Study On The MRTF-A/miR-155/SOX1 Pathway Mediating Gastric Cancer Migration And Invasion

Background:Gastric cancer(GC)remains one of the most common causes of cancer-related death worldwide,and is the fourth leading cause of cancer-related death in the world.Although chemotherapy,radiotherapy and surgical techniques for gastric cancer have improved substantially in the past few decades,the prognosis of gastric cancer patients is not optimistic,and the recurrence rate and metastasis rate remain high,resulting in the median survival time of these patients is only about half a year.Metastasis is a complex pathophysiological process,which begins with the migration and invasion of cancer cells to surrounding tissues and lymphatic system.Cell migration is related to a variety of signaling pathways and transcription factors,and plays an important role in many biological processes.Therefore,it is necessary to better understand the targeted molecular mechanism related to the occurrence and metastasis of gastric cancer,so as to achieve early detection and treatment of diseases,and guide the clinical to determine more effective treatment methods for patients.MicroRNA(miRNA)is a kind of endogenous small coding RNA,which is about20 –25 nucleotides in length and can regulate gene expression.MiRNAs are a group of coding RNAs that specifically bind to the 3 '-noncoding region of mRNA of target genes and negatively regulate the expression of target genes at the transcriptional level.MiRNA plays an important role in cell growth,movement,invasion and stress response,as well as in tumor growth,invasion and metastasis.Therefore,it is necessary to better understand the molecular pathways of miRNA regulation,which is expected to provide a theoretical basis for inhibiting the invasion and metastasis of gastric cancer,and develop new targeted strategies for the treatment of gastric cancer.Previous studies X have shown that miR-155 promotes the occurrence and metastasis of gastric cancer.It has been shown that miR-155 promotes cell growth and migration by negatively regulating TGF?R2.MiR-155 down regulates the proliferation of gastric cancer cells by interacting with the target site of 3'UTR of TGF?R2.MRTF-A(also known as MKL-1)plays a key role in cell proliferation,differentiation,migration and apoptosis.MRTF-A is a coactivator of SRF(serum response factor),which is expressed in many tissues.In the nucleus,the MRTF-A-SRF complex binds to casgbox in the promoter of the target gene to activate the transcription of the target gene.Y(SRY)-box(SOX)protein family is a group of transcription factors,which contains highly conserved HMG DNA binding domains.Sox family members play an important role in embryonic development,postnatal development and stem cell regulation.In addition,some members of SOX family are closely related to the development of cancer.For example,SOX1 plays a role in cervical cancer and ovarian cancer;SOX1 is detected in advanced esophageal squamous cell carcinoma(ESCC)and highly invasive tumor tissues;SOX10 plays a promoting role in the occurrence of hepatocellular carcinoma(HCC);SOX1 inhibits the growth and invasion of breast cancer cells by inhibiting Wnt/?-Catenin signaling pathway.This study aims to explore the role of miR-155 in the occurrence,invasion and metastasis of gastric cancer and its mechanism.Objectives:In this paper,gastric cancer cell lines(SGC-7901 and MGC-803)were used as the cytological basis of in vitro experiments.The research objects were selected as MRTFA,miR-155 and SOX1.The regulatory relationship of the three in the growth of gastric cancer was verified by relevant in vitro experiments.The mechanism of MRTF-A regulating Sox-1 on the metastasis and invasion of gastric cancer cells by mediating miR-155 was preliminarily analyzed,and animal experiments were attempted to verify the mechanism It can also affect the growth of gastric cancer in the complex internal environment,so as to further deepen the understanding of the invasion and migration of gastric cancer cells,and provide a new idea and theoretical basis for the clinical treatment of gastric cancer.Methods:1.The effect of MRTF-A on the expression of miR-155Firstly,the full-length cDNA of MRTF-A was amplified by PCR,and the amplified product was transfected into pcDNA3.1 plasmid to synthesize pcdna3.1-MRTF-A plasmid.Pcdna3.1-MRTF-A and si-MRTF-A were transfected into SGC-7901 and MGC-803 cells respectively.The mRNA and protein expression levels of MRTF-A and miR-155 were detected by RT qPCR and Western blot.Then the activity of miR-155 promoter was detected by luciferase reporter assay under two experimental conditions,so as to further study the effect of MRTF-A on the activity of miR-155 promoter.Subsequently,SGC-7901 cells and MGC-803 cells were treated with ccg-1423(inhibiting nuclear translocation of MRTF-A)and cytochalasin D(stimulating nuclear translocation of MRTF-A),respectively.The mRNA and protein expression levels of MRTF-A and miR-155 were detected again.2.The effect of MRTF-A on the transcriptional activity of miR-155It has been reported that MRTF-A forms a complex with SRF to promote the transcription of target genes,and usually binds to the downstream casgbox site.We used Si SRF to knock out SRF gene in SGC-7901 and MGC-803 cells,and then detected the expression of SRF mRNA and protein in SGC-7901 and MGC-803 cells by RT qPCR and Western blot.RT qPCR was used to detect the expression level of miR-155 in the cells after the expression level of SRF was significantly decreased.Then the diluted chromatin fragments were incubated with MRTF-A antibody,RNAPII antibody,ach3k9 antibody and rabbit Ig G(negative control)overnight.After washing and elution,the chip product was purified,and the content of miR-155 promoter in the product was detected by RT-qPCR.3.MRTF-A regulates miR-155 activity by participating in Wnt signaling pathwayWe stimulated SGC-7901 cells with Li Cl(Wnt signaling pathway stimulant),siMRTF-A and Li Cl + si-MRTF-A at a concentration of 2.5 mm.After 24 hours,the mRNA and protein expression levels of MRTF-A and miR-155 in SGC-7901 cells were XII detected by RT qPCR and Western blot,respectively.It was confirmed that MRTF-A was involved in Wnt signaling pathway,and the expression of miR-155 was regulated by the expression of MRTF-A.We treated SGC7901 cells with 10 ? m Wnt3 a,siMRTF-A and Wnt3 a + si-MRTF-A respectively for 24 hours.RT qPCR and Western blot were used again The mRNA and protein expression levels of MRTF-A and miR-155 were detected by blot.It was confirmed that MRTF-A was involved in Wnt signaling pathway,and the expression of miR-155 was regulated by the expression of MRTF-A.Similarly,after SGC-7901 cells were transfected with Si-?-Catenin,the mRNA and protein expression levels of ?-Catenin,MRTF-A and miR-155 were detected.All tests were set up with corresponding negative control group..4.MiR-155 regulates the metastasis and invasion of GC cells by targeting SOX1geneFirst,we transfected SGC-7901 and MGC-803 cells with miR-155 mimics,miR-155 inhibitor and negative control respectively,and then detected the expression level of SOX1 in SGC-7901 and MGC-803 cells by RT qPCR and Western blot.In order to prove that MRTF-A binds to miR-155 promoter and affects the activity of miR-155 promoter,we performed luciferase reporter experiment,and cloned miR-155 into PGL3 plasmid without promoter to prepare luciferase reporter plasmid(wild type).A luciferase reporter plasmid containing mutant miR-155 promoter(mutant)was constructed by mutated calg(from cattttgg to aattttgg).Pcdna3.1-MRTF-A plasmid and si-MRTF-A were transfected into SGC-7901 gastric cancer cells respectively,and the luciferase activity of miR-155 promoter was detected.Similarly,to prove that miR-155 targets SOX1,the 3 'untranslated region of human SOX1 gene was cloned into PGL3 plasmid without promoter to prepare wild-type SOX1 luciferase reporter plasmid,and the SOX1 gene with variant was cloned into PGL3 plasmid without promoter to prepare mutant SOX1 luciferase reporter plasmid.The cells were transfected with miR-155 mimics and miR-155 inhibitor for 24 hours.Luciferase activity was measured by luciferase assay system.Next,we examine whether MRTF-A regulates SOX1 through miR-155.SGC-7901 and MGC-803 cells were transfected with si-MRTF-A,miR-155 mimics,siMRTF-A + miR-155 mimics and blank control respectively.Then the expression levels of MRTF-A and SOX1 were detected by Western blot.Then,we transfected SGC-7901 cells with SOX1 overexpression plasmid,blank plasmid,si-SOX1 plasmid and blank knockout plasmid respectively,and carried out cell invasion and migration experiments respectively,so as to prove SOX1's effect on invasion and migration of gastric cancer cells.5.The effect of MRTF-A on the expression of miR-155 and the invasion and migration of tumor cellsWe transfected SGC-7901 cells with SOX1 plasmid,miR-155 mimics,SOX1 overexpression plasmid+miR-155 mimics and blank plasmid respectively,and studied the invasion and migration of SGC-7901 cells.Then,SGC-7901 was transfected with si-MRTF-A,miR-155 mimics,si-MRTF-A+miR-155 mimics and blank control respectively.Then,the invasion and migration experiments showed that MRTF-A could regulate the migration and invasion of SGC-7901 cells by regulating miR-155.6.To study the effect of miR-155 on the growth of gastric cancer in vivoMale BALB/C nude mice aged 4-5 weeks were randomly assigned to control group or experimental group(5 mice in each group).SGC-7901 cell line and miR-155 inhibitor transfected SGC-7901 cell line were subcutaneously injected into the back of nude mice.Tumor formation was monitored every 3 or 4 days by measuring the maximum and minimum diameter of the formed tumor.After euthanasia,the tumors were removed completely and the wet weight of each tumor was examined to verify the role of miR-155 in solid tumors.In addition,we detected the expression level of SOX1 in each group by immunohistochemistry to further confirm the effect of miR-155 on tumor growth.In addition,we also analyzed the survival of tumor bearing mice to confirm the effect of miR-155 on tumor growth.Results:1.After MRTF-A was transfected into SGC-7901 and MGC-803 cells,RT-qPCR and Western blot showed that the protein and RNA expression levels of MRTF-A were increased,and the miR-155 expression level was significantly increased;after siXIV MRTF-A was transfected into SGC-7901 and MGC-803 cells,the MRTF-A level was significantly decreased,and the miR-155 RNA level was significantly decreased.Luciferase assay showed that miR-155 promoter activity was significantly increased after MRTF-A overexpression in SGC-7901 cells.In addition,MRTF-A deletion inhibited mir155 promoter activityCCG-1423 inhibited miR-155 expression and promoter activity in SGC-7901 and MGC-803 cells.Transcription of miR-155 requires nuclear localization of MRTF-A.Treatment of gastric cancer cells with cytochalasin D increased the expression of miR-155 and promoter activity in SGC-7901 and MGC-803 cells.These results indicate that MRTF-A can promote the transcription of miR-155.2.MRTF-A forms a complex with SRF to promote the transcription of target genes,which usually binds to the calgbox site in the promoter of downstream genes.After SRF gene was knocked out in SGC-7901 and MGC-803 cells by Si SRF,SRF mRNA and protein levels were significantly decreased.Depletion of SRF resulted in the decrease of miR-155 RNA level in SGC-7901 and MGC-803 gastric cancer cells.This suggests that the MRTF-A-SRF complex promotes miR-155 transcription by binding to the calg box.The next chip test showed that MRTF-A antibody effectively precipitated the DNA fragment containing carrg in miR-155 promoter,which indicated that MRTF-A had physical association with miR-155 promoter.Acetylation of histone H3K9 has been reported to activate transcription.The same results showed that acetylated histone H3K9 increased the binding of miR-155 promoter after MRTF-A overexpression.In addition,after MRTF-A overexpression,RNA polymerase II showed increased aggregation to miR-155 promoter.These results suggest that MRTF-A plays a direct role in the activation of miR-155 promoter.3.After treatment with Li Cl,the expression of miR-155 in SGC-7901 cells was induced by Li Cl,which was significantly reversed by MRTF-A knockout.When SGC7901 cells were treated with Wnt3 a,the results showed that Wnt3 a induced the expression of miR-155,while MRTF-A knockout significantly reversed this trend.The mRNA and protein levels of ?-Catenin in SGC-7901 cells transfected with Si-?-Catenin were significantly decreased.In these ?-Catenin deficient cells,the levels of MRTF-A and miR-155 were significantly decreased,indicating that the expression of miR-155 requires the involvement of ?-Catenin.These results indicate that Wnt/?catenin regulates the expression of miR-155 through MRTF-A.4.MiRNA targeting analysis using targetscan website showed that miR-155 was identified as a potential regulator of SOX1 expression.After transfection of mir155 mimics into SGC-7901 and MGC803,the expression level of miR-155 increased,while the expression level of SOX1 mRNA and protein decreased significantly.In contrast,the mRNA and protein levels of SOX1 were significantly up-regulated after miR-155 inhibition.In order to confirm that SOX1 is the direct target of miR-155,luciferase reporter gene was detected.The results showed that co-transfection of PGL3-SOX1 and miR-155 mimics decreased the luciferase activity,but had no effect on the mutant.Co transfection of PGL3-SOX1 and miR-155 inhibitor significantly increased the luciferase activity,but did not affect the mutant.These data suggest that SOX1 is a direct target of miR-155 in gastric cancer cells.Next,we examined whether MRTF-A regulated SOX1 gene in gastric cancer cells through miR-155.The inhibition of MRTFA significantly inhibited SOX1 expression.Co-transfection with miR-155 mimics could significantly reverse the tumor inhibitory effect of miR-155 mimics on gastric cancer cells.SOX1 overexpression plasmid and si-SOX1 plasmid were transfected into SGC-7901 gastric cancer cells respectively.Migration and invasion test showed that SOX1 overexpression inhibited the migration and invasion of SGC-7901 cells.These results suggest that miR-155 regulates the migration and invasion of gastric cancer by targeting SOX1 gene.5.SGC-7901 cells were transfected with SOX1 overexpression plasmid,mir155 mimics,SOX1 overexpression plasmid + miR-155 mimics and blank plasmid respectively.The results of cell invasion and migration showed that SOX1 overexpression significantly inhibited the migration and invasion of gastric cancer cells.Co-treatment with miR-155 mimics significantly reversed the tumor inhibition in SGC-7901 cells.SGC-7901 cells were transfected with si-MRTF-A,miR-155 mimics,siMRTF-A + miR-155 mimics and blank control respectively.The invasion and migration experiments showed that the decrease of MRTF-A significantly inhibited the migration and invasion of gastric cancer cells.Co-transfection with miR-155 mimics could significantly reverse the tumor inhibition of SGC-7901 cells,suggesting that miR-155 activity is necessary for MRTF-A-induced cell migration and invasion.In conclusion,these findings suggest that MRTF-A regulates the expression of miR-155 and is related to the migration and invasion of gastric cancer cells.6.The tumor growth rate of SGC-7901 cells transfected with miR-155 inhibitor was significantly slower than that of SGC-7901 cells without miR-155 inhibitor.The weight gain of tumor transfected with miR-155 inhibitor was also slower than that of control group.In addition,miR-155 inhibitor transfected SGC-7901 cells increased the expression of SOX1.Survival analysis showed that the down-regulation of miR-155 in gastric cancer was helpful to prolong the survival time.These data suggest that downregulation of miR-155 inhibits the development of gastric cancer.Conclusions:1.MRTF-A played a positive role in the regulation of miR-155 expression in GC cells.2.MRTF-A promoted the transactivity of miR-155 through the CArG box.3.MRTF-A was crucial for miR-155 activation induced by Wnt signaling.4.MiR-155 regulated GC migration and invasion by targeting the SOX1 gene.5.MRTF-A regulated the expression of miR-155,which is related to migration and invasion.6.MiR-155 downregulation inhibited gastric cancer growth in a nude mouse xenograft model.