| Backgroud and ObjectiveGastric cancer is a serious malignant tumor threatening the survival and health of all human beings,and Asia is one of the high-incidence areas.In China,the incidence number of gastric cancer ranks first in Asia and the incidence and mortality rate are among the top three in all malignant tumors.Due to the early symptoms are not clear and lack of standardized physical examination,the majority of patients with gastric cancer already have metastasis,which lead to poor prognosis.Therefore,explore the molecular mechanism of metastasis and search for effective molecular markers may provide a new point for the prediction,diagnosis and treatment of gastric cancer metastasis.Epithelial-Mesenchymal Transition(EMT)is one of the most important mechanisms for tumor metastasis.Tumor cells lose epithelial polarity and acquire mesenchymal polarity,accompanied with the loss of epithelial markers such as E-cadherin,ZO-1 and the elevation of mesenchymal markers such as vimentin,N-cadherin.et al.Cancer stem cells(CSCs)are small population of cancer cells with the characteristics of self-renewal,tumor initiation,and chemotherapy resistance,and“Sternness”is generated to feature those molecular signatures of CSCs,including markers such as CD 133,CD44,Sox2.et al.Studies have found EMT was the important process to acquire stemness.Though undergoing EMT,turmor cells gain better motility,invasion,metastasis and induce chemoresistance.The mechanism of EMT is complicated and the microenvironment of tumor is closely related to EMT.Transforming growth factor ?(TGF?)in the tumor microenvironment is the most important EMT promoting factor and can regulate EMT through the classical Smad pathway to form transcriptional complex,which induce EMT.TGF? can also regulate non-Smad-dependent signaling pathways such as PI3K/AKT pathway,MAPK pathway,NF-kB pathway,thus mediating EMT.Therefore,further elucidating the molecular mechanism of TGF?-mediated EMT may provide a new strategy for blocking EMT and solving tumor metastasis.MicroRNAs(miRNAs)are members of the non-coding RNA family,ranging from 19 to 25 nucleotides in length.Studies have found that more than half of the human genome encodes genes are regulated by microRNAs and play important roles in various biological behaviors such as proliferation,invasion,angiogenesis,and apoptosis.Previous studies indicate that microRNA can regulate EMT and act as a downstream effector of TGF? and participate in its biological behavior.MiR-577 is a tumor-related gene discovered in recent years.MiR-577 is abnormally expressed in many tumors such as glioma,osteosarcoma and colorectal cancer.However,the expression,biological function and regulatory molecules mechanism of miR-577 in gastric cancer s is still not clear.Through analysis of TCGA(The Cancer Genome Atlas)database,we found that miR-577 was significantly upregulated in gastric cancer.In vitro and in vivo studies confirmed that miR-577 promoted gastric cancer invasion,metastasis and EMT but did not affect cell proliferation.Through a variety of cytokine screening,we found that TGF? significantly induced the expression of miR-577.Transcriptional analysis confirmed that NF-?B P65 was the transcription factor of miR-577.TGFP activated NF-?B,activated NF-?B transferred into nucleus and regulated the expression of miR-577.Through bioinformatics analysis and experimental validation,we confirmed that SDPR was the downstream target gene of miR-577.SDPR directly binded with ERK and inhibited phosphorylation of ERK,which affected ERK-IKK?/?-IKB?-P65 pathway,lead to the suppress of P65 phosphorylation and formed a positive feedback to inhibit transcription of miR-577.Combined with bioinformatics analysis and experimental validation,we found that miR-577 and its target gene SDPR are important mechanism for TGF?-mediated EMT,and form a positive feedback loop to promote gastric cancer metastasis.The present study aims to explore how miR-577 and its target genes participate in TGF?mediated EMT,and provide experimental evidence for precise therapy by targeting miR-577 and SDPR in the future.Methods1.The expression of miR-577 in GC cells,tissues and clinical paraffin-embedded samples(1)By analyzing the microRNA microarrays in TCGA ST AD(The Cancer Genome Atlas Stomach Adenocarcinoma)database,the differential microRNAs of GC were screened,and miR-577 was one of the differentially expressed genes and was highly expressed in gastric cancer.(2)The expression of miR-577 in thirty-six fresh gastric cancer specimens and their adjacent paracancerous normal mucosa tissues,seven gastric cancer cell lines(AGS,SGC7901,MGC803,MGC823,BGC803,MKN45)and immortalized gastric epithelial cells GSE-1 were detected by fluorescence quantitative RT-PCR.(3)The expression of miR-577 in 153 GC specimens with complete follow-up visit information was examined by in situ hybridization(ISH)method.The relationship between miR-577 and prognosis,clinical pathological factors was analyzed2.Investigating the biological behaviors of miR-577 in vivo and in vitro(1)Transfecting miR-577 AgomiR/AntagomiR was used to overexpress or silence the expression of miR-577 in GC cells.The effect of miR-577 on the proliferation of GC cells was detected by MTT assay,Edu cell proliferation assay and cloning formation assay.The stable overexpression of miR-577 cells and their control cells were constructed in lentivirus vector with Luciferase signal,nude mice subcutaneous injection was used to analyze the effect of miR-577 on GC cell proliferation in vivo.(2)Transfecting miR-577 AgomiR/AntagomiR was used to overexpress or silence the expression of miR-577 in GC cells.The effect of miR-577 on cell migration and invasion was detected by Transwell assay.The stable overexpression of miR-577 cells and their control cells were constructed in lentivirus vector with Luciferase signal amd tail vein injection of nude mice.was used to analyze the effect of miR-577 on lung Colonization of GC cells and survival of mice in vivo(3)After transfection of miR-577 AgomiR/AntagomiR,the effect of miR-577 on sphere formation and chemotherapy sensitivity were detected by Tumorspheres assay and MTT assay.The cells were divided into NC group,miR-577 group,NC+oxaliplatin group and miR-577+oxaliplatin group,the effects of miR-577 on apoptosis were detected by flow cytometry and Western Blot was used to detect the change of apoptosis related indicators.3.MiR-577 was involved in TGF?-induced EMT and stemness(1)The morphology change after transfection of miR-577 AgomiR/AntagomiR was observed under inverted microscope.Western Blot,immunohistochemistry and immunofluorescence were used to detect change of EMT and sternness markers.(2)GC cells were treated with TGFP for OH,24H,48H,inverted microscope was used to observe the morphology change,Transwell assay was used to detect the change of cell migration,Western Blot was used to detect the change of EMT and sternness markers,RT-PCR was used to detect the change of miR-577.(3)GC cells were divided into TGF? control group,TGF? treatment group and TGF?+AntagomiR treatment group,inverted microscope was used to observe the morphology change,Transwell assay was used to detect the change of cell migration,Western Blot was used to detect the change of EMT and sternness markers.(4)The Genomics website UCSC and the transcription factor database PROMO were used to analyze the transcription factor of miR-577.Analysis indicated that NF-kB P65 was the transcription factor of miR-577 and 3 binding sites were found.TGF?treated gastric cancer cells for OH and 24H,Western Blot was used to analyze the expression of P-P65 and the localization of NF-kB P65 was observed by immunofluorescence.(5)GC cells were treated with NF-kB inhibitor QNZ and BAY 11-7082,the expressions of P-P65 and P65 were detected by Western Blot,and the expression of miR-577 was detected by real-time RT-PCR.SiRNAs targeting NF-kB P65 were used and the change of miR-577 was detected by quantitative RT-PCR.(6)The promoter luciferase reporter vectors were constructed and divided into PGL4+ P65 group,WT +P65 group,MUTAB + P65 group,MUTAC +P65 group,MUTBC+P65 group,MUTABC + P65 group,The changes of luciferase activity were detected.According to A,B and C,three sites were designed specific probe,and CHIP experimentwas used to verify that NF-kB P65 directly binded to these promoter locus.4.miR-577 associated target gene screening and identifying(1)Target genes of miR-577 were analyzed by miRDB,PITA,RNA22 and Targetscan,TCGA analysis the expression and abundance of target gene in GC.Quantitative RT-PCR analyze change of target genes after transfection of miR-577;3'UTR region of target gene was constructed in luciferase vector to verify the direct binding of miR-577 and 3'UTR region.(2)RT-PCR was used to analyze the expresision of target gene SDPR in 30 cases of pathologically diagnosed gastric cancer tissue samples and adjacent normal gastric mucosa tissue expression,and analyze the relevance of SDPR and miR-577.(3)The expression of SDPR in 153 GC specimens with complete follow-up visit information was examined by immunohistochemical,the relationship between SDPR and prognosis,clinical pathological factors was analyzed.(4)SiRNAs were used to silence SDPR expression and vector was chosen to overexpress SDPR.Inverted microscope was used to observe the morphology change,Transwell assay was used to detect the change of cell migration,Western Blot was used to detect the change of EMT markers.(5)Immunofluorescence was used to research subcellular localization of SDPR and ERK,and CHIP was chosen to prove direct binding effect,the change of ERK/NF-?B pathway was detected by Western blot.(6)After SDPR suppression or overexpression,RT-PCR was used to analyze the change of miR-577.(7)GC cells were divided into Mock group,NC group,miR-577 group,miR-577 +SDPR group,the changes of migration of gastric cancer cells were detected by and Transwell assay,and the changes of EMT markers and ERK/NF-?B pathway were detected by Western blot.5.Statistical analysisSPSS 20.0 software was used for data analysis.The expression of miR-577 in fresh paired GC tissues was analyzed by Wilcoxon rank-sum test.The expression of miR-577 in ??+? stage tissues and ?+? stage tissues,metastatic group(Ml)and non-metastatic group(MO)was analyzed by Kruskal-Wallis test.The relationship between the expression of miR-577 and clinical pathological factors was analyzed by Pearson's chi-squared(x2)test.Survival curves was drawn by Kaplan Meier method and the statistical differences in different group was analyzed by Log-Rank test Trans well assay,Edu assay,olony formation assay,nude mice subcutaneous injection were analysed through two-tailed independent-samples t-test when compared with two group of mean±standard deviation(SD),and when groups over two,One-Way ANOVA was proffered.Before the analysis of variance,the Levene method was used to test the homogeneity of the variance,and the LSD method was used when variance had no difference.Dunnett's T3 method was used when the variance had difference.Results1.MiR-577 is upregulated in GC tissues and cell lines(1)TCGA microarray analysis reveals high expression of miR-577 in GCBy analyzing the TCGA GC microRNA microarray,In 42 paired GC tissues,29 cases found with the up-regulation of miR-577.(2)miR-577 is up-regulated in GC tissues and cellsAccording to the RT-PCR results of 36 paired GC tissues,miR-577 was upregulated in gastric cancer tissues compared with match normal tissues,and the expression of miR-577 in tumor stage ? + ? group was higher than that in ? + ?group.And miR-577 expression was higher in metastasis group(M1)than in non-metastasis group(MO)group.At the cellular level,the expression miR-577 was significantly upregulated in 6 GC cells compared with mmortalized gastric mucosal epithelial cells GSE-1.(3)miR-577 is upregulated in paraffin-embedded GC tissues and associated with poor prognosisThe expression level of miR-577 was analyzed by in situ hybridization(ISH)from a large cohort of 153 archived GC tissues.The results showed miR-577 was upregulated in GC and was significantly correlated with TNM stage,tumor invasion,Lymph node metastasis,Distant metastasis.High expression of miR-577 indicated poor prognosis in I-III stage DFS and IV stage OS.The above experiments show that miR-577 is highly expressed in GC and is associated with patient metastasis and poor prognosis.It may be an independent predictor of metastasis and prognosis in patients with gastric cancer.2.MiR-577 does not affect proliferation but induce metastasis and chemoresistance(1)miR-577 does not affect GC cells proliferation in vitro and in vivoAfter overexpress or silence miR-577,MTT assay,Edu cell proliferation assay and cloning formation assay showed miR-577 had no effect on cell proliferation,nude mice subcutaneous injection in vivo get the same results.(2)miR-577 promotes metastasis in vitro and in vivoWith the overexpression of miR-577 in vitro,Transwell assay showed enhance migration and invasion,however,miR-577 silence suppressed migration and invasion.With the stable expression of miR-577,the lung colonization of GC cells increased and the survival of mice was shorter.(3)miR-577 induces stenmess and chemoresistance to oxaliplatin in GCTurnorspheres assay revealed that miR-577 could promote sphere formation ability,MTT assay indicated that miR-577 overexpression lead to chemoresistance to oxaliplatin while miR-577 supression increased chemosensitivity.Flow cytometry found miR-577 inhibited oxaliplatin induced apoptosis and decreased apoptosis related markers Cleaved-caspase3 and Cleaved-caspase7 were seen by Western Blot.The above experiments show that miR-577 does not affect the proliferation of gastric cancer cells,but promotes cancer cells migration,invasion,lung colonization ability,sphere formation ability,and induce chemotherapy resistance.3.miR-577 participates TGF?-induced EMT and stemness(1)MiR-577 promotes EMT and sternnessWith the overexpression of miR-577 in MGC803 cells,cells displayed a spindle-shaped form,but the MKN45 cells with miR-577 supression present a spheroid-shaped morphology.Further immunoblot assays showed that miR-577 suppressed the expression of epithelial markers(E-cadherin)while stimulated the mesenchymal markers(Vimentin,N-cadherin,MMP9)and sternness markers(SOX2,CD44).Moreover,miR-577 silence reversed the expression of EMT-associated proteins.(2)MiR-577 is induced by TGF?GC cells were treated with TGF? OH,24H,48H and gradually present spindle-shaped morphology and stretch out pseudopodia,accompanied with increasing of the cell migration.Western Blot results showed that E-cadherin,an epithelial marker,was down-regulated,and mesenchyrmal markers such as Vimentin,N-cadherin,MMP9 and sternness markers CD44,SOX2 were up-regulated.RT-PCR results showed that TGF? stimulation significantly increased miR-577 expression in a time-dependent manner.(3)Interference with miR-577 reverses TGF?-induced EMT and sternnessGC cells were treated with TGF? and miR-577 interference,inverted microscope found TGF? induced spindle-shaped morphology was reversed by miR-577 supression,and the migration ability of cells was also restored.Western blot showed the effect of TGF? on EMT markers and sternness markers were also reversed by miR-577 interfering.(4)NF-KB P65 directly transcribes miR-577 expressionAccording to A,B,C three binding sites,the luciferase reporter gene vectors were designed.Luciferase reporter assay showed that all three binding sites A,B and C could enhance the luciferase activity.CHIP also confirmed that NF-?B P65 could bind with A,B and C binding sites.in both MGC803 And MKN45 cells.This part of the research shows that miR-577 is a new target of TGF?.TGF?regulates tumor EMT and sternness by regulating the nuclear translocation of NF-?B P65 and increase the transcription level of miR-577.4.SDPR is a direct target of miR-577(1)SDPR is a candidate target of miR-577Through the combination of miRDB,PITA,RNA22,and Targetscan databases,104 candidate target genes were screened out,and then the gene expression and abundance were searched by TCGA.Seven candidate genes were selected for further investigation:LM07,CCDC68,KLF9,Smad4,KLF4,KAT6B,and SDPR.After overexpression of miR-577,RT-PCR revealed that SDPR was most significantly downregulated.The dual luciferase reporter assay confirmed that miR-577 binds directly to both sites of the 3'UTR region of SDPR.(2)SDPR is downregulated in GC tissues and negatively correlated with miR-577RT-PCR analysis of 30 pairs of GC and paracancerous tissues showed that SDPR was significantly lower in GC,the expression of SDPR in patients with stage? + ? was lower than in stage ? + ?(P<0.01).Statistical analysis showed that there was a negative correlation between miR-577 and SDPR expression in fresh gastric cancer(R=-0.442,P = 0.015).(3)SDPR is downregulated in clinical specimens and correlates with better prognosisImmunohistochemical stain of SDPR in 153 paraffin-embedded GC tissues with complete follow-up data showed that SDPR was lower expressed in tumors than normal gastric mucosa tissues,and the expression decreased with the progress of tumor stage.Statistical analysis also found that miR-577 high expression GC tissues,the expression of were SDPR lower.And DFS analysis of stage ?-? patients and OS analysis of stage ? patients found that high expression of SDPR correlated with better prognosis.Synergistic analysis found that miR-577 high+SDPR low expression group had the worst prognosis,and miR-577 low+ SDPR high had the best prognosis.(4)SDPR inhibits EMT,sternness and tumor metastasisAfter interference of SDPR in MGC803 cells,the morphology of the cells changed into spindle-shaped,and cell migration and invasion ability,sphere formation ability were increased.Western Blot results showed that the epithelial marker E-cadherin decreased,mesenchymal markers and sternness markers were up-regulated,which suggested that interference with SDPR could promote EMT.However,vector-mediated SDPR overexpression get the opposite trend.(5)SDPR inhibits ERK/NF-?B pathway.Immunofluorescence assay showed co-subcellular localization of SDPR and ERK,CHIP assay proved direct binding effects between SDPR and ERK,SDPR suppression lead to phosphorylation of ERK,IKK?/?,IKB?,and P65,which indicated SDPR inhibited ERK/NF-?B pathway.(6)SDPR medidated d ERK/NF-?B positive feedback loop to inhibit miR-577NF-?B p65 was the transcription factor of miR-577,and SDPR inhibited ERK/NF-?B pathway,these results indicated a positive feedback loop in GC.RT-PCR found SDPR suppression lead to miR-577 upregulation and SDPR overexpression inhibited miR-577 expression.(7)Restoring SDPR expression reverses miR-577-induced EMTTranswell experiments showed that overexpression of miR-577 promoted GC cell migration,whereas restored SDPR expression reversed miR-577 induced cell migration;Western Blot results showed that overexpression of miR-577 up-regulated mesenchymal markers Vimentin,N-cadherin and MMP9,and sternness markers CD44,SOX2,however,restoration of SDPR expression reversed these effects.Similarly,the same conclusion was detected in the ERK/NF-?B pathway.This part shows that SDPR as the target gene of miR-577,directly binds and inhibits the phosphorylation of ERK and inhibits the ERK/NF-?B pathway in gastric cancer cells,leading to inhibition of EMT and sternness on the one hand,and on the other hand,inhibiting miR-577 transcription through a positive feedback loop.Conclusions:1,MiR-577 is upregulated in GC and associated with turmor metastasis and poor prognosis,which indicated miR-577 may play an oncogene in GC.2,MiR-577 does not affect proliferation but enhances migration,invasion and lung colonization of GC cells in vitro and in vivo.3,TGF? induces miR-577 expression by NF-?B medidated transcription,and overexpression of miR-577 leads to EMT and sternness.4,SDPR is a direct target gene of miR-577 and inhibits ERK/NF-?B pathway,on the one hand,inhibits EMT and sternness,on the other hand,medidates miR-577 expression though a positive feedback. |
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