| Background & Aims:The aberrant classical mi RNAs are considered to play significant roles in tumor progression,while it remains unclear for non-classical mi RNAs(named Drosha-independent mirtrons)in the process of various biology.Here,we aim to reveal the key role of a non-classical mirtronic mi R-4646-5p in gastric cancer metastasis.Methods:(1)Abnormally highly expressed mi RNAs in Drosha knocked-down gastric cancer cells were identified by mi RNA chips.Among them,13 potential non-classical mi RNAs(Drosha-independent mirtrons)were screened out by literature review and biometric analysis.And the expression of 3 classic mi RNAs and 13 potential mirtrons in the chip were verified by q RT-PCR,and mirtronic mi RNA-4646-5p with the largest expression was selected as the study objective.Then,its expression was verified by q RT-PCR in Drosha-silenced gastric cancer cells.The expression of mi RNA-4646-5p in gastric tumor samples and its relationship with the prognosis and metastasis of gastric cancer patients were verified using star BASE data,TCGA STAD data and our cohort of GC patients.The effect of mi RNA-4646-5p on the invasion and metastasis of gastric cancer cells was evaluated in vivo and in vitro.(2)The relationship between mi RNA-4646-5p and its host gene Abhd16 a was explored using the UCSC database.The formation of mi RNA-4646-5p by the specific splicing of intron 3 of its host gene Abhd16 a was confirmed by intron mutation experiments.Furthermore,the expression of host gene Abhd16 a in Drosha-silenced gastric cancer cells was tested by q RT-PCR and Western Blot.The Human Splicing Finder online tool was used to analyze the potential splicing factors SRSF1,SRSF2 and SRSF5 on intron 3 of the host gene Abhd16 a.The expression of splicing factors in Drosha-silenced gastric cancer cells was detected by q RT-PCR and up-regulated SRSF2 was picked out.By knocking down SRSF2 in Drosha-silenced gastric cancer cells,change in mi RNA-4646-5p expression was detected with q RT-PCR.The clinical relevance of Drosha,SRSF2,ABHD16 A and mi R-4646-5p was evaluated using tumor samples collected from patients with gastric cancer.(3)The potential target genes of mi RNA-4646-5p were predicted in the mi RDB and Target Scan databases,and compared with the down-regulated genes in the Drosha knockdown gene chip of gastric cancer,and five potential target genes PHD3,LRG1,EPHA3,SMAD9 and COL23A1 were screened out.Next,the potential target gene expression changes in Drosha knockdown gastric cancer cells,mi RNA-4646-5p overexpression or knockdown GC cells were detected by q RT-PCR,and PHD3 with the most significant difference was selected.Furthermore,the relationship between mi RNA-4646-5p and the target gene PHD3 was verified using luciferase reporter experiment.The effects of mi R-4646-5p and its target PHD3 on the ubiquitination level of HIF1 A were evaluated with IP-WB or with protease inhibitors.The regulatory effect of HIF1 A as a transcription factor on Abhd16 a has been confirmed using biometric analysis,CHIP and dual luciferase report experiments.HIF1 A was further overexpressed in GC cells,and the expression changes of host genes Abhd16 a and mi RNA-4646-5p were detected by q RT-PCR.(4)The relationship between the expression of ABHD16 A and the prognosis and metastasis of GC patients were evaluated using the TCGA database.By constructing ABHD16 A over-expressing cell line,Drosha and ABHD16 A double knocked-down GC cell lines,the efficiency of ABHD16 A knockdown or overexpression was verified using Western Blot.Furthermore,the effect of ABHD16 A on the invasiveness of GC cells was evaluated by the Transwell assay.The changes in lipid metabolites caused by ABHD16 A and mi RNA-4646-5p were analyzed by LC/MS-MS,and abnormal accumulation of lyso-phosphatidylserine(lyso-PS)was found out.By exogenously adding lyso-PS to GC cells,the changes in the invasion ability of gastric cancer cells are detected by Transwell assay.And in vivo,using liver and lung metastasis models to explore the effect of ABHD16 A and lyso-PS on the invasion ability of GC cells.(5)Gastric cancer cells are processed by exogenous lyso-PS,and changes in downstream pathways are analyzed using RNA-Seq and bioinformatics.The expression of GPR34,P2Y10 and GPR174 in GC cells were detected by RT-PCR.Using the Rho binding domain(RBD)of the effector protein Rhotekin as a probe,the effect of mi R-4646-5p/ABHD16A/lyso-PS/GPR34/Gi axis on the activation of Rho A was evaluated.The relationship between HIF1 A and Rho A expression was tested by CHIP and dual luciferase experiments.Then,the effect of mi R-4646-5p/PHD3/HIF1 A axis on Rho A expression was tested by Western Blot.Furthermore,the effects of Rho A activation and up-regulation on LIMK/cofilin phosphorylation levels and GC cell invasiveness were evaluated using Western Blot and Transwell experiments.In the liver and lung metastases tissue of mice in vivo,changes in Rho A/LIMK/cofilin were verified by Western Blot,and changes in lyso-PS levels were detected by LC/MS-MS.In clinic,the downstream pathways of mi R-4646-5p or ABHD16 A overexpression are enriched by GSEA with TCGA database.In the samples of GC patients with metastasis,the level of GPR34 and Rho A was detected by q RT-PCR and the expression of ABHD16A/GPR34/HIF1A/Rho A/p-LIMK/P-cofilin was detected by immunohistochemistry.Results:(1)Drosha knockdown or low-expression in gastric cancer cells was accompanied by abnormal elevation of non-classical mirtrons,especially mirtronic mi R-4646-5p.The mirtronic mi R-4646-5p was highly expressed in GC and positively related to poor prognosis and metastasis of gastric cancer.mi R-4646-5p promoted the invasion and metastasis of gastric cancer cells in vivo and in vitro.(2)mi R-4646-5p was a 5′-tail mirtron,which was formed by intron-3specific splicing of its host gene Abhd16 a.And the m RNA and protein levels of ABHD16 A were increased in Drosha knocked-down GC cells.High level of splicing factor SRSF2 in Drosha decreased GC cells promoted the intron specific splicing of Abhd16 a,leading to an up-regulated mirtronic mi R-4646-5p in GC.(3)PHD3 was a direct target gene of mi R-4646-5p and down-regulated in Drosha KD cells.mi R-4646-5p could mitigate ubiquitination of HIF1 A by inhibiting the target gene PHD3,thus lead to an enhanced HIF1 A in Drosha-knockdown gastric tumor cells.The expression of ABHD16 A had the same trend as HIF1 A done in these GC cells.HIF1 A can act as a transcription factor of Abhd16 a and feedback promoted expression of the host gene Abhd16 a and mi R-4646-5p.(4)High expression of ABHD16 A in GC correlated with poor prognosis and metastasis.In vitro experiments showed that ABHD16 A overexpression in gastric cancer cells promoted the invasion and metastasis of gastric cancer cells.Metabonomics results showed that ABHD16 A participated in the lipid metabolism of gastric cancer,causing the accumulation of the metabolite lyso-PS.The accumulation of lyso-PS mediated by mi R-4646-5p/ABHD16 A promoted the invasion and metastasis of gastric cancer cells in vitro and in vivo.(5)mi R-4646-5p/PHD3/HIF1 A axis up-regulated Rho A transcription and the Rho A activation can be triggered by mi R-4646-5p/ABHD16A-mediated lipid metabolite lyso-PS accumulation in gastric cancer cells.mi R-4646-5p/PHD3/HIF1A-mediated up-regulation of Rho A and mi R-4646-5p/ABHD16A/lyso-PS stimulated activation of Rho A synergistically trigger LIMK/cofilin signaling to promote GC metastasis in vitro and in vivo.GSEA enrichment plots showed that mi R-4646-5p and ABHD16 A expression was positively correlated with Rho-GTPase and cytoskeleton related signaling pathways.The expression of ABHD16 A,HIF1A,Rho A and GPR34,and phosphorylated LIMK and cofilin are increased in human metastatic gastric tumors.Conclusion:(1)The reduced Drosha in gastric cancer is accompanied with an increase of Drosha-independent mirtrons,especially mi R-4646-5p,which is associated with gastric cancer metastatic malignancy and poor prognosis.(2)The elevated mirtronic mi R-4646-5p is a specific splicing product of intron-3 of the host gene Abhd16 a with the aid of enhanced SRSF2.(3)mi R-4646-5p stabilizes HIF1 A by targeting PHD3 and feedback promotes expression of the host gene Abhd16 a and mi R-4646-5p itself.(4)ABHD16A,as a novel phosphatidylserine-specific lipase,plays an essential role to fuel gastric cancer metastasis via regulating lyso-PS accumulation in lipid metabolism.(5)Rho A triggered LIMK/cofilin signaling to promote GC metastasis is closely related with mi R-4646-5p/PHD3/HIF1 A regulated high expression of Rho A and lyso-PS/GPR34 depended activation of Rho A in GC cells. |
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