MSCs-regulated LncRNA MACC1-AS1 Promotes Stemness And Chemoresistance In Gastric Cancer Through Fatty Acid Oxidation

BackgroundCancer cells that possess the character of stem cells are called cancer stem cells(CSCs)or tumor initiating cells(TICs),a small cluster of cells that are in quiescent or dormant state and have the capacity of self-renewal and multi-differentiation.They are one of the key factors for tumor initiation and are identified as the 'seed cells' for tumor recurrence.'Sternness' refers to these collective functions of cancer stem cells.Sternness plays a pivotal role in chemoresistance.Mesenchymal stem cells(MSCs),derived from mesoderm,are non-hematopoietic adult stem cells,which are the important components of tumor environment.Emerging evidences indicate MSCs play a significant role in tumor progress,especially tumor sternness and chemotherapy resistance.However,the underlying mechanism is obscure.Besides glucose and glutamine,fatty acid is another important energy source for tumor cells.Fatty acid oxidation(FAO)in mitochondria provides 2.5 times as much ATP in comparison with oxidation of glucose.It is reported that FAO is able to promote tumor sternness and MSCs can induce chemotherapy by release of polyunsaturated fatty acids.Thus,MSCs may promote sternness and chemoresistance through FAO.Long non coding RNA(LncRNA)MACC1-AS1 is the antisense LncRNA of metastasis-associated in colon cancer 1(MACC1).We found that MACC1-AS1 expression was evidently enhanced in gastric cancer(GC)cells interacted with MSCs and MACC1-AS1 overexpression promoted FAO and ATP production.Based on these findings,we speculated MACC1-AS1 play an important role in MSC-induced sternness and chemoresistance dependent on FAO.Hepothesis1.MSCs promote sternness and chemoresistance in GC cells.2.FAO plays an import role in MSCs-induced stemness and chemoresistance.3.MSCs-regulated LncRNA MACC1-AS1 promotes sternness and chemoresistance through FAO.Research contents1.The effect of MSCs on stemness and chemoresistance in GC cells.We firstly detected the mRNA and protein level of stemness-associated markers CD133?OCT4?SOX2?LIN28 by q-PCR and western blotting in GC cells with or without MSCs co-culture.Flow cytometric analysis was then employed to detect for the population of CD44 positive(CD44+)GC cells,one of the stemness markers,with or without co-culture with MSCs.Moreover,sphere formation assay was applied to assess the self-renewal capacity of GC cells after MSCs co-culture.Clone formation assay was used to evaluate the ability of clone formation of GC cells with or without co-culture with MSCs when treated with 5-FU and OX.In vivo limiting dilution assay(LDA)was used to construct subcutaneous transplanted model in nude mouse.GC cells diluted serially to the desired doses,and then co-injected subcutaneously with MSCs into nude mouse.The number of tumors was counted to appraise the tumor initiation of MSCs.2.The effect of FAO on MSCs-induced stemness and chemoresistance.After demonstration of the promoted effect of MSCs on sternness and chemoresistance in GC cells,we further investigated the role of FAO in this process.Firstly,q-PCR and western blotting were used to detect the expression of FAO enzymes in GC cells culture either alone or with MSCs.Then,the rate of fatty acid P oxidation and ATP production were employed after co-culture with MSCs.Moreover,GC cells were transfected with siCPT1 sequences.We detected the mRNA and protein level of sternness-associated markers CD 133?OCT4?SOX2?LIN28 by q-PCR and western blotting in GC cells after silencing CPT1 and MTT assay was applied to sensitivity of GC cells to 5-FU and OX after silencing CPT1.Lastly,rescues experiments were applied to assess the role of FAO on MSCs-induced sternness and chemoresistance with 100 ?mol/1 ETX for 48h.3.The regulation of MACC1-AS1 on MSCs-induced sternness and chemoresistance.q-PCR was used to detect mRNA level of MACC1-AS1 in GC cells,sphere of GC cells alone or culture with MSCs,tumor formatted from GC cells or MSCs in combined with GC cells.Then expression of sternness-associated markers by q-PCR and western blotting,sphere formation capability by sphere formation assay,resistance to 5-FU and OX by clone formation assay and MTT assay were evaluated after stable transfection with MACC1-AS1.4.The effect of FAO on MACC1-AS1-induced sternness and chemoresistance.Firstly,q-PCR and western blotting were used to detect the expression of FAO enzymes in GC cells table transfected with MACC1-AS1.The rate of FAO and ATP production were also evaluated after stable transfection with MACC1-AS1.Then mRNA and protein level of sternness-associated markers was measured when GC cells were overexpressed MACC1-AS1 and with or without 100 ?mol/1 ETX for 48h.Sphere formation capability by sphere formation assay and sensitivity to 5-FU and OX by MTT assay were evaluated after stable transfection with MACC1-AS1 with or without ETX.5.The mechanism of MACC1-AS 1-induced stemness and chemoresistance through FAO.To further investigate the mechanism of MACC1-AS 1-induced sternness and chemoresistance through FAO,we found that miR-145-5p was one of the target miRNAs of MACC1-AS1 predicted by LncRNASNP database.To confirm their regulation,q-PCR was used to detect the miR-145-5p expression after MACC1-AS1 overexpression.Next,the role of miR-145-5p on sternness and chemoresistance was further studied.Additionally,whether overexpression miR-145-5p could reverse the role of MACC1-AS1 on sternness and chemoresistance was conformed by detecting expression of sternness-associated markers,sphere formation capability and sensitivity to 5-FU and OX.6.The effect of MSCs on chemoresistance in vivo after inhibition of FAOTo evaluate the effect of MSCs on chemoresistance in vivo after inhibition of FAO,MKN45 cells alone or MKN45 together with MSCs were subcutaneously implanted into right flank of nude mice.Mice were divied into four groups:GC?GC+FOLFOX?GC+FOLFOX+MSC?GC+FOLFOX+MSC+ETX.Tumor volume was detected every 3 days.Expressions of sternness-associated marker OCT4,key enzyme of CPT1,ki-67 and cleaved caspase 3 were measured by immunohistochemical staining.Structures of hearts,livers,spleens,lungs and kidneys were showed by hematoxylin and eosin(H&E)staining.Results1.MSCs promote stemness and chemoresistance in GC cells.After co-culture with MSCs,sternness-associated markers of GC cells were enhanced in mRNA and protein levels.The population of CD44 positive(CD44+)GC cells evaluated by flow cytometric analysis was significantly increased.Moreover,sphere formation ability of GC cells was obviously increased and the sensibility to 5-FU and OX were remarkably decreased in presence of MSCs.Accordingly,MSCs can promote sternness and chemoresistance in GC cells.In vivo,compared to GC injection alone,the presence of admixed MSCs increased the tumor-initiating frequencies.2.Fatty acid oxidation plays an important role in MSC induced-sternness and chemoresistance.We found that expression of FAO enzymes was increased when GC cells was co-culture with MSCs.Consistently,FAO rate and ATP level of GC cells were enhanced in present of MSCs.Silence of CPT1 suppressed sternness and chemoresistance of GC cells.ETX reversed the expression of sternness-associated markers and the ability of sphere formation induced by MSCs.Sensitivity to 5-FU and OX were increased in GC cells with MSCs when ETX was added.Besides,inhibition of FAO with ETX impairs ATP production in GC cells co-cultured with MSCs.3.MACC1-AS1 is induced by MSCs and contributes to stemness and chemoresistance.Expression of MACC1-AS1 is obviously increased both in GC cells and sphere of CG cells after co-culture with MSCs.Additionally,compared to injection of GC cells alone,expression of MACC1-AS1 in subcutaneous tumor of nude mice was significantly increased in GC cells admixed MSCs.Sternness-associated markers expression and sphere formation capability of GC cells were enhanced after stable transfection with MACC1-AS1.Moreover,overexpression of MACC1-AS1 facilitated the ability of clone formation and reduced growth inhibition when treated with 5-FU and OX.4.The role of MACC1-AS1 on stemness and chemoresistance is dependent on fatty acid oxidation.Overexpression of MACC1-AS1 increased the expression of FAO enzymes,Consistent results can be obtained from detection of FAO rate and ATP production.ETX reversed the promoted role of MACC1-AS1 on expression of stemness-associated markers.Also,sphere formation assay and MTT assay showed that ETX treatment abrogated the promoted effect of MACC1-AS1 on the ability of self-renewal and reversed the inhibited effect of MACC1-AS1 on the sensitivity of 5-FU and OX,respectively.Moreover,ETX remarkably reduced the acceleration of MACC1-AS1 on mitochondria ATP production.5.MACC1-AS1 promoted stemness and chemoresistance through fatty acid oxidation is mediated by miR-145-5p.miR-145-5p was one of the target miRNAs of MACC1-AS1 predicted by LncRNASNP database.miR-145-5p expression was significantly decreased after overexpression of MACC1-AS1.Moreover,miR-145-5p significantly repressed stemness and chemoresistance in GC cells.Overexpression miR-145-5p obviously abolished stemness,chemoresistance and FAO induced by MACC1-AS1.6.Inhibition of fatty acid oxidation attenuates MSC-induced chemoresistance in vivo.Intraperitoneal administration of FAO inhibitor ETX attenuates MSC-induced chemoresistance in vivo.Immunohistochemical staining showed that expression of stemness-associated marker OCT4,key enzyme of CPT1 and ki-67 are obviously decreased with ETX,and the apoptotic marker cleaved caspase 3 was markedly increased.H&E stained sections of hearts,livers,spleens,lungs and kidneys showed that the structures had not significantly different between groups with and without ETX.ConclusionMSCs-regulated LncRNA MACC1-AS1 promotes sternness and chemoresistance in gastric cancer through fatty acid oxidation.