Study Of Transcription Factor SP1-Induced MicroRNA-146b-3p Facilitates The Progression And Metastasis Of Colorectal Cancer Via Regulating FAM107A

Part Ⅰ MicroRNA-146b-3p facilitates the progression and metastasis of colorectal cancer via regulating FAM107AAims: We screened the highly associated miR-146b-3p in the tissue samples of colorectal cancer patients,and analyzed the effects of the dysregulated expression of miR-146b-3p on proliferation,invasion and migration.The downstream target gene FAM107 A that miR-146b-3p may act on was discussed,and the regulatory mechanism was verified and clarified.We provide a new idea for the treatment of colorectal cancer.Methods: We used bioinformatics software for preliminary screening and verification,and Array Express(E-MTAB-4036)and GEO(GSE53159)databases for overlapping analysis of miRNAs.We cultured colorectal tissues and colorectal cancer cell lines(NCM460,DLD1,SW48,SW620 and Lo Vo)in vitro,and detected the expression levels of corresponding miRNAs in CRC tissues and cell lines by using q RT-PCR.We collected the tumor tissue samples of CRC,including CRC focus tissues,corresponding paracancerous normal tissues,metastatic and metastatic normal lymph nodes,and analyzed the relationship between miR-146b-3p expression and age,gender,lymph node metastasis and TNM stage of colorectal cancer patients.The impact of miR-146b-3p on CRC cell proliferation,migration,and invasion were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)cell proliferation assay and transwell migration and invasion assay.The impact of miR-146b-3p on CRC cell cycle and apoptosis was investigated using flow cytometry.Male BALB/c nude mice were selected to establish an animal model to detect tumor growth and analyze the influence of miR-146b-3p expression level.Overlapping analysis of DEGs using GSE50117 and GSE156355 with predicted targets and validated targets through miRWalk database.We cultured colorectal tissues and colorectal cancer cell lines(NCM460,DLD1 and SW48)in vitro,and detected the expression levels of the corresponding target gene in CRC tissues and cell lines by using q RT-PCR.In the collected CRC tissue samples,the relationship between the expression of FAM107 A and the age,gender,lymph node metastasis and TNM stage of colorectal cancer patients was analyzed.We explored the effect of upregulated miR-146b-3p expression on cyclin D1 expression by performing western blotting.We performed luciferase assay to validate whether miR-146b-3p directly targets 3′UTR of FAM107 A.We analyzed the effects of miR-146b-3p/ FAM107 A axis expression on the proliferation,migration,invasion,cell cycle and apoptosis of CRC cells by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)cell proliferation assay and transwell migration and invasion assay.Results:Using bioinformatics software for preliminary screening,we found 13 DEMs in CRC tissues compared with non-neoplastic tissues based on the overlapping miRNA analysis using the Array Express(E-MTAB-4036)and GEO(GSE53159)databases.we performed RT-q PCR to assess the relative expression of the miR-146b-3p、miR-1183 and miR-940 miRNAs in CRC tissues and CRC cell lines(NCM460,DLD1,SW48,SW620,and Lo Vo).The results demonstrate that among the miRNAs,miR-146b-3p was overexpressed in the four CRC cell lines than in NCM460 cells(Figure 1D);We also found that the expression levels of miR-146b-3p was significantly higher in DLD1 and SW48 cells than that in the SW620 and Lo Vo cells.Therefore,DLD1 and SW48 cells were selected for the subsequent analysis.It was found that 70.4% of the patients with lymph node metastasis were positive for miR-146b-3p,and 20.0% of the patients without lymph node metastasis were positive for miR-146b-3p,by collecting CRC tissues to analyze the expression of miR-146b-3p.According to the classification of colorectal cancer TNM staging,the positive expression of miR-146b-3p in stage Ⅰ patients accounted for 9.1%,Ⅱ period patients accounted for 56.3%,Ⅲ period patients accounted for 80.0%;Moreover,the expression of miR-146b-3p in CRC tissues was higher than that in NC tissues(P<0.05).The impact of miR-146b-3p on CRC cell proliferation,migration,and invasion were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)cell proliferation assay and transwell migration,invasion assay and flow cytometry.We found that the overexpression of miR-146b-3p promoted the proliferation,invasion and migration of DLD1 and SW48 cells,while the down-regulation of miR-146b-3p significantly inhibited the proliferation,invasion and migration of CRC cells.Overexpression of miR-146b-3p significantly promoted the cell cycle and weakened the apoptosis of colorectal cancer cells,whereas down-regulation of miR-146b-3p led to the opposite results.Analyzing the influence of miR-146b-3p expression level by establishing animal experimental model and detecting tumor growth:upregulation of miR-146b-3p expression promoted the occurrence and development of colorectal cancer in animal experiments in vivo.Next,we explored the possible mechanisms by which miR-146b-3p promotes CRC based on the overlapping analysis of DEGs using GSE50117 and GSE156355 with predicted targets and validated targets through miRWalk database.Performing q RT-PCR to detect the expression levels of corresponding target genes in CRC Ctissues and cell lines(NCM460,DLD1 and SW48),we found that the expression levels of three target genes were downregulated in CRC tissues than in the normal tissues,and the expression of FAM107 A was most significantly downregulated in DLD1 and SW48 cells compared with the NCM460 cells;In addition,according to the classification of lymph node metastasis,33.3% of patients with lymph node metastasis and 73.3% of patients without lymph node metastasis were positive for FAM107 A in colorectal cancer tissue samples.According to the classification of colorectal cancer TNM staging,the positive expression of FAM107 A in stage Ⅰ patients accounted for 81.8%,Ⅱ period patients accounted for 50.0%,Ⅲ period patients accounted for 20.0%;The expression of FAM107 A in CRC tissues was lower than that in NC tissues(P<0.05).We performed luciferase assay to validate whether miR-146b-3p directly targets 3′UTR of FAM107 A.Our results showed that the luciferase activity of the FAM107A-WT-3′-UTR plasmid in the DLD1 and SW48 CRC cell lines was evidently reduced in response to upregulated miR-146b-3p expression,and was significantly increased in response to downregulated miR-146b-3p expression.However,the luciferase intensity of FAM107AMUT-3′-UTR plasmid did not change.Analyzing the expression of miR-146b-3p/FAM107 A axis by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)cell proliferation assay and transwell migration,invasion assayc and flow cytometry,we found that the overexpression of miR-146b-3p enhanced the proliferation,invasion,and migration of DLD1 and SW48 cells,which was significantly reversed by FAM107 A downregulation,as well as reversed the effects of overexpression of miR-146b-3p on the facilitated cell cycle and blocked the apoptosis of CRC cells(P<0.05).We explored the effect of upregulated miR-146b-3p expression on cyclin D1 expression by performing western blotting.We observed that miR-146b-3p overexpression significantly increased cyclin D1 expression,which could be reversed by FAM107 A overexpression.Meanwhile,miR-146b-3p had no effect on the expression of cyclin A.Conclusions: Our study outcomes suggest that miR-146b-3p acts as an oncogene in CRC.It targets FAM107 A to regulate cell proliferation,cell cycle progression,and apoptosis.Part Ⅱ Transcription factor SP1-induced microRNA-146b-3p facilitates the progression and metastasis of colorectal cancer via regulating FAM107AAims: To investigate the upstream regulatory factors and regulatory mechanisms of MiR-146b-3p/FAM107 A axis in colorectal cancerMethods: To identify the upstream regulatory molecules that modulate miR-146b-3p expression,we used JASPAR database to predict TFs regulating miR-146b-3p promoter.In vitro cultured CRC cell tissues and cell lines(NCM460,DLD1,SW48),the expression level of TFS was predicted by q RT-PCR.The effect of SP1 on the expression of FAM107 A,Cyclin D1 and Cyclin A in the two CRC cells was assessed via western blot assay.The effect of SP1 upregulation on miR-146b-3p/FAM107 a axis was assessed via q RT-PCR.The impact of SP1 upregulation on CRC cell proliferation,migration,and invasion was analyzed using the transwell assay.Ch IP assay was performed to explore whether SP1 transcriptionally regulated miR-146b-3p expression.Binding sites of SP1 on the miR-146b-3p promoter region were predicted using JASPAR database,which was verified by luciferase assay.Results: To investigate the upstream regulation of miR-146b-3p expression,TFs modulating miR-146b-3p promoter were predicted using JASPAR database and q RT-PCR assay was performed to assess the expression levels of the top five potential TFs(SP1、ZSCAN4、ZNF263、RBPJ and EGR3)that may regulate miR-146b-3p expression.Our results indicated that the expression levels of SP1 were most significantly upregulated in DLD1 and SW48 cells compared with NCM460 cells.We explored the effect of SP1 on the expression of FAM107 A,cyclin D1,and cyclin A in the two CRC cell lines by performing western blotting.The results indicated that the SP1 upregulation significantly elevated cyclin D1 expression and reduced FAM107 A expression in both the CRC cell lines.In contrast,SP1 had no effect on cyclin A expression.The effect of SP1 upregulation on miR-146b-3p/FAM107 A axis was assessed via q RTPCR.The impact of SP1 upregulation on CRC cell proliferation,migration,and invasion was analyzed using the transwell assay.We found that miR-146b-3p expression levels were visibly enhanced by SP1 upregulation.These results also indicate that high SP1 expression intensified proliferation,invasion,and migration of CRC cells,which could be significantly reversed by miR-146b-3p inhibitor(P < 0.05).Ch IP assay was performed to explore whether SP1 transcriptionally regulated miR-146b-3p expression.Binding sites of SP1 on the miR-146b-3p promoter region were predicted using JASPAR database.Results of the Ch IP assay confirmed that the region at 1091-1100 bp upstream from the promoter region of the pre-miR-146b-3p was targeted by SP1.Additionally,luciferase activity in the WT miR-146b-3p promoter was notably upregulated in response to SP1 overexpression,which was remarkably reduced by miR-146b-3p knockdown.Interestingly,no significant difference in luciferase activity was observed when the SP1 binding sites at 1091-1100 bp were mutated.These results indicate that SP1 directly regulates miR-146b-3p at the transcription level.Conclusions: Our results suggest that SP1-induced miR-146b-3p facilitates CRC progression by targeting FAM107 A.