| BACKGROUND&OBJECTIVEColorectal carcinoma(CRC)is one of the most common malignant cancers in digestive system.China Forum on Epidemiology and Prevention of CRC in 2017 noted that the incidence of CRC ranked the third in the incidence of malignant tumors in China,following lung cancer and gastric cancer.Its mortality rate was the 5th,following lung cancer,liver cancer,gastric cancer and esophageal cancer.The effect of early treatment of CRC is good,but the effect of late treatment is relatively poor.The recurrence and metastasis of CRC patients are the main causes of death due to lack of effective predictive molecules and therapies.Therefore,finding new early diagnostic markers and therapeutic targets for CRC is the key to control the occurrence and development of CRC.Tumorigenesis is a complex biological process involved in multilevel reaction,multi-factor action and multi-gene.The researches had been focused on tumor related genes coding protein gene in decades.However,with the continuous development of high-throughput sequencing technology,large amount of long noncoding RNA(LncRNA)were detected,their important value in activities of life was also revealed gradually.Providing a new perspective and important source for the study of new tumor related gene and has become a research hotspot.HOXD-AS1,an LncRNA,has been found to have abnormal expression in many cancers,including neuroblastoma,bladder cancer,prostate cancer and liver cancer,and to involve in the tumor cell proliferation,invasion and metastasis,tumor angiogenesis and other malignant biological processes.Our previous study found that the expression level of HOXD-AS1 in CRC was significantly down-regulated,suggesting that its declined influence may be closely related to the occurrence and development of CRC.Therefore,studying the molecular mechanism of abnormal influence of HOXD-AS1 in CRC will provide a new way to elucidate the pathogenesis,diagnosis and treatment of CRC.METHODS1.In situ hybridization and immunehistochemistry were used to detect the expression of HOXD-AS1 and HOXD3 in CRC tissues.Meanwhile,we analyzed the relationship between their expression level and clinicopathological parameters of CRC patients.2.CCK-8 and colony formation experiments were used to detect cell proliferation in vitro.Wound healing and transwell invation assay were used to detect migration and invasion ability in vitro.In vivo,the tumorigenesis and metastasis abilities were measured by subcutaneous tumorigenesis assary and splenic capsule injection of nude mice.3.RNA-pull down combined with mass spectrometry and RIP experiments were used to screen and assess the target proteins combined with HOXD-AS1.ChIP and luciferase reporter system were applied to explore the mechanisms of HOXD3 expression regulated by HOXD-AS1.4.The downstream molecule of HOXD3 was measured by ChIP and luciferase reporter system,to further elucidate the molecular pathways that HOXD-AS1 plays in CRC.RESULTS1、The expression of LncRNA HOXD-AS1 in CRC and its relationship with clinicopathological factors of patients with CRCWe design HOXD-AS1 specific probe for in situ hybridization to detect the expression level of HOXD-AS1 in CRC tissue and normal tissue.It was found that HOXD-AS1 located in the cell nucleus.In addition,HOXD-AS1 was high expressed in 76.92%normal intestinal epithelium tissues.However,only 36.04%CRC tissues had the high expression of HOXD-AS1.The expression of HOXD-AS1 was found down-regulated in CRC tissue,compared with normal mucosa tissues(χ2 = 48.01,P<0.001).Low expression of HOXD-AS1 was significantiy correlated with tumor differentiation(P = 0.013),T stage(P = 0.001),lymph node metastasis(P = 0.001),and distant metastasis(P = 0.028),but there was no significant relevance with sex,age,tumor size or tumor location(P>0.05).Furthermore,Kaplan-Meier analysis reveals that CRC patients with lower HOXD-AS1 expression exhibited shorter overall survival time than that of with higher HOXD-AS1 expression(92.19± 5.42 vs 74.32± 4.58,log-rank P<0.001).Multivariate COX analysis showed that the low expression of HOXD-AS1 was an independent factor in the prognosis of patients with CRC(HR = 0.581,P=0.043).2.The expression of HOXD3 in CRC and its relationship with clinicopathological factors of patients with CRCWe ordered specific immunohistochemical antibody,and analyzed the expression of HOXD3 in CRC and normal intestinal epithelium tissues.We found that HOXD3 located in the cell nucleus.In addition,HOXD3 was low expressed in 78.1%normal intestinal epithelium tissues.However,81.06%CRC tissues had high expression of HOXD3.The expression of HOXD3 was found up-regulated in CRC tissue,compared with normal mucosa tissues(r2 = 42.572,P<0.001).High expression of HOXD3 was significantiy correlated with tumor lymph node metastasis(P = 0.016)and distant metastasis(P = 0.046),but there was no significant relevance with sex,age,tumor size or tumor location(P>0.05).Furthermore,Kaplan-Meier analysis shown that CRC patients with higher HOXD3 expression exhibited shorter overall survival time than that of the CRC patients with lower HOXD3 expression in clinical samples(75.29±6.82 vs.94.32 ± 5.23,log-rank P = 0.014).Multivariate COX analysis of the prognosis of CRC patients showed that high expression of HOXD3 was an independent factor in the prognosis of patients with CRC(HR =1.862,P = 0.004).3.HOXD-AS 1 promotes CRC progression partly by regulating HOXD3 expression.We performed rescue experiment in SW620 cell with co-transfection of pcDNA3.1-HOXD-AS1 and pcDNA3.0-HOXD3.The funtional experiments showed that:CCK-8 assay showd that when overexpression of HOXD-AS1 the proliferation of CRC was inhibited(F= 256.356,P<0.001),whereas overexpression of HOXD3 the proliferation of CRC was enhanced(F= 91.495,P<0.001).The plate clone formation assay showed that when overexpression of HOXD-AS1 the number was decreased(t= 1.09,P<0.001),however overexpression of HOXD3 the colony number was increased(t= 7.223,P<0.001).Wound healing assay showed that overexpression of HOXD-AS1 the migration of SW620 was decreased significantly(t = 7.363,P = 0.0018),however overexpression of HOXD3 increased the ability of migration(t= 12.09,P<0.001).Transwell showed that overexpresion of HOXD-AS1 the number of cells that migrated from the polycarbonate membrane was decreased(t = 71363,P= 0.0018)while overexpression of HOXD3 the number of migration cells was increased(t=8.571,P= 0.001).The results of subcutaneous tumor formation in nude mice showed that the proliferation of SW620/HOXD-AS1 subcutaneous tumors was significantly slower than that of SW620/Control cells(F= 24.977,P<0.001),but that after re-expression of HOXD3,the proliferation of SW620/HOXD-AS1+HOXD3 cells significantly increased(F＝19.427,P<0.001).The results of nude mice liver metastasis model showed that SW620-HOXD-AS1 cells exhibited fewer intrahepatic metastasis(=0.046);while HOXD3 overexpression partly rescued the metastasis ability suppressed by HOXD-AS1 overexpression(P = 0.014).4.The molecular mechanism of HOXD-AS1 regulates HOXD3 expression in CRC.4.1 Identification of binding proteins with HOXD-AS1We performed RNA-pull down assay to identify the protein partner of HOXD-AS1.The band specially enriched in HOXD-AS1 subjected to mass spectrometry,which identified the interaction between HOXD-AS1 and SUZ12.SUZ12 is a component of PRC2 complex,the other key molecular of which is EZH2.To examine whether HOXD-AS1 can also binding EZH2,EZH2 was detected in the RNA-pull down products by immunoblotting using anti-EZH2,which confirmed the interaction between HOXD-AS1 and EZH2.Moreover,we conducted RIP assay using anti-EZH2 antibodies and anti-SUZ12 antibodies,and found that HOXD-AS1 can bind to both EZH2 and SUZ124.2 Effect of SUZ12 and EZH2 on the expression of HOXD3The SUZ12 and EZH2 genes that in the SW620 cell line were interfered respectively,the expression of HOXD3 mRNA in the SW620cells was detected by Real-Time PCR.The results shown that SW620 cells with knockdowned SUZ12 or EZH2 displayed an increased expression of HOXD3(FSUZ12= 7.294,PSUZ12=0.011;FEZH2 = 14.49,PEZH2-0.0096).4.3 HOXD-AS1 can regulated HOXD3 expression by interacting with PRC2 complex to induce H3K27me3.We conducted chromatin immunoprecipitation(ChIP)assays for HOXD3 promoter(-2019～-23 nt)using anti-SUZ12,anti-EZH2 and anti-H3K27me3 antibodies,and respectively detected the enrichment of each of the seven segments(P1-P7)of HOXD3 promoter with seven paired primers.We found that SUZ12,EZH2 and H3K27me3 are enriched in the promoter of HOXD,especially P1,P3,P5,P6.After overexpression of HOXD-AS1,the enrichment of SUZ12,EZH2 and H3K27me3 in P1 and P3 increased significantly.Taken together,these results suggested that HOXD-AS1 can suppressed HOXD3 expression by interacting with PRC2 complex.5.HOXD3 activates transcription of ITGB3 geneHOXD3 is a transcription factor and has been reported to be positive correlation with expression of integrin subunit beta 3(ITGB3).We predicted the HOXD3-bind region of ITGB3 promoter by JASPAR and preformed ChIP assays.The result of ChIP confirmed the binding of HOXD3 to the ITGB3 promoter.Moreover,duel-luciferase report showed that HOXD3 could bind ITGB3 promoter in both the HOXD3 and ITGB3 elements group and activate luciferase both in SW620 and 293T cells.Collectively,we found that HOXD3 could promote the transcription of ITGB3 in CRC.CONCLUSION1.The expression level of HOXD-AS1 was down-regulated in CRC tissues and correlated with poor prognosis in patients with CRC.2.The expression level of HOXD3 was up-regulated in CRC tissues and correlated with metastasis in CRC patients and higher HOXD3 expression positively correlated with poor Prognosis.3.HOXD-AS1 expression was negatively associated with HOXD3 expression.Meanwhile,overexpression of HOXD-AS1 could inhibit the proliferation and migration of CRC cell in vitro and in vivo,depending on HOXD3.4.HOXD-AS1 interacting with PRC2 complex could induce H3K27me3 of HOXD3 promoter and suppress HOXD3 expression.5.HOXD3 acts as a transcription factor to activate the transcription of ITGB3,and three of them form HOXD-AS1/HOXD3/ITGB3 regulatory axis to inhibit the occurrence and development of CRC. |
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