BACKGROUND Accumulating evidence has revealed that several long non-coding ribonucleic acids(lncRNAs)are crucial in the progress of hepatocellular carcinoma(HCC).AIM To classify a long non-coding RNA,i.e.,lncRNA W5,an...BACKGROUND Accumulating evidence has revealed that several long non-coding ribonucleic acids(lncRNAs)are crucial in the progress of hepatocellular carcinoma(HCC).AIM To classify a long non-coding RNA,i.e.,lncRNA W5,and to determine the clinical significance and potential roles of lncRNA W5 in HCC.METHODS The results showed that lncRNA W5 expression was significantly downregulated in HCC cell lines and tissues.Analysis of the association between lncRNA W5 expression levels and clinicopathological features suggested that low lncRNA W5 expression was related to large tumor size(P<0.01),poor histological grade(P<0.05)and serious portal vein tumor thrombosis(P<0.05).Furthermore,Kaplan-Meier survival analysis showed that low expression of lncRNA W5 predicts poor overall survival(P=0.016).RESULTS Gain-of-loss function experiments,including cell counting kit8 assays,colony formation assays,and transwell assays,were performed in vitro to investigate thebiological roles of lncRNA W5.In vitro experiments showed that ectopic overexpression of lncRNA W5 suppressed HCC cell proliferation,migration and invasion;conversely,silencing of lncRNA W5 promoted cell proliferation,migration and invasion.In addition,acting as a tumor suppressor gene in HCC,lncRNA W5 inhibited the growth of HCC xenograft tumors in vivo.CONCLUSION These results showed that lncRNA W5 is down-regulated in HCC,and it may suppress HCC progression and predict poor clinical outcomes in patients with HCC.LncRNA W5 may serve as a potential HCC prognostic biomarker in addition to a therapeutic target.展开更多
BACKGROUND Investigating molecular biomarkers that accurately predict prognosis is of considerable clinical significance.Accumulating evidence suggests that long noncoding ribonucleic acids(lncRNAs)are frequently aber...BACKGROUND Investigating molecular biomarkers that accurately predict prognosis is of considerable clinical significance.Accumulating evidence suggests that long noncoding ribonucleic acids(lncRNAs)are frequently aberrantly expressed in colorectal cancer(CRC).AIM To elucidate the prognostic function of multiple lncRNAs serving as biomarkers in CRC.METHODS We performed lncRNA expression profiling using the lncRNA mining approach in large CRC cohorts from The Cancer Genome Atlas(TCGA)database.Receiver operating characteristic analysis was performed to identify the optimal cutoff point at which patients could be classified into the high-risk or low-risk groups.Based on the Cox coefficient of the individual lncRNAs,we identified a ninelncRNA signature that was associated with the survival of CRC patients in the training set(n=175).The prognostic value of this nine-lncRNA signature was validated in the testing set(n=174)and TCGA set(n=349).The prognostic models,consisting of these nine CRC-specific lncRNAs,performed well for risk stratification in the testing set and TCGA set.Time-dependent receiver operating characteristic analysis indicated that this predictive model had good performance.RESULTS Multivariate Cox regression and stratification analysis demonstrated that this nine-lncRNA signature was independent of other clinical features in predicting overall survival.Functional enrichment analysis of Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology terms further indicated that these nine prognostic lncRNAs were closely associated with carcinogenesis-associated pathways and biological functions in CRC.CONCLUSION A nine-lncRNA expression signature was identified and validated that could improve the prognosis prediction of CRC,thereby providing potential prognostic biomarkers and efficient therapeutic targets for patients with CRC.展开更多
BACKGROUND Primary biliary cholangitis(PBC)is a chronic and slowly progressing cholestatic disease,which causes damage to the small intrahepatic bile duct by immunoregulation,and may lead to cholestasis,liver fibrosis...BACKGROUND Primary biliary cholangitis(PBC)is a chronic and slowly progressing cholestatic disease,which causes damage to the small intrahepatic bile duct by immunoregulation,and may lead to cholestasis,liver fibrosis,cirrhosis and,eventually,liver failure.AIM To explore the potential diagnosis and staging value of plasma S100 calcium binding protein A6(S100A6)messenger ribonucleic acid(mRNA),LINC00312,LINC00472,and LINC01257 in primary biliary cholangitis.METHODS A total of 145 PBC patients and 110 healthy controls(HCs)were enrolled.Among them,80 PBC patients and 60 HCs were used as the training set,and 65 PBC patients and 50 HCs were used as the validation set.The relative expression levels of plasma S100A6 mRNA,long noncoding ribonucleic acids LINC00312,LINC00472 and LINC01257 were analyzed using quantitative reverse transcription-polymerase chain reaction.The bile duct ligation(BDL)mouse model was used to simulate PBC.Then double immunofluorescence was conducted to verify the overexpression of S100A6 protein in intrahepatic bile duct cells of BDL mice.Human intrahepatic biliary epithelial cells were treated with glycochenodeoxycholate to simulate the cholestatic environment of intrahepatic biliary epithelial cells in PBC.RESULTS The expression of S100A6 protein in intrahepatic bile duct cells was up-regulated in the BDL mouse model compared with sham mice.The relative expression levels of plasma S100A6 mRNA,log10 LINC00472 and LINC01257 were upregulated while LINC00312 was down-regulated in plasma of PBC patients compared with HCs(3.01±1.04 vs 2.09±0.87,P<0.0001;2.46±1.03 vs 1.77±0.84,P<0.0001;3.49±1.64 vs 2.37±0.96,P<0.0001;1.70±0.33 vs 2.07±0.53,P<0.0001,respectively).The relative expression levels of S100A6 mRNA,LINC00472 and LINC01257 were up-regulated and LINC00312 was down-regulated in human intrahepatic biliary epithelial cells treated with glycochenodeoxycholate compared with control(2.97±0.43 vs 1.09±0.08,P=0.0018;2.70±0.26 vs 1.10±0.10,P=0.0006;2.23±0.21 vs 1.10±0.10,P=0.0011;1.20±0.04 vs 3.03±0.15,P<0.0001,respectively).The mean expression of S100A6 in the advanced stage(III and IV)of PBC was up-regulated compared to that in HCs and the early stage(II)(3.38±0.71 vs 2.09±0.87,P<0.0001;3.38±0.71 vs 2.57±1.21,P=0.0003,respectively);and in the early stage(II),it was higher than that in HCs(2.57±1.21 vs 2.09±0.87,P=0.03).The mean expression of LINC00312 in the advanced stage was lower than that in the early stage and HCs(1.39±0.29 vs 1.56±0.33,P=0.01;1.39±0.29 vs 2.07±0.53,P<0.0001,respectively);in addition,the mean expression of LINC00312 in the early stage was lower than that in HCs(1.56±0.33 vs 2.07±0.53,P<0.0001).The mean expression of log10 LINC00472 in the advanced stage was higher than those in the early stage and HCs(2.99±0.87 vs 1.81±0.83,P<0.0001;2.99±0.87 vs 1.77±0.84,P<0.0001,respectively).The mean expression of LINC01257 in both the early stage and advanced stage were up-regulated compared with HCs(3.88±1.55 vs 2.37±0.96,P<0.0001;3.57±1.79 vs 2.37±0.96,P<0.0001,respectively).The areas under the curves(AUC)for S100A6,LINC00312,log10 LINC00472 and LINC01257 in PBC diagnosis were 0.759,0.7292,0.6942 and 0.7158,respectively.Furthermore,the AUC for these four genes in PBC staging were 0.666,0.661,0.839 and 0.5549,respectively.The expression levels of S100A6 mRNA,log10 LINC00472,and LINC01257 in plasma of PBC patients were decreased(2.35±1.02 vs 3.06±1.04,P=0.0018;1.99±0.83 vs 2.33±0.96,P=0.036;2.84±0.92 vs 3.69±1.54,P=0.0006),and the expression level of LINC00312 was increased(1.95±0.35 vs 1.73±0.32,P=0.0007)after treatment compared with before treatment using the paired t-test.Relative expression of S100A6 mRNA was positively correlated with log10 LINC00472(r=0.683,P<0.0001);serum level of collagen type IV was positively correlated with the relative expression of log10 LINC00472(r=0.482,P<0.0001);relative expression of S100A6 mRNA was positively correlated with the serum level of collagen type IV(r=0.732,P<0.0001).The AUC for the four biomarkers obtained in the validation set were close to the training set.CONCLUSION These four genes may potentially act as novel biomarkers for the diagnosis of PBC.Moreover,LINC00472 acts as a potential biomarker for staging in PBC.展开更多
Background:Bile acids(BAs)facilitate the progression of gastric intestinal metaplasia(GIM).Long non-coding RNAs(lncRNAs)dysregulation was observed along with the initiation of gastric cancer.However,how lncRNAs functi...Background:Bile acids(BAs)facilitate the progression of gastric intestinal metaplasia(GIM).Long non-coding RNAs(lncRNAs)dysregulation was observed along with the initiation of gastric cancer.However,how lncRNAs function in GIM remains unclear.This study aimed to explore the role and mechanism of lncRNA PVT1 in GIM,and provide a potential therapeutic target for GIM treatment.Methods:We employed RNA sequencing(RNA-seq)to screen dysregulated lncRNAs in gastric epithelial cells after BA treatment.Bioinformatics analysis was conducted to reveal the regulatory mechanism.PVT1 expression was detected in 21 paired biopsies obtained under endoscopy.Overexpressed and knockdown cell models were established to explore gene functions in GIM.Molecular interactions were validated by dual-luciferase reporter assay,RNA immunoprecipitation(RIP),and chromatin immunoprecipitation(Ch-IP).The levels of relative molecular expression were detected in GIM tissues.Results:We confirmed that lncRNA PVT1 was upregulated in BA-induced GIM model.PVT1 promoted the expression of intestinal markers such as CDX2,KLF4,and HNF4α.Bioinformatics analysis revealed that miR-34b-5p was a putative target of PVT1.miR-34b-5p mimics increased CDX2,KLF4,and HNF4αlevels.Restoration of miR-34b-5p decreased the pro-metaplastic effect of PVT1.The interactions between PVT1,miR-34b-5p,and the downstream target HNF4α were validated.Moreover,HNF4αcould transcriptionally activated PVT1,sustaining the GIM phenotype.Finally,the activation of the PVT1/miR-34b-5p/HNF4α loop was detected in GIM tissues.Conclusions:BAs facilitate GIM partially via a PVT1/miR-34b-5p/HNF4α positive feedback loop.PVT1 may become a novel target for blocking the continuous development of GIM and preventing the initiation of gastric cancer in patients with bile reflux.展开更多
BACKGROUND Gastric cancer(GC)is a type of cancer which causes high cancer-related mortality.Surgical operation and systematic chemical therapies are primary choices for the treatment of GC patients with advanced stage...BACKGROUND Gastric cancer(GC)is a type of cancer which causes high cancer-related mortality.Surgical operation and systematic chemical therapies are primary choices for the treatment of GC patients with advanced stages,however,the 5-year overall survival is only around 30%.AIM To investigate the role of mesenchymal stem cell(MSC)-derived long non-coding RNAs(lncRNA)NKILA in fatty acid oxidation and chemoresistance in GC cells,mediated through the miR-485-5p/STAT3 pathway.METHODS GC cell lines(AGS and MKN45)were co-cultured with human bone marrowderived MSCs were cultured.The MSC identity was confirmed by flow cytometry(CD73,CD90,CD105>95%positive,CD34,CD45 negative).Co-culture of GC cells and MSCs was performed in Transwell plates,where MSCs were placed in the upper chamber and GC cells in the lower chamber for 72 hours.For transfections,pcDNA-NKILA vectors,shSTAT3,and miR-485-5p mimics were utilized.Colony formation,apoptosis assays(Annexin V/PI staining),sphere formation,and flow cytometry were performed to evaluate cell proliferation,stemness,and chemoresistance.qPCR was used to analyze gene expression(Sox2,Oct4,CD133,LIN28,NKILA),and Western blotting assessed protein levels of stemness markers.Luciferase reporter assays were conducted to confirm miR-485-5p/STAT3 interactions,and biotin-labeled RNA pulldown was used to assess RNA-protein binding.Fatty acid oxidation was evaluated using a CPT1 activity assay andβ-oxidation rate detection.ATP levels were measured to assess the energetic status of GC cells.Clinical GC tissue samples were collected from patients at our hospital for validation.RESULTS MSCs were found to enhance the stemness and chemoresistance of GC cells.Co-culturing MKN45 and AGS cells with MSCs significantly increased sphere-forming ability and the expression of key cancer stem cell markers(SOX2,Oct4,LIN28,CD133),indicating that MSCs promote stem-like properties.Flow cytometry confirmed an enrichment of CD44+and CD133+subpopulations in MSC-treated GC cells.Additionally,MSC co-culture reduced chemotherapy-induced apoptosis and enhanced cell proliferation,suggesting a protective role in chemotherapy resistance.MSC-derived lncRNA NKILA further promoted stemness and chemoresistance,enhancing expression of stem cell markers and protecting cells from oxaliplatin and 5-FU-induced apoptosis.MSC co-culture also induced fatty acid oxidation in GC cells,as shown by increased CPT1 activity,β-oxidation rates,and ATP levels.NKILA mediated these effects by upregulating STAT3,which was confirmed to regulate fatty acid oxidation and chemoresistance.NKILA’s interaction with miR-485-5p further promoted STAT3 expression and fatty acid oxidation,reinforcing its role in maintaining stemness and enhancing chemoresistance.CONCLUSION MSCs enhance the stemness and chemoresistance of GC cells by secreting lncRNA NKILA,which promotes fatty acid oxidation through STAT3 activation.NKILA modulates the miR-485-5p/STAT3 axis,thereby increasing energy metabolism and supporting cancer stem cell properties.Targeting NKILA or the miR-485-5p/STAT3 pathway offers potential therapeutic strategies to overcome chemoresistance in GC.展开更多
基金Supported by National High Technology Research and Development Program of China,No.2015AA020924Natural Science Foundation of Beijing,China,No.7202194 and No.7162185.
文摘BACKGROUND Accumulating evidence has revealed that several long non-coding ribonucleic acids(lncRNAs)are crucial in the progress of hepatocellular carcinoma(HCC).AIM To classify a long non-coding RNA,i.e.,lncRNA W5,and to determine the clinical significance and potential roles of lncRNA W5 in HCC.METHODS The results showed that lncRNA W5 expression was significantly downregulated in HCC cell lines and tissues.Analysis of the association between lncRNA W5 expression levels and clinicopathological features suggested that low lncRNA W5 expression was related to large tumor size(P<0.01),poor histological grade(P<0.05)and serious portal vein tumor thrombosis(P<0.05).Furthermore,Kaplan-Meier survival analysis showed that low expression of lncRNA W5 predicts poor overall survival(P=0.016).RESULTS Gain-of-loss function experiments,including cell counting kit8 assays,colony formation assays,and transwell assays,were performed in vitro to investigate thebiological roles of lncRNA W5.In vitro experiments showed that ectopic overexpression of lncRNA W5 suppressed HCC cell proliferation,migration and invasion;conversely,silencing of lncRNA W5 promoted cell proliferation,migration and invasion.In addition,acting as a tumor suppressor gene in HCC,lncRNA W5 inhibited the growth of HCC xenograft tumors in vivo.CONCLUSION These results showed that lncRNA W5 is down-regulated in HCC,and it may suppress HCC progression and predict poor clinical outcomes in patients with HCC.LncRNA W5 may serve as a potential HCC prognostic biomarker in addition to a therapeutic target.
基金National Natural Science Foundation of China,No.81860433the Natural Science Youth Foundation of Jiangxi Province,No.20192BAB215036+2 种基金the Foundation for Fostering Young Scholar of Nanchang University,No.PY201822National Natural Science Foundation of China,No.81960359the Key Technology Research and Development Program of Jiangxi Province,No.20202BBG73024.
文摘BACKGROUND Investigating molecular biomarkers that accurately predict prognosis is of considerable clinical significance.Accumulating evidence suggests that long noncoding ribonucleic acids(lncRNAs)are frequently aberrantly expressed in colorectal cancer(CRC).AIM To elucidate the prognostic function of multiple lncRNAs serving as biomarkers in CRC.METHODS We performed lncRNA expression profiling using the lncRNA mining approach in large CRC cohorts from The Cancer Genome Atlas(TCGA)database.Receiver operating characteristic analysis was performed to identify the optimal cutoff point at which patients could be classified into the high-risk or low-risk groups.Based on the Cox coefficient of the individual lncRNAs,we identified a ninelncRNA signature that was associated with the survival of CRC patients in the training set(n=175).The prognostic value of this nine-lncRNA signature was validated in the testing set(n=174)and TCGA set(n=349).The prognostic models,consisting of these nine CRC-specific lncRNAs,performed well for risk stratification in the testing set and TCGA set.Time-dependent receiver operating characteristic analysis indicated that this predictive model had good performance.RESULTS Multivariate Cox regression and stratification analysis demonstrated that this nine-lncRNA signature was independent of other clinical features in predicting overall survival.Functional enrichment analysis of Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology terms further indicated that these nine prognostic lncRNAs were closely associated with carcinogenesis-associated pathways and biological functions in CRC.CONCLUSION A nine-lncRNA expression signature was identified and validated that could improve the prognosis prediction of CRC,thereby providing potential prognostic biomarkers and efficient therapeutic targets for patients with CRC.
基金National Natural Science Foundation of China,No.81871723.
文摘BACKGROUND Primary biliary cholangitis(PBC)is a chronic and slowly progressing cholestatic disease,which causes damage to the small intrahepatic bile duct by immunoregulation,and may lead to cholestasis,liver fibrosis,cirrhosis and,eventually,liver failure.AIM To explore the potential diagnosis and staging value of plasma S100 calcium binding protein A6(S100A6)messenger ribonucleic acid(mRNA),LINC00312,LINC00472,and LINC01257 in primary biliary cholangitis.METHODS A total of 145 PBC patients and 110 healthy controls(HCs)were enrolled.Among them,80 PBC patients and 60 HCs were used as the training set,and 65 PBC patients and 50 HCs were used as the validation set.The relative expression levels of plasma S100A6 mRNA,long noncoding ribonucleic acids LINC00312,LINC00472 and LINC01257 were analyzed using quantitative reverse transcription-polymerase chain reaction.The bile duct ligation(BDL)mouse model was used to simulate PBC.Then double immunofluorescence was conducted to verify the overexpression of S100A6 protein in intrahepatic bile duct cells of BDL mice.Human intrahepatic biliary epithelial cells were treated with glycochenodeoxycholate to simulate the cholestatic environment of intrahepatic biliary epithelial cells in PBC.RESULTS The expression of S100A6 protein in intrahepatic bile duct cells was up-regulated in the BDL mouse model compared with sham mice.The relative expression levels of plasma S100A6 mRNA,log10 LINC00472 and LINC01257 were upregulated while LINC00312 was down-regulated in plasma of PBC patients compared with HCs(3.01±1.04 vs 2.09±0.87,P<0.0001;2.46±1.03 vs 1.77±0.84,P<0.0001;3.49±1.64 vs 2.37±0.96,P<0.0001;1.70±0.33 vs 2.07±0.53,P<0.0001,respectively).The relative expression levels of S100A6 mRNA,LINC00472 and LINC01257 were up-regulated and LINC00312 was down-regulated in human intrahepatic biliary epithelial cells treated with glycochenodeoxycholate compared with control(2.97±0.43 vs 1.09±0.08,P=0.0018;2.70±0.26 vs 1.10±0.10,P=0.0006;2.23±0.21 vs 1.10±0.10,P=0.0011;1.20±0.04 vs 3.03±0.15,P<0.0001,respectively).The mean expression of S100A6 in the advanced stage(III and IV)of PBC was up-regulated compared to that in HCs and the early stage(II)(3.38±0.71 vs 2.09±0.87,P<0.0001;3.38±0.71 vs 2.57±1.21,P=0.0003,respectively);and in the early stage(II),it was higher than that in HCs(2.57±1.21 vs 2.09±0.87,P=0.03).The mean expression of LINC00312 in the advanced stage was lower than that in the early stage and HCs(1.39±0.29 vs 1.56±0.33,P=0.01;1.39±0.29 vs 2.07±0.53,P<0.0001,respectively);in addition,the mean expression of LINC00312 in the early stage was lower than that in HCs(1.56±0.33 vs 2.07±0.53,P<0.0001).The mean expression of log10 LINC00472 in the advanced stage was higher than those in the early stage and HCs(2.99±0.87 vs 1.81±0.83,P<0.0001;2.99±0.87 vs 1.77±0.84,P<0.0001,respectively).The mean expression of LINC01257 in both the early stage and advanced stage were up-regulated compared with HCs(3.88±1.55 vs 2.37±0.96,P<0.0001;3.57±1.79 vs 2.37±0.96,P<0.0001,respectively).The areas under the curves(AUC)for S100A6,LINC00312,log10 LINC00472 and LINC01257 in PBC diagnosis were 0.759,0.7292,0.6942 and 0.7158,respectively.Furthermore,the AUC for these four genes in PBC staging were 0.666,0.661,0.839 and 0.5549,respectively.The expression levels of S100A6 mRNA,log10 LINC00472,and LINC01257 in plasma of PBC patients were decreased(2.35±1.02 vs 3.06±1.04,P=0.0018;1.99±0.83 vs 2.33±0.96,P=0.036;2.84±0.92 vs 3.69±1.54,P=0.0006),and the expression level of LINC00312 was increased(1.95±0.35 vs 1.73±0.32,P=0.0007)after treatment compared with before treatment using the paired t-test.Relative expression of S100A6 mRNA was positively correlated with log10 LINC00472(r=0.683,P<0.0001);serum level of collagen type IV was positively correlated with the relative expression of log10 LINC00472(r=0.482,P<0.0001);relative expression of S100A6 mRNA was positively correlated with the serum level of collagen type IV(r=0.732,P<0.0001).The AUC for the four biomarkers obtained in the validation set were close to the training set.CONCLUSION These four genes may potentially act as novel biomarkers for the diagnosis of PBC.Moreover,LINC00472 acts as a potential biomarker for staging in PBC.
基金supported by grants from the National Natural Science Foundation of China(Nos.82170560,82200567,and 82330110)the Booster Plans of Xijing Hospital(No.XJZT21L07)Healthcare lnnovation Capability Enhancement Plan in Shaanxi Province(No.2024TD-06).
文摘Background:Bile acids(BAs)facilitate the progression of gastric intestinal metaplasia(GIM).Long non-coding RNAs(lncRNAs)dysregulation was observed along with the initiation of gastric cancer.However,how lncRNAs function in GIM remains unclear.This study aimed to explore the role and mechanism of lncRNA PVT1 in GIM,and provide a potential therapeutic target for GIM treatment.Methods:We employed RNA sequencing(RNA-seq)to screen dysregulated lncRNAs in gastric epithelial cells after BA treatment.Bioinformatics analysis was conducted to reveal the regulatory mechanism.PVT1 expression was detected in 21 paired biopsies obtained under endoscopy.Overexpressed and knockdown cell models were established to explore gene functions in GIM.Molecular interactions were validated by dual-luciferase reporter assay,RNA immunoprecipitation(RIP),and chromatin immunoprecipitation(Ch-IP).The levels of relative molecular expression were detected in GIM tissues.Results:We confirmed that lncRNA PVT1 was upregulated in BA-induced GIM model.PVT1 promoted the expression of intestinal markers such as CDX2,KLF4,and HNF4α.Bioinformatics analysis revealed that miR-34b-5p was a putative target of PVT1.miR-34b-5p mimics increased CDX2,KLF4,and HNF4αlevels.Restoration of miR-34b-5p decreased the pro-metaplastic effect of PVT1.The interactions between PVT1,miR-34b-5p,and the downstream target HNF4α were validated.Moreover,HNF4αcould transcriptionally activated PVT1,sustaining the GIM phenotype.Finally,the activation of the PVT1/miR-34b-5p/HNF4α loop was detected in GIM tissues.Conclusions:BAs facilitate GIM partially via a PVT1/miR-34b-5p/HNF4α positive feedback loop.PVT1 may become a novel target for blocking the continuous development of GIM and preventing the initiation of gastric cancer in patients with bile reflux.
文摘BACKGROUND Gastric cancer(GC)is a type of cancer which causes high cancer-related mortality.Surgical operation and systematic chemical therapies are primary choices for the treatment of GC patients with advanced stages,however,the 5-year overall survival is only around 30%.AIM To investigate the role of mesenchymal stem cell(MSC)-derived long non-coding RNAs(lncRNA)NKILA in fatty acid oxidation and chemoresistance in GC cells,mediated through the miR-485-5p/STAT3 pathway.METHODS GC cell lines(AGS and MKN45)were co-cultured with human bone marrowderived MSCs were cultured.The MSC identity was confirmed by flow cytometry(CD73,CD90,CD105>95%positive,CD34,CD45 negative).Co-culture of GC cells and MSCs was performed in Transwell plates,where MSCs were placed in the upper chamber and GC cells in the lower chamber for 72 hours.For transfections,pcDNA-NKILA vectors,shSTAT3,and miR-485-5p mimics were utilized.Colony formation,apoptosis assays(Annexin V/PI staining),sphere formation,and flow cytometry were performed to evaluate cell proliferation,stemness,and chemoresistance.qPCR was used to analyze gene expression(Sox2,Oct4,CD133,LIN28,NKILA),and Western blotting assessed protein levels of stemness markers.Luciferase reporter assays were conducted to confirm miR-485-5p/STAT3 interactions,and biotin-labeled RNA pulldown was used to assess RNA-protein binding.Fatty acid oxidation was evaluated using a CPT1 activity assay andβ-oxidation rate detection.ATP levels were measured to assess the energetic status of GC cells.Clinical GC tissue samples were collected from patients at our hospital for validation.RESULTS MSCs were found to enhance the stemness and chemoresistance of GC cells.Co-culturing MKN45 and AGS cells with MSCs significantly increased sphere-forming ability and the expression of key cancer stem cell markers(SOX2,Oct4,LIN28,CD133),indicating that MSCs promote stem-like properties.Flow cytometry confirmed an enrichment of CD44+and CD133+subpopulations in MSC-treated GC cells.Additionally,MSC co-culture reduced chemotherapy-induced apoptosis and enhanced cell proliferation,suggesting a protective role in chemotherapy resistance.MSC-derived lncRNA NKILA further promoted stemness and chemoresistance,enhancing expression of stem cell markers and protecting cells from oxaliplatin and 5-FU-induced apoptosis.MSC co-culture also induced fatty acid oxidation in GC cells,as shown by increased CPT1 activity,β-oxidation rates,and ATP levels.NKILA mediated these effects by upregulating STAT3,which was confirmed to regulate fatty acid oxidation and chemoresistance.NKILA’s interaction with miR-485-5p further promoted STAT3 expression and fatty acid oxidation,reinforcing its role in maintaining stemness and enhancing chemoresistance.CONCLUSION MSCs enhance the stemness and chemoresistance of GC cells by secreting lncRNA NKILA,which promotes fatty acid oxidation through STAT3 activation.NKILA modulates the miR-485-5p/STAT3 axis,thereby increasing energy metabolism and supporting cancer stem cell properties.Targeting NKILA or the miR-485-5p/STAT3 pathway offers potential therapeutic strategies to overcome chemoresistance in GC.
