The Transcription Factor HOXB9 Inhibits MiR-203 And Up-regulates SNAI2 Expression To Promote The Invasion And Metastasis Of Primary Hepatocellular Carcinoma

BackgroundPrimary hepatocellular carcinoma(HCC)is one of the most common and harmful malignant tumors.About 780,000 people die of liver cancer every year.The main causes of liver cancer-related death are the high invasiveness and metastasis of cancer cells.Therefore,it is important to clarify the mechanism of HCC’s invasion and metastasis and explore the corresponding treatment methods to further improve the survival rate of HCC patients.HOXB9(homeobox B9)is a member of the B subgroup of Hox gene.Recently,studies have found that HOXB9 is closely related to progression of cancer and is the key factor to promote the invasion and metastasis of cancer cells.Our previous research confirmed that overexpression of HOXB9 can promote invasion and metastasis of HCC.However,the downstream effector gene of HOXB9 in promoting invasion and metastasis of HCC and its regulatory mechanism are still unclear.SNAI2(Snail family transcriptional repressor 2)is a typical epithelial-mesenchymal transition transcription factor(EMT-TF).SNAI2 promotes invasion and metastasis of cancer cells by increasing EMT.In liver cancer,the expression of SNAI2 is increased and correlated with poor prognosis,it is a biomarker for predicting tumor metastasis.Studies have confirmed that the abnormal expression of SNAI2 in tumors is affected by many factors,including micro RNA.SNAI2 is a target gene of miR-203,and miR-203 inhibits invasion and metastasis of liver cancer by reducing the expression of SNAI2.ObjectiveBasing on the above theory,we speculate that SNAI2 is the downstream effector gene of HOXB9 in promoting invasion and metastasis of HCC,and HOXB9 upregulates SNAI2 expression by inhibiting miR-203.In order to prove our hypothesis,this study intends to analyze the correlation between HOXB9 and SNAI2 by detecting the expression of them in HCC tissue.Then,a variety of in vitro cell experiments and in vivo experiments are used to explore whether HOXB9 can promote invasion and metastasis of HCC by regulating SNAI2.Furthermore,to clarify whether HOXB9 regulate the expression of SNAI2 by miR-203.Finally,the molecular mechanism of HOXB9 inhibiting miR-203 is explored by using CHIP,luciferase reporter gene assays and immunoprecipitation experiments.Our research will provide a new target and theoretical basis for the treatment of HCC metastasis.Methods1.Quantitative real-time PCR(qRT-PCR)and western blotting were used to detect the expression of HOXB9 and SNAI2 in 48 fresh HCC tissues and corresponding adjacent tissues,the correlation between HOXB9 and SNAI2 was statistically analyzed.Immunohistochemistry(IHC)were used to detect the protein expression levels of HOXB9 and SNAI2 in 128 HCC specimens and corresponding adjacent specimens.The clinical medical records and prognosis of 128 HCC patients were collected,the correlation between HOXB9 or SNAI2 expression levels and survival prognosis of HCC patients were analyzed.2.Sh RNA lentivirus plasmid targeting HOXB9 were used to construct HCC cell lines that stably reducing HOXB9 and the HOXB9-overexpressing lentivirus were used to construct HCC cell lines that stably upregulating HOXB9.The expression changes of SNAI2 were detected by qRT-PCR and western blotting.Real time cell analysis(RTCA),transwell invasion assays and orthotopic liver transplantation model in nude mice were used to analyze the influence of HOXB9 alteration on the invasion and metastasis of HCC cells.SNAI2 overexpression plasmid was transfected into HOXB9-silenced cells to up-regulate SNAI2 expression,shRNA targeting SNAI2 was transfected into HOXB9-overexpressing cells to down-regulate SNAI2 expression,and then,western blotting was used to detect the expression changes of SNAI2.Real time cell analysis(RTCA),transwell invasion assays and orthotopic liver transplantation model in nude mice were used to analyze the changes of invasion and metastasis ability of HCC cells in each group and verify that HOXB9 promoted invasion of HCC cells by increasing the expression of SNAI2.3.CHIP-seq and luciferase reporter gene assay were used to verify whether HOXB9 directly regulates the transcription of SNAI2.4.The expression of HOXB9 was down-regulated or up-regulated in HCC cells,and the expression changes of miR-203 were detected by qRT-PCR.Inhibiting miR-203 in HOXB9-silenced cells or increasing miR-203 in HOXB9-overexpressing cells,the expression of SNAI2 in each group was detected to verify that HOXB9 affected SNAI2 expression by regulating miR-203.5.CHIP-qPCR and luciferase reporter gene assay were used to verify whether HOXB9 directly inhibits miR-203 transcription.6.CHIP-qPCR,luciferase reporter gene and qRT-PCR were used to determine whether up-regulation or down-regulation of EZH2 can change the trimethylation level of the 27 th lysine of histone H3(H3K27me3)in the miR-203 promoter region and affect the transcription and expression of miR-203.CHIP-qPCR was used to detect the effect of down-regulating or up-regulating HOXB9 on H3K27me3 level in miR-203 promoter region.CHIP-qPCR,qRT-PCR and luciferase reporter gene assay were used to detect the effect of EZH2 inhibition on miR-203 transcription and expression in HOXB9-overexpressing cells.Co-immunoprecipitation(co-IP)and GST-pull down were used to verify the interaction between HOXB9 and EZH2.CRISPR-Cas9 technology was used to construct HOXB9-knocked out HCC cells,and CHIP-qPCR was used to detect whether EZH2 was recruited to miR-203 promoter region depending on HOXB9,and then affected histone methylation and inhibited miR-203 transcription and expression.Results1.The results of qRT-PCR and western blotting showed that the expression of HOXB9 and SNAI2 in HCC tissues was higher than that in adjacent tissues.Correlation analysis showed that the expression of HOXB9 was positively correlated with that of SNAI2.The results of immunohistochemical(IHC)staining also confirmed that the expression of HOXB9 and SNAI2 in HCC specimens was higher than that in adjacent tissues.Moreover,the high expression of HOXB9 and SNAI2 is related to the malignant clinical features of HCC,which are manifested as advanced tumor TNM staging,more venous invasion and tumor microsatellite formation.Kaplan-Meier analysis showed that HCC patients with high expression of HOXB9 and SNAI2 had the lowest overall survival rate.2.Down-regulation of HOXB9 in HCC cells leads to a decrease in the expression of SNAI2 and is accompanied by a decline in cell’s invasion and metastasis abilities.While overexpression of HOXB9 can up-regulate the expression of SNAI2 and the invasion and metastasis of cells.Overexpression plasmid of SNAI2 upregulates the expression of SNAI2 in HOXB9-silenced HCC cells.The results of in vitro and in vivo experiments showed that the upregulation of SNAI2 can rescue the decline of cancer cells’ invasion and metastasis caused by HOXB9 inhibition.On the contrary,shRNA targeting SNAI2 can reduce the expression of SNAI2 in HOXB9-overexpressing HCC cells and weaken the increase of invasion and metastasis caused by HOXB9 overexpression.3.CHIP-seq results showed that HOXB9 was not enriched in the promoter region of SNAI2,and luciferase reporter gene experiment showed that HOXB9 did not regulate the transcriptional activity of SNAI2 promoter.4.Reducing the expression of HOXB9 upregulates miR-203 expression,while overexpressing HOXB9 inhibits miR-203 expression.Inhibition of miR-203 in HOXB9-silenced HCC cells can restore the expression of SNAI2 and the ability of invasion and migration;up-regulation of miR-203 in HOXB9-overexpressing HCC cells can inhibit the increase of SNAI2 expression and the invasion and migration ability of cells.5.CHIP-seq results showed that HOXB9 was enriched in the promoter region of miR-203.Luciferase reporter gene assays and qRT-PCR results showed that HOXB9 inhibited the transcription and expression of pri-miR-203.6.Overexpression of EZH2 upregulates the level of H3K27 in miR-203 promoter region and inhibits the transcription and expression of miR-203.On the contrary,inhibition of EZH2 resulted in the decrease of H3K27me3 level in the promoter region of miR-203 and up-regulation of transcription and expression of miR-203.The level of H3K27me3 in miR-203 promoter region was decreased in HOXB9-silencing cells and increased in HOXB9-overexpressing cells.Inhibition of EZH2 in HOXB9-overexpressing cells can reduce the level of H3K27me3 and up-regulate the transcription and expression of miR-203.IP and GST-pull down experiments confirmed that HOXB9 directly binds to EZH2,and the binding region is WD-Binding domain of EZH2.In HCCLM3 cells,the enrichment region of EZH2 in miR-203 promoter is the same as that of HOXB9,but EZH2 cannot be enriched in miR-203 promoter anymore when HOXB9 was knocked out in HCC cells.Even if EZH2 is increased,the transcription and expression of miR-203 cannot be inhibited in HOXB9-knocked out cells.However,after the expression of HOXB9 was restored,EZH2 could be re-enriched in the promoter region of miR-203,and up-regulation of EZH2 can inhibit the transcription and production of miR-203.Conclusions1.HOXB9 promotes the invasion and metastasis of HCC cells by inhibiting miR-203 to up-regulate the expression of SNAI22.EZH2 is a key factor for HOXB9 to inhibit miR-203 transcription3.HOXB9 increases the level of H3K27me3 in the promoter region of miR-203 by recruiting EZH2 to inhibit the transcription of miR-203.