| Background and objectiveGastric cancer is a common malignant tumor of the digestive tract,which seriously threatens the health of our residents.The onset of gastric cancer is occult.When it is discovered,tumor metastasis and disease progression often occur,and the best time for treatment are missed.Therefore,comprehensive treatment such as tumor chemotherapy plays a dominant role in the treatment of gastric cancer,but its effect is still negatively affected by chemotherapy resistance.Therefore,it is of great clinical significance to elucidate the molecular mechanisms involved in drug resistance to gastric cancer and to find therapeutic targets for inhibiting or even reversing drug resistance in gastric cancer.Chemotherapy is an indispensable part of comprehensive treatment of tumors,but it often fails due to multidrug resistance,leading to an increase in cancer-related mortality.The efficacy of multidrug resistance research on tumor chemotherapy is becoming more and more important as multidrug resistance is increasing.Multi-drugs resistance(MDR)is a phenomenon that occurs frequently after long-term chemotherapy,leads to refractory cancer,and can induce tumor recurrence.Numerous studies have shown that there is a close and complex relationship between multidrug resistance and autophagy.Autophagy-associated gene(ATG)plays an important role in autophagy,and ATG14 plays an important role in autophagy.Under normal conditions,Beclin 1 forms a complex with hVps34,ATG 14 and p150.When the cell is in a stressful environment,Bcl-2 dissociates from the Beclin 1-hVps34-ATG14-p150 complex,inducing autophagy expression.How to regulate the synthesis and expression of ATG 14 protein is the key to the autophagy process,and the stability of mRNA and the transcriptional efficiency of translation are crucial for proteins.This paper mainly discusses the inhibition of autophagy from the stability of mRNA.Both long-chain non-coding RNA(LncRNA)and microRNA(miRNA)can affect the stability of mRNA.LncRNA can increase the stability of mRNA and the transcriptional efficiency of translation by binding to the positive strand of mRNA.It can also mask the target of miRNA enzyme in its positive strand,thus preventing the degradation of mRNA by miRNA.miRNAs mainly affect the stability of mRNA by binding to the 3’UTR region of the target gene,or regulate the expression of target genes at the post-transcriptional level.In addition,studies have shown that miRNAs may target autophagy-associated genes as targets,and together with Lnc regulate the function of autophagy-related genes,leading to cell resistance.Through TCGA(The Cancer Genome Atlas)database analysis,we found that LncRNA EIF3J-AS1 is significantly up-regulated in poor prognosis of melanin,colorectal cancer,prostate cancer and other tumors,but less in gastric cancer.In vitro and in vivo studies have confirmed that EIF3J-AS1 promotes autophagy in gastric cancer and can produce multidrug resistance,and ATG 14,an autophagy-related protein,plays a key role in multidrug resistance.Through bioinformatics analysis and experimental verification,we confirmed that EIF3J-AS1 and ATG14 have binding sites,and found that miRNA mir-188-3p interacts with the above two genes and targets ATG 14.Combined with bioinformatics analysis and experimental verification,we found that EIF3-AS1 regulates ATG 14 by adsorbing miR-188-3p.EIF3J-AS1 can inhibit the inhibition of ATG 14 mRNA by miR-188-3p by competitive binding to miR-188-3p,and promote the expression of ATG14,thereby mediating autophagy and chemoresistance.This study wants to explore how EIF3J-AS1 and mir-188-3p can participate in the regulation of autophagy,provide experimental theoretical basis for reversing multidrug resistance,and seek new targets for sensitizing chemotherapy and improving patient prognosis.Experimental methods and results1.Autophagy mediates chemoresistanceIn this chapter,Western blot analysis showed that the autophagy level of tumor cells increased with the stimulation time.The ratio of autophagy markers did not change significantly after the addition of autophagy inhibitors.The electron microscopy confirmed the self-stimulation in tumor cells after stimulation with chemotherapy drugs.The number of phagosomes was significantly higher than before stimulation.MTT experiments confirmed that the oxaliplatin/5-Fu resistant strain constructed in the laboratory was more tolerant to oxaliplatin/5-Fu than the sensitive strain.The level of autophagy in the successfully constructed resistant strains was higher than that in the sensitive strains by Western blot.Both confocal microscopy and transmission electron microscopy suggest the formation of autophagosomes.Autophagy inhibitor chloroquine reversed the tolerance of drug-resistant strains to chemotherapeutic drugs,suggesting that tumor cells are resistant to chemotherapeutic drugs and are associated with autophagy.2.EIF3J-AS1 promotes chemotherapy resistance in gastric cancer(1)Gene chip screening in drug-resistant strains expressed abundance change multiples twice times and ranked above the top five lncRNA,respectively,ACTG1P4,HLA-J,RPL23P3,ANXA2P3 and EIF3J-AS1,and qPCR experiments to detect the abundance of gene expression in wild and drug-resistant cell lines;EIF3J-AS1 was confirmed to be a gene that mediates chemoresistance by MTT assay after silencing RNA.Further analysis of the data by GEO and KM plottet database,high expression of EIF3J-AS1 suggest a poor prognosis of chemotherapy patients,suggesting that EIF3J-AS1 is a key drug resistance gene.(2)Data screening of GEO’s genetic datasets,EIF3J-AS1 is highly expressed in a variety of tumor-resistant strains,and in the clinical database,the prognosis of patients has a negative correlation with its expression;at the genetic level,The drug-resistant and wild-type strains were verified by qPCR experiments,and the results showed that the expression of EIF3J-AS1 in the drug-resistant strain was up-regulated.It was confirmed by MTT assay that if EIF3J-AS1 was silenced,its tolerance to chemotherapeutic drugs was greatly reduced.To further validate the function of EIF3J-AS1,a plate cloning experiment and a mouse tumor formation model were used,which together showed that the growth of tumor cells was significantly inhibited after EIF3J-AS1 was silenced.3.EIF3J-AS1 promotes the mechanism of chemotherapy resistance in gastric cancer(1)In vitro,Western blot was used to explore the protein level of NC(transient strain of EIF3J-AS1 not silenced)and SI(transient strain of EIF3J-AS1 silenced)to verify the different concentrations of chemotherapy drugs and autophagy inhibitors/Under the stimulating condition of agonist,EIF3J-AS1 and autophagy have a promoting relationship;by electron microscopy and confocal fluorescence,the visual expression of autophagosome increases is related to EIF3J-AS1,indicating that EIF3J-AS1 has a promoting effect on the autophagy level of gastric cancer cells.(2)At the data analysis level,the IFGA Nature 2014 and TCGA Provisional databases were used for analysis.The EIF3J-AS1 cell line was obtained by constructing and transfecting the plasmid,and the mRNA of EIF3J-AS1 and ATG14 was confirmed by qPCR,Western blot and MTT assays.The expression amount showed a significant positive correlation.Flow cytometry confirmed that ATG14 enhanced cell resistance and may be associated with its inhibition of cell apoptosis.EIF3J-AS1 and ATG14 jointly regulate the autophagy level of tumor cells and the resistance to chemotherapy drugs.(3)ATG14 participates in EIF3J-AS1 regulation of drug resistance and autophagyThe cell line was transfected into pcDNA(empty),EIF3J-AS1(containing EIF3J-AS1 plasmid,EIF3J-AS1 overexpressing cell line),EIF3J-AS1+siRNAl,EIF3J-AS1+siRNA2(transfection silencing ATG14)Four groups of EIF3J-AS1 overexpressing cell lines were confirmed by MTT and clone formation experiments.Silencing ATG14 partially reversed the drug resistance and autophagy effect of EIF3J-AS1.Silencing of ATG14 partially reverses the chemoresistance of EIF3J-AS1 overexpression,and ATG14 is also involved in EIF3J-AS1-mediated drug resistance and autophagy.4.EIF3J-AS1 promotes the stability of ATG14 mRNA(1)Gene sequencing revealed that ATG14 mRNA and EIF3J-AS1 have numerous binding sites.It was confirmed by FISH experiments that EIF3J-AS1 is in the cytoplasm,and it is considered that it may affect the biological function of ATG14 mRNA by regulating the stability of mRNA.The qPCR assay was used to detect the intracellular mRNA content in each time period,suggesting that EIF3J-AS1 may slow down the degradation of mRNA by specific binding to ATG14 mRNA.The mutant Mut1 of EIF3J-AS1 was made to be unable to bind to ATG14 mRNA.It was confirmed by MTT,clone formation and protein level,and immunofluorescence that the cells that could not bind to the ATG14 mRNA-binding mutant showed a significant decrease in resistance to chemotherapeutic drugs and autophagy.(1)It was confirmed by gene sequencing that there is a binding site between miR-188-3p and EIF3J-AS1 and ATG14,so there is a possibility of structural interaction.qPCR and Western blot confirmed that the content of mir-188-3p was negatively correlated with the expression of ATG14 mRNA and its protein,suggesting that miR-188-3p can inhibit the expression of ATG14 and reduce the intracellular content of mRNA.(2)The ATG14 3’UTR(mt),Mutant 3’ UTR(mt)luciferase vector was constructed with psiCHECK2 as a no-load to change its binding ability to miR-188-3p.The luciferase gene reporter experiment confirmed that the luciferase activity of the mt group was more than double that of the wt group,suggesting that ATG14 is the target gene of miR-188-3p,and there is a direct binding effect between the two.On the protein marker level and fluorescence confocal microscopy,it was found that the autophagy decreased in the cell line transferred to MiR-188-3p,suggesting that MiR-188-3p has the function of autophagy inhibition.(3)Sequencing suggests that there are also base pairs that are bound to each other between EIF3J-AS1 and mir-188-3p.MTT assay confirmed that miR-188-3p partially restored EIF3J-AS1-induced chemoresistance,and constructed a mutant plasmid Mut2 with decreased binding to mir-188-3p.MTT2 transfectants were found to be resistant to chemotherapeutic drugs by MTT assay.Decreased,suggesting that drug resistance may be achieved by the combination of miR-188-3p and EIF3J-AS1.Western blot analysis of autophagy protein markers and immunofluorescence showed that the level of autophagy in MUT2 was lower than that in EIF3J-AS1,suggesting that mir-188-3p can reverse EIF3J-AS1-induced autophagy by binding to EIF3J-AS1.Conclusion1.Tumor cells in the case of chemotherapy drug stimulation will occur drug resistance,and its drug resistance strains in the high level of autophagy,inhibit the reversal of chemotherapy resistance after autophagy,inhibition of autophagy may be a breakthrough in the target of tumor resistance.2.EIF3J-AS1 after silence to promote tumor cells to produce drug resistance,and its abundance and poor prognosis related.3.EIF3J-AS1 through the stabilization of ATG14 MRNA to promote the autophagy of tumor cells and resistance to chemotherapy drugs4.EIF3J-AS1 competitive combination of mir-188-3p,the release of mir-188-3p to ATG14 mRNA inhibition,promote ATG14 expression,so as to mediate autophagy and chemotherapy resistance. |
PDF Full Text Request