The Preliminary Research Of Downstream Target Proteins By Sox2 And Its Effects On Proliferation And Migration In Colon Cancer Cells

Background and purposeColorectal cancer is one of the most common malignant tumor in China and even in the whole world,the morbidity and mortality of which is still on the rise in the whole world.The incidence of colorectal canceris increased by 4.2%one year in China(in Shanghai,for example),which is higher than the global average..According to the statistics,the increase of colorectal cancer patients has been about 4.7%every year since 1991.The increase of morbidity in females and the shift to right-sided colon cancer is also the new trend for colorectal cancer in recent years.A synergistic effect caused by environmental,dietary,lifestyle and genetic factors are responsible for colorectal cancer.Colorectal cancer is treated by surgery combinedwith radiation therapy and chemotherapy with about 50%-70%surviving 5-year after treatment.while treatment outcomes for advancedcolorectal cancer patients is poor.Occurrence and development of colorectal cancer is an extremely complex biological phenomenon,with multiple genes involved,many factors interaction and diverse stages experienced.Colorectal cancer has been characterized by obscure etiology,no obvious symptoms in the early stages and easy tometastasize,which is the main cause of poor prognosis and high mortality.Therefore,the study of colorectal cancer has been focused on the basic research in development and mechanism of colorectal cancer,further investigation on molecular mechnism of colorectal cancer and methods for effective prevention and treatment.In the prophase study,our group detect the expression level of hsa-miR-200c in colorectal primary tumors is higher than the lymph node metastasis in colorectal cancer primary focal organizations carcinoma,which is verified by the experimental miR200c promotes colorectal cancer cell proliferation and migration ability.The miRNA target genes act mainly through negative regulation,in order to explore the relationship hsa-miR-200c gene associated with dry Sox2,Bmil,Oct4,Gfi1 mRNA expression,we conducted fluorescence quantitative RT-PCR test,found that in four dry related genes,Sox2 mRNA expression level is low in low transfer cell line SW480,HT29 and HCT116,and is higher in the high transfer of cell lines SW620.The results is inversely proportional in the expression level compared with hsa-miR-200c,so we preliminary speculate Sox2 as the target genes of miR-200c.Further applicating luciferase report system proved that hsa-miR-200c combined directly with Sox2 3 ’UTR area,confirmed the hsa-miR-200c can negatively regulate the expression of Sox2,so we determine to take Sox2 as one of the important goals for us.The earliest SOX(sry-related high mobility group box contatining)gene family was found in the mice lacking the Y chromosome.The gene family widely distributed in many organisms.so far,more than 30 genes or gene segments has been found in the family.Sox2 is a member of theSOX family of transcription factors,which have been shown to play key roles in various important developmental events,such as development of embryonic stem cells,organofaction and neural differentiation.Recent researches have revealed that Sox2 is closely related to the occurrence,development and its abnormal expression of a variety of tumors in the human body,which atttracts more and more attention.While Sox2 is closely related to colorectal cancer occurrence and development,researches show that Sox2 can promote the development of colorectal cancer,Sox2 highly expresses in the colon cancer and is the prognosis of candidate biomarkers,and are closely related to lymph node metastasis and distant metastasis of colorectal cancer.A growing number of studies have found that Sox2 interacted with NANOG and OCT3/4 stem cells transcription factors involve in occurrence of EMT in colorectal cancer cells,promoting the formation of colorectal cancer stem cells,and also could induce the drug resistance of colorectal cancer cells.But which downstream protein it can regulate and how to control the downstream protein to exert its biological function are still needs further research.Sox2 plays its biological function mainly by activating its downstream target genes.Therefore,the identification of target genes is the key to reveal Sox2’ role in tumor formation and biological action mechanism.In order to further explore Sox2’ s role in colorectal cancer and identification of the downstream proteins adjusted by Sox2,our work first identified Sox2 downstream regulation protein S3a and ENO1 by Proteomics technology and mass spectrum analysis in colorectal cancer SW480 cells,and further raised and lowered Sox2 in colorectal cancer SW480 and SW620 cell line,then verified the adjustment Sox2 on S3a and ENO1,and found Sox2 could promote colorectal cancer cell proliferation and migration ability.In the same time,considering the prevous work we did about regulation of the downstream target genes are on the protein level,as Sox2 serves as a kind of important transcription regulatory factors,we also want to further study Sox2 downstream proteins directly on transcription regulation.Literature shows that,through the CHIP CHIP screening,Sox2 can combine with HDAC9 gene directly at the transcriptional level,in order to further validate Sox2’ s adjustment on HDAC9.we used Sox2 overexpression and interference cells to verify the changes of HDAC9 expression level,and HDAC9 overexpression and interference cells to verify HDAC9’ s role in colorectal cance.The main contents are as follows:1.Screening and confirming Sox2 related downstream regulatory proteins and observed the effect of Sox2 overexpression in colorectal cancer cells on proliferation and migration.2.Establish Sox2 stable overexpression and interference cell lines,further validating Sox2 regulation function on downstream protein,clearing Sox2 effects on proliferation and migration in colorectal cancer cells.3.Establish HDAC9 overexpression and interference cell,to explore the mutual regulation relationship between HDAC9 and Sox2,observing HDAC9’s influence on proliferation and migration in colorectal cancer cells.The subject is aim at reveal a new Sox2 expressive regulation mechanism,clarifying Sox2 and downstream proteins’ role in evolution of colorectal cancer,and provide new targets for clinical treatment for colorectal cancer.Methods1.Screening and verificating Sox2 downstream proteins in colorectal cancerUse coomassie brilliant blue staining and silver nitrate staining combined with mass spectrometry analysis method for preliminary screening and validating Sox2 alters significantly related proteins in SW480 cells,the downstream regulation proteins expression are validated by the fluorescent quantitative PCR and western blotting.2.Sox2’s effect on proliferation and migration in colorectal cancer CellUsing Cell Counting Kit-8(CCK8)experiment to observe Sox2 influence on Cell proliferation,Transwell experimental observing Sox2 influence on migration ability,the influence of relevant statistical analysis was carried on.3.Establish SW620 stable expression and instantaneous interference Sox2 cells(1)Using pCDNA3.0/Sox2 vector to build Sox2 stable overexpression SW620 cell lines,QPCR and Western blottingting validate Sox2 expression efficiency;(2)Using synthetic Sox2 instantaneous SiRNA interference fragment,NC control group,transfected by Lipofectamine2000 to transfect SW620 cells.After transfection validating Sox2 interference efficiency within 48h by using QPCR and Western blottingting;(3)Using Cell Counting Kit-8(CCK8)and Transwell experiment to test Sox2 overexpression and interference effect on proliferation and migration ability in SW620 Cells;(4)Using QPCR and Western Blotting to validate the downstream protein expression after Sox2 overexpression and interference in colorectal cancer cells.4.Build HDAC9 overexpression and interference cells,validating HDAC9’s influence on proliferation and migration in colorectal cancer cells and its effects on Sox2 expression(1)Utilize the plasmid vector to build HDAC9 overexpression LS174T and HCT116 cell lines,QPCR and Western blottingting validating HDAC9 expression efficiency;(2)Make use of HDAC9 synthetic instantaneous SiRNA interference fragment,NC group,Lipofectamine2000 transfecting HT29 and SW620 cells.After transfection,QPCR and Western blottingting validate HDAC9 interference efficiency;(3)Using Cell Counting Kit-8(CCK8)and Transwell experiment to observe the influence HDAC9 overexpression and interference on proliferation and migration in colorectal cancer Cell;(4)After HDAC9 overexpression and interference observing the effect on the Sox2 expression.Results1.Preliminarily screen and verificate Sox2 downstream regulatory proteins in Colorectal cancer cells.(1)SDS-PAGE electrophoresis combined with coomassie brilliant blue staining results indicate that after overexpressing and disrupting Sox2 in SW480 cells,found that relative to other group,a strong expression stripe occurring in Sox2 interference region,cut strong expression strip sending to the pathologic physiology teaching and research section of southern medical university for mass spectrometry,the results are:40 s ribosomal protein(S3a).(2)The SDS-PAGE electrophoresis combined with silver nitrate staining,after scanning we analysis of two strongly expression stripes in Sox2 overexpression area in SW480 cells,sending strong expression stripes to southern medical university pathophysiology teaching and research section for mass spectrometry,the results are as follows:the Alpha-enolase(ENO1),Gama-actin.(3)The Real-time PCR(QPCR)and western blotting confirmed S3a expression decreased(F value:10.596,P value=0.004)and ENO1 expression increased(F value:14.416,P value=0.003)after Sox2 overexpression,while after Sox2 interference,the expression of S3a increased(F value:13.857,P value=0.009)and ENO1 expression reduced(F value:10.860,P value<0.001),while the Western blottingting showed Sox2 expression changes had no meaning on Gama-actin expression.2.Investgate Sox2 overexpressing effects on proliferation and migration in colorectal cancer cell.Cell Counting Kit-8(CCK8)confirmed after overexpressing Sox2,SW480-Sox2 group Cell growth accelerated significantly comparing with the control group(P<0.05).Transwell detected cell migration ability,its results show that the number of SW480-Sox2 cell group migration increased significantly(P<0.01)more than the control group at 48 h.3.The identification of Sox2 overexpression in SW620 cells and the chang e of downstream regulatory proteins,the influence on proliferation and mig ration of colorectal cancer cells.3.1 The identification of Sox2 overexpression SW620 cell line and the chan ge of S3a and ENOl expressionAfter stable expression Sox2 in SW620 cells,QPCR and western blotting detect the stable expression efficiency,results show successfully stable overexpressed Sox2,SW620 cell morphology has certainly changed,from short spindle into a small circle.After overexpressing Sox2,verification of QPCR and western blotting show S3a expression decreases,the difference was statistically significant(F value:6.874,P value=0.002)),ENO1 expression level increases,the difference was statistically significant(F value:4.974,P value=0.027).3.2 SW620 Sox2 overexpression effect on biological behavior of colorectal cancer in vivo.Cell Counting Kit-8(CCK8)confirmed,after overexpressing Sox2,SW620-Sox2 group Cell growth speed accelerated significantly(P<0.05)comparing with the control group.Transwell results show that the migration number of SW620-Sox2 cell group increased significantly more than the control group(P<0.01).4.The identification of SW620 Sox2 interference cell lines and the change of downstream regulatory proteins,effects on proliferation and migration ability of colorectal cancer cells4.1 The identification of Sox2 interference in SW620 cell line and the S3a and ENO1 expression changeAfter SW620 cells transient interfering Sox2,QPCR and westernblotting detect transient interference effect,results show that successful interference of Sox2,after Sox2 interference,S3a expression level increases,the difference was statistically significant(F value:0.042,P value=0.022),ENO1 expression level decreases,the difference was statistically significant(F value:1.288,P value=0.016).4.2 SW620 Sox2 interference’s effect on biological behavior of colorectal ca ncer in vivoAfter Sox2 transient interference in SW620,according to the results of Cell Counting Kit-8(CCK8),cell growth rate reduced relative to the control group(P<0.05).According to the results of Transwell,SW620 Sox2 cell migration amount is less than the control group,Differences are statistically significant(P<0.01).5.The identification of Instantaneous interference and overexpression of HDAC9,the HDAC9 impact on the proliferation and migration of colorectal cancer cells and the effect on Sox2 expression5.1 Identificate the HDAC9 expression after Sox2 overexpression and interferenceAfter Sox2 stable overexpression in SW620 cells,QPCR and western blotting show HDAC9 expression increased,difference was statistically significant(F value:9.647,P = 0.006).After Sox2 interference in SW620 cell,QPCR and western blotting show HDAC9 expression decreased,difference was statistically significant(F value:7.567,P = 0.002).5.2 The identification of HDAC9 Overexpression cell and HDAC9’s impact on proliferation and migration of colorectal cancer cellUsing Synthetic HDAC9 expression plasmid to build HCT116 and LS174T overexpressing HDAC9 cells,western blotting and QPCR experiment tested the expression result,the results show successful HDAC9 overexpression;After expressing HDAC9,CCK8 confirmed that cell growth slowed significantly(P<0.01).Transwell experiment tested HDAC9’s influence on cell migration ability,the result shows that HDAC9 overexpression cell migration number is less than the control group(P<0.01).5.3 The identification of instantaneous HDAC9 interference cell and effects on proliferation and migration of colorectal cancer cellUsing Synthetic HDAC9 SiRNA fragments to build instantaneous HDAC9 interference HT29 and SW620 cells,western blotting and QPCR experiment testing results,the results showed that compared with the control group,the SiRNA segments interfered HDAC9 successfully.CCK8 experiments confirmed that cell growth speed accelerated(P<0.01)after HDAC9 interference;Transwell experiment proved cell migration increased than the control group(P<0.01)after HDAC9 interference.5.4 The influence on Sox2 expression of HDAC9 overexpression and interferenceAfter overexpressing HDAC9,according to the results of QPCR and western blotting,the expression of Sox2 decreased,difference was statistically significant(F value:13.869,P = 0.01).Successful jamming HDAC9 genes,according to the results of QPCR and western blotting,the expression of Sox2 increased,difference was statistically significant(F value:12.954,P = 0.037).Conclusion1.Sox2 can negatively regulate the expression of S3a,positively regulate ENO1 expression;2.Sox2 can promote proliferation and migration ability of colorectal cancer cell;3.Sox2 can positively regulate HDAC9 expression;4.HDAC9 inhibit the proliferation and migration ability of colorectal cancer cell.Innovation points of our research:1.Successfully screened Sox2 downstream regulation protein S3a and ENO1,their relationship with Sox2 has not yet been reported.2.Preliminarily clarify HDAC9’s role in colorectal cancer and its role in colorectal cancer has not been reported.3.Certificate Sox2 can positively regulate the expression of HDAC9.