The Role And Mechanism Of HDAC2 In Colorectal Cancer Metastasis

Background:Colorectal cancer is one of the leading malignant tumors worldwide.Despite the great progress in current treatment of CRC,the five-year survival rate of CRC patients is still low.Metastasis is the main cause of treatment failure in colorectal cancer.So,it is of great significance to clarify the molecular regulation mechanism of colorectal cancer metastasis.Colorectal cancer metastasis is a multi-factor and multi-step process.Cancer cells need to escape from the limitation of basement membrane,intravasate into blood vessels and colonize at the distal organs.Epithelial mesenchymal transition(EMT)is a biological process under physiological or pathological conditions in which epithelial cells lose tight junctions and adherent junctions,increase migration ability,improve anti apoptotic ability,produce a large number of extracellular matrixs,increase invasion ability,and finally obtain mesenchymal phenotype.At present,EMT is considered as the initial step of tumor metastasis,and promoting EMT often leads to the enhancement of tumor metastasis.EMT is regulated by numerous factors in different ways,and non-coding RNA(ncRNA)is one of them.Non-coding RNA refers to RNA that does not encode protein,mainly including microRNA(miRNA,19-22 nt)and long noncoding RNA(lncrna,more than 200 nt).MiRNAs regulate EMT by targeting transcription factors such as Twistl,snail 1/2 and ZEB1/2.Lncrna can form specific secondary structure by base complementary pairing or folding,which can provide molecular binding sites to DNA,other RNAs and proteins to regulate EMT.It has been confirmed that lncRNA such as H19,ZEB1-AS1,lncRNA-ATB,lncRNA HIT,MEG3,lncRNA Hh,MALAT1 and Hotair play important roles in EMT.Apart from ncRNAS,a few HDACs have been found to be involved in the regulation of EMT and tumor metastasis.HDACs are enzymes that catalyze the deacetylation of histones to repress gene expression.HDAC1 could interact with transcription factor TCF12 to promote EMT and gallbladder cancer metastasis.SIRT1 could bind to transcription factor ZEB1 and inhibit the expression of E-cadherin to promote EMT and pancreatic cancer metastasis.However,the role and molecular mechanism of HDACs in the regulation of colorectal cancer metastasis remains to be largely unknown.Objective:The purpose of this study was to determine whether HDACs play a role in the regulation of colorectal cancer metastasis and reveal the specific molecular mechanism.The details are as follows:(1)Screen out HDACs related to colorectal cancer metastasis;(2)Verify the regulatory role of HDAC2 in colorectal cancer metastasis;(3)Screen out and verify the downstream targets of HDAC2 in regulating EMT and colorectal cancer metastasis;(4)Clarify the specific molecular mechanism of HDAC2 regulating downstream targets;(5)Clarify the specific molecular mechanism of HDAC2 downstream targets regulating EMT and colorectal cancer metastasis;(6)Verify the role and molecular mechanism of HDAC2 in colorectal cancer metastasis in vivo.Methods:1.Public cancer database Oncomine were queried to obtain the expression data of HDACs in colorectal cancer primary sites and metastases;GEPIA tool was used to analyze the relationship between HDACs expression and the survival time of colorectal cancer patients;The expression of HDAC2 in matched clinical samples of colorectal cancer was detected by immunohistochemical staining;The expression of HDAC2 and E-cadherin in colorectal cancer cells were detected by qPCR and WB;The correlation between HDAC2 and E-cadherin expression in CRC was analyzed by Starbase.2.HDAC2 was knocked out in colorectal cancer cell DLD1and microarray analysis was used to detected gene expression changes;Cytoskeleton staining was used to detect cell morphological changes in DLD1 HDAC2 KO cells;Transwell and RTCA assay were used to detect migration motility of colorectal cancer cells after knocking out or knocking down HDAC2;WB and immunofluorescence staining were used to detect the protein expression changes of EMT markers in CRC cells after HDAC2 knocking out or knocking down.3.Non-coding RNA chip was used to detect the expression changes of ncRNA in DLD1 HDAC2 KO cells;Lncrna H19 was selected as the downstream target gene of HDAC2;qPCR was used to detect the expression of H19 in CRC cells after HDAC2 knocking out and knocking down;siRNA was used to knock down H19 in DLD1 HDAC2 KO cells and SW620 cells;The changes of migration motility in colorectal cancer cells after H19 knocking down were detected by Transwell and RTCA,while the protein expression changes of EMT marker was detected by WB.4.The interactions between HDAC2,Histone and H19 promoter were detected by chromatin immunoprecipitation assay;The changes of histone acetylation after HDAC2 knocking out in DLD1 were detected by WB;Luciferase reporter gene assay and immunoprecipitation assay were used to verify the regulation effect of interaction between HDAC2 and transcription factor SP1 on the activity of H19 promoter;qPCR was used to detect the expression change of H19 after knocking down SP1 in CRC cells;Transwell chamber assay was used to detect the change of migration ability in CRC cells after interfering with SP1 expression;WB was used to detect the protein expression changes of EMT marker in SP1 knock down CRC cells.5.QPCR was used to detect the expression of MMPs in DLD1 HDAC2 KO cells;WB was used to detect the expression of MMP14 in colorectal cancer cells after knockout of HDAC2,knockdown of HDAC2,interference with H19 and interference with SP1;Transwell chamber experiment was used to detect the migration ability of CRC cells after interference with MMP14;WB was used to detect the protein expression of EMT markers in MMP14 knock down CRC cells;Starbase database was used to analyze the expression correlation between H19 and MMP14 in CRC;The binding sites of H19 and MMP14 mRNA with miR-22-3p respectively were analyzed using starbase tools;RIP assay was used to detect the interaction between H19 and miR-22-3p;qPCR assay was used to detect the expression of H19 in CRC cells transfected with miR-22-3p mimics;Transwell and WB were used to detect the chang of EMT phenotype after transfecting miR-22-3p mimics in CRC cells.6.CRC cells labeled with Lnc were injected into immunodeficient mice through tail vein to verify role of HDAC2 in CRC metastasis in vivo;H&E,FISH and immunohistochemical staining were used to verify the molecular mechanism of HDAC2 in regulating CRC metastasis.Results:1.Public database showed that compared with primary site CRC samples,the expression of HDAC1 and HDAC2,but not HDAC3 and HDAC8 is significantly lower in metastases.Notably,of all HDACs(1-3,8),Kaplan-Meier analyses indicated that only low expression of HDAC2 is correlated with poor survival of CRC patients;Immuno-histochemical staining results showed that the expression of HDAC2 was lower in 12 cases of 22 paired clinical colorectal cancer samples;Mining TCGA database using analyzing tools from starBase database,we found that the expression of CDH1(E-cadherin),a well-accepted marker negatively associated with EMT and metastasis,is positively correlated with the expression of HDAC2 in CRC(including 471 COAD samples and 167 READ samples).In addition,the expression of HDAC2 displays a lower level in highly invasive CRC cells.2.The expression of1555 mRNAs changed significantly in DLD1 HDAC2 KO cells.Pathway analysis of deregulated genes in KO cells showed strong enrichment of genes involved in pathways crucial for cancer metastasis;47 EMT related genes were significantly changed;DLD1 HDAC2 KO cells displayed a non-polarized and spindle-shaped morphology,their migration ability was also increased and the expression of E-cadherin was decreased,while fibronectin and itga5 were significantly increased;HDAC2 knockdown in another CRC cell line SW480 also leads to the conversion of epithelial to mesenchymal features,including increased migratory ability and up-regulated fibronectin and ITGA5.3.Non-coding RNA microarray results showed that lncRNA H19 was the most upregulated ncRNA after HDAC2 knockout,qPCR showed that H19 did increase significantly after HDAC2 knockout or knockdown in colorectal cancer cells;when knock down H19 in DLD1 HDAC2 KO cells by siRNA,their migratory ability was decreased,and the expression of E-cadherin was increased,while fibronectin and itga5 were significantly decreased;Similar results were observed in another colorectal cancer cell line SW620 when the expression of H19 was knocked down.4.The results of chromatin immunoprecipitation showed that HDAC2 could bind to the promoter of H19.The acetylation level of histone H3K27 in DLD1 cells was increased after HDAC2 was knocked out,and the acetylation level of histone H3K27 in the promoter region of H19 was also increased when HDAC2 was knocked out.Overexpression of HDAC2 decreased the activity of H19 promoter,while HDAC2 knockdown decreased the activity of H19 promoter.The effect of HDAC2 on the activity of H19 promoter disappeared after the Spl binding site was mutated.The results of immunoprecipitation showed that HDAC2 had strong interaction with Spl in DLD1 and SW480 cells;when we knocked down Spl by siRNA in colorectal cancer cells,the expression of H19 increased,the migration ability of cells increased,the expression of E-cadherin decreased,while the expression of fibronectin and itga5 increased significantly.5.QPCR results showed that MMP14 was the most upregulated MMPs after HDAC2 knockout;WB showed that MMP14 expression increased after HDAC2 knockout,HDAC2 knockdown,and Sp1 knockdown in colorectal cancer cells,but decrease after H19 knockdown;Knocking down MMP14 by siRNA in colorectal cancer cells,cell migration ability decreased,the expression of E-cadherin increased while fibronectin decreased.The expression of H19 and MMP14 was positively correlated in colorectal cancer;Both H19 and MMP14 mRNA had two potential binding sites with miR-22-3p;RIP confirmed that H19 could bind with miR-22-3p;when colorectal cancer cells were transfected with miR-22-3p mimics,the expression level of H19 did not change,cell migration ability decreased,MMP14 expression decreased,E-cadherin expression increased,while fibronectin and itga5 decreased significantly.6.The lung metastasis model of colorectal cancer established by tail vein injection showed that the lung metastasis ability of colorectal cancer was significantly increased after HDAC2 knockout,and this increase was significantly decreased after H19 knockdown;the expression of E-cadherin was decreased and MMP14 was increased after HDAC2 knockout,and the changes of E-cadherin and MMP14 were decreased after H19 knockdown;Compared with SW480 cells,SW620 cells had stronger lung metastasis ability.Immunohistochemical staining showed that the expression of HDAC2 and E-cadherin in SW620 was lower,but the expression of MMP14 was higher.Conclusion:1.HDAC2 is downregulated in CRC metastases and low expression of HDAC2 is correlated with poor survival of CRC patients.Low expression of HDAC2 promotes EMT and colorectal cancer metastasis;2.Lncrna H19 is a downstream target gene for HDAC2 to negatively regulate EMT and colorectal cancer metastasis;3.HDAC2 inhibits the expression of H19 by binding to transcription factor Spl,anchoring to H19 promoter and catalyzing the deacetylation of histone H3K27;4.H19 can promote EMT and colorectal cancer metastasis by adsorbing miR-22-3p and relieving its inhibitory effect on MMP14.