higher expression levels in HCC cell lines (Huh7, Hep3B) and tumor tissues than in liver cell lines (L-02, QSG-7701) and non-tumor tissues, respectively. Performed additional RIP assays and confirmed that four of the genes (RP11-4O1.2, RP11-119F7.5, RP11-448G15.3, RP11-517B11 .7) bound RPS6. We obtained the full length of the four gene sequences from the UCSC database and analyzed the open reading frames by ORF Finder;to determine the translation potential of the four candidate small open reading frames (smORFs), we subcloned a FLAG epitope tag into the C-terminal of the four selected smORFs before the stop codon, and the fusion sequences were then cloned into three different plasmid vectors (pSPT19, pcDNA3.1, and PEGFP-N1). We performed coupled transcription and translation reactions and found that the pSPT19 plasmids encoded small peptidesin vitro. After then transfected the pcDNA3.1 constructs into Huh7 cells, and a single 7.2 kDa micro-peptide was encoded from the candidate smORF of RP11.119F7.5. We transfected the recombinant pEGFP-N1 plasmids with smORFs in HCC cells, and western blot analysis revealed a band above GFP in the RP11.119F7.5 recombinant plasmid lane. The coding potential of the RP11-119F7.5 vector was also confirmed by immunofluorescence assay. Fluorescencein situ hybridization assay revealed that RP11-119F7.5 was localized in the cytoplasm and nucleoplasm of HCC cells. Gene ontology enrichment analysis showed that the micro-peptide–interacting proteins were mainly involved in extracellular exosomes. We also found the identified proteins were involved in several biological functions like protein binding, poly(A) RNA binding, translational initiation, and the nuclear-transcribed mRNA catabolic process. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed the peptide-interacting proteins might participate in several critical pathways including ribosome, biosynthesis of amino acids, carbon metabolism, biosynthesis of antibiotics, glycolysis and gluconeogenesis, pathogenicEscherichia coli infection and influenza A.Conclusion: Our study revealed a novel micro-peptide translated by ncRNA RP11-119F7.5, highlighting the coding ability and potential role of ncRNAs in HCC.
基金This work was supported by the Shanghai Science and Technology Committee of China(No.18XD1405300)the State Key Program of National Natural Science Foundation of China(No.81730076)the Program of Shanghai Fourth People’s Hospital of China(No.SY-XKZT-2020-1009).
文摘Objective:Liver cancer stem cells(CSCs)are the culprits of hepatocellular carcinoma metastasis and recurrence.Only by eliminating tumor stem cells can malignant tumors be fundamentally cured.This study aimed to identify the role and underlying mechanism of aberrant Collagen Type XIV Alpha 1 Chain(COL14A1)overexpression in liver CSCs,and improve understanding of the molecular basis of hepatocellular carcinoma metastasis and recurrence.Methods:First,quantitative real-time polymerase chain reaction was used to confirm aberrant high-expression of COL14A1 in liver CSCs.Next,interference experiments were performed to determine the key role of COL14A1.To explore the mechanism of COL14A1 overexpression in liver CSCs,putative microRNA(miRNAs)targeting COL14A1 were analyzed using the miRTarBase database.Next,quantitative real-time polymerase chain reaction,western blotting,and luciferase reporter assays were performed to verify the interaction between miR-7108-3p and COL14A1.Lastly,key target proteins of the COL14A1-extracellular-regulated signal kinase(ERK)signaling pathway were identified through western blotting analysis.This study was approved by the Ethics Committee of Shanghai Fourth People’s Hospital,Tongji University School of Medicine,China(approval No.2019tjdx17)on February 21,2019.Results:COL14A1 is abnormally highly expressed in liver CSCs,which is necessary for liver CSCs to maintain their self-renewal capability.Mechanistically,COL14A1 is post-transcriptionally regulated by miR-7108-3p in a negative manner.Low expression of miR-7108-3p increased translation of COL14A1,which subsequently activated ERK signaling,ultimately maintaining the self-renewal and stem cell-like properties of liver CSCs.Conclusion:COL14A1,which is negatively regulated by miR-7108-3p,was found to play a crucial role in maintaining the selfrenewal and stem cell-like properties of liver CSCs through activation of ERK signaling.
