| Part1 Generation of Drosha-independent microRNA6778-5p and its effects on gastric cancer stem cellsObjective:Our previousstudyfound that there are a set of upregulated non-canonical miRNAs in Drosha-knocked down gastric cancer(GC)MGC-803 cells by miRNA array analysis.Knockdown of Drosha did not completely reverse the malignant features of gastric cancer.Due to the aberrantnon-canonical miRNAs in Drosha-knocked downgastriccancercells,wewonderedwhetherthese Drosha-independent miRNAs play an important role in Drosha-silenced malignant behavior of gastric cancer cells(eg,gastric cancer stem cell maintenance).Therefore,our research aims to:(1)To investigate whether microRNA6778-5p(miR6778-5p)is a Drosha-independent microRNA.(2)To explore the generated mechanism of miR6778-5p.(3)To investigate the effect of miR6778-5pon gastric cancer stem cells.Methods:To validate accuracy of the microarray data,the expressions of up-regulated miRNAs were further verified in Drosha-knocked down(Drosha KD)gastric cancer MGC-803 cells by qRT-PCR.Using bioinformatics,we analyzed candidate miRNAs.Using UCSC Genome Browser Home(http://genome.ucsc.edu/),we find host genes for candidate miRNA.QRT-PCR detects the expression of candidate miRNA and their host genes in different Drosha KD gastric cancer cell lines.Plasmid construction methods for splicing analysis.The splicing mutation of host gene was verified by Real-time PCR(RT-PCR),and the expression of miRNA after host gene mutation was by qRT-PCR.The sequences of miR6778-5p were synthesized to constructto construct miR6778-5poverexpressedviruses.Interferencesequencesof miR6778-5p were designed and recombined into lentiviral vectors to constructmiR6778-5pknockdownviruses.Overexpressedand knockdown viruses were transfected into Drosha wild-type cells of gastric cancer(MGC-803/Drosha WT and SGC-7901/Drosha WT)and Drosha knockout cells of gastric cancer(MGC-803/Drosha KD and SGC-7901/Drosha KD)to establish stable cell lines.The expression of miR6778-5p was detected by qRT-PCR.The expression of Drosha,CD44and miR6778-5p in gastric cancer tissues and corresponding adjacent tissues were verified by qRT-PCR.Gastric cancer stem cells(GCSCs)were cultured to observethe formation efficiencies of GCSC spheres and spheres size of indicated over-expressed and knocked down cell lines.Gastric cancer stem cells(1×10~5)were subcutaneously injected into4-week-old female nude mice,and observe the tumorigenesis ability in vivo.Results:we discovered more than 10 aberrant non-canonical miRNAs in Drosha-knocked down gastric cancer cells,and we found miR6778-5p was significantly increased following Drosha knocked-down.Using bioinformatics,miR6778-5p,may a 5'tail mirtron,is derived fromintron 5 of SHMT1.The expression of miR6778-5p was decreased when splicing site mutation.The overexpress and knockdown cell lines were successfully constructed.There were no significant changes of CD44between Drosha lower and higher expressing gastric tumors.Compared to gastric tumors with high expression of Drosha,the expressionof miR6778-5p was higher in gastric tumors with low expression of Drosha.In Drosha wild-type(Drosha WT)and Drosha KDgastric cancer stem cells,knockdown of miR6778-5p reduced cancer stem cell(CSC)formation efficiencies and CSC sphere volume,and knocked-down Drosha and miR6778-5p simultaneously in gastric cancer cells,the formation ability of stem cell is more obvious.Overexpress miR6778-5p in gastric cancer cells enhances the formation ability of stem cell.Compared with the MGC-803/Drosha KD group,the formation ability of gastric cancer stem cellwas reduced in MGC-803/Drosha KD/miR6778-5p KDgroup.Conclusions:Silencing Drosha,we found the presence of non-canonicalmicroRNAs(e.g.miR6778-5p)ingastriccancer cell.MiR6778-5p,a 5'tail mirtron,is generated by splicing of intron 5from SHMT1.MiR6778-5p enhances gastric cancer stem cells stemness.Part2 Molecular mechanism of the effect of micro RNA6778-5p on gastric cancer stem cellsObjective:To explore the mechanism of miR6778-5p enhanced gastric cancer stem cells stemness.Methods:Using bioinformatics,the target genes of miR6778-5p were predicted.The mRNA levels of candidate target genes in Drosha KD gastric cancer cells were validated.Target of miR6778-5p were determined by Luciferase reporter assay,q RT-PCR and western blot(WB).Observing the formation ability of gastric cancer stem cells(GCSCs) with overexpress or knockdown target gene.Observing the formation ability of gastric cancer stem cells with overexpress or knockdown SHMT1.Review literature to verify the regulation of miR6778-5p on SHMT1.Treating gastric cancer stem cells with Serine and observing the formation ability of GCSCs.Using isotope-tracking,we observe the transition of mitochondria and cytoplasmic carbon metabolism in gastric cancer cells.QRT-PCR detectedthe expression of miR6778-5p,SHMT1 and CD44 in gastric cancer tissues and adjacent normal tissues,5-FU sensitive gastric cancer tissues and 5-FU-resistant gastric cancer tissues.The gastric cancer stem cells were treated with 5-FU,and observing the formation ability of gastric cancer stem cells.The indicating GCSCs were injected subcutaneouslymice.Results: YWHAE is a target of microRNA6778-5p(miR6778-5p)by Luciferase reports,q RT-PCR and western blot(WB).YWHAE impedes the formation ability of GSCSs,while SHMT1 enhances the formation ability of GSCSs.miR6778-5p can positively regulate SHMT1 by targeted inhibition of YWHAE.Serine can restore the formation ability of GCSCs.Using Isotope tracking,more 10-formyl-THF and M + 1 d TTP in Drosha WTGCSC,however,more N5,N10-methylenetetrahydrofolate(5,10-m THF)and M + 2 d TTP in Drosha KD GCSC.Knockdown SHMT2 in Drosha WT gastric cancer cells reduced M + 1 d TTP,however,knockdown miR6778-5p,SHMT1 or SHMT2 in Drosha KD gastric cancer cells,did not affected the content of M + 1 d TTP.In contrast,loss of miR6778-5p,SHMT1 or SHMT2 had little effect on M + 2 d TTP in Drosha WT GCSCs,but knocking down miR6778-5p or SHMT1 significantly reduced M + 2 d TTP in Drosha KD GCSCs.High levels of miR6778-5p,SHMT1,and CD44 were detected in gastric cancer tissues compared to para-cancerous tissues;higher levels of miR6778-5p,SHMT1 and CD44 were detected in 5-FU-resistant gastric tumors compared to 5-FU-sensitive tumors.Compared with Drosha KD GCSC,5-FU reduced GCSC formationin Drosha WT GCSC.Knocking down miR6778-5p or SHMT1 in Drosha WT celldid not significantly reduce CSC formation.However,loss of miR6778-5p or SHMT1 in Drosha KD cell significantly reduced the formation ability of GCSC under 5-FU treatment.Conclusions:miR6778-5p can positively regulate SHMT1 by targeted inhibition of YWHAE,thus affecting the formation of gastric cancer stem cells.Furthermore,miR6778-5p-SHMT1 signal axis contributes to maintain GCSCs via the cytoplasmic one carbon metabolisms. |
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