The Effect And Mechanism Of Sex-determining Region Y Box Protein 9 On Hepatocarcinogenesis

?Background and Objective? Hepatocellular carcinoma?HCC?accounts for 70-90% of primary liver cancer and it is a rapidly-growth malignant tumor with early matastasis and poor survival.Although significant progress has been achieved over the past decades,the outcomes of patients with late-stage HCC are still unsatisfactory.Thus,many researchers begin to work on interferencing the initiation of HCC.But until now,the molecular mechanisms invoving the HCC initiation are still elusive.The molecular pathogenesis of HCC remains to be defined,and novel diagnostic and therapeutic techniques need to be developed.Sex-determining region Y box protein 9?SOX9?is an important transcription factor that controls cell fate decision during the development and homeostasis of a broad range of tissues as a member of SRY family.SOX9 is regarded as a stem/progenitor cell maker in some tissues.As to the liver,SOX9 is mainly expressed in biliary epithelial cells and seldom expressed in hepatocytes under physiological condition.As is known to all,the development of hepatocellular carcinoma closely relates to the presence of chronic liver disease.When the liver suffers from chronic injury,hepatocytes will express the SOX9 and contributes to the regeneration of hepatocytes and biliary epithelial cells as stem/progenitor cells.SOX9 is expressed in a wide range of cancers,and its deletion prevents tumorigenesis in prostate and pancreatic mouse cancer models.In addition,as is reported,SOX9 expression is elevated in the HCC tissue and predicts poor outcomes.Of note,many kinds of tumors originate from stem cells,such as skin,hematopoietic system and intestine.Recently,Mu et al have reported that hepatocellular carcinoma originates from hepatocytes and not from the progenitor/biliary compartment.Thus,the malignant transformation of hepatocytes seems to be the early stage in HCC initiation.Collectively,whether SOX9+ hepatocytes assoiating with the initiation of HCC and the role of SOX9 playing in hepatocarcinogenesis need to be clarified.Based on the research background above,this study aims to explore the role of hepatocytes which expressed SOX9 as well as the effect and mechanism of SOX9 in hepatocarcinogenesis.?Methods? 1?The induction of HCC model?1?STZ-HFD model STZ-HFD model was induced in male mice by a single subcutaneous injection of 200 ?g STZ?V900890-100 mg,Sigma,MO,USA?at 2 days after birth and fed with HFD at 4 weeks old.After chowing HFD for 18 weeks,HCC could be induced in wild-type mice.?2?DEN×1 model Mice were injected i.p.with DEN?25 mg/kg BW?only once on day 14 after birth.About 8-9 mouths later,HCC could be induced in wild-type mice.?3?DEN×25 model All the mice were injected i.p.with DEN?35 mg/kg BW?once a week since mice were 5-week-old.HCC could be induced in wild-type mice after 21 injections?about 26-week-old?.2.Investigate whether primary HCC originates from hepatocytes which expressed SOX9?1?Generation of Sox9-Flp/Ai65 D mice To generate the knockin mice,IRES-Flp-poly A cassette was introduced into the UTR ahead of the third exon.The tail DNA was extracted and the genotype was detected by PCR.Then the mice were crossed with Ai65 D mice to obtain Sox9-Flp/Ai65 D mice,which could be marked with Td Tom specificly in the SOX9-expressed hepatocytes after the injection of AAV8-TBG-Cre.?2?The expression of Td Tom in livers of Sox9-Flp/Ai65 D mice The adult mice age 8-12 weeks old were choosed.Then,all the mice were injected i.v.with AAV8-TBG-Cre?2×1011genome copies?and were sacrificed two weeks later.The tail DNA was extracted and the genotype was detected by PCR.The expression of Td Tom,HNF4?,CK19 and Desmin in the liver tissues was detected by Immunofluorescence.To detect whether the amount of SOX9-expressed hepatocytes vary with time,the 14-,30-and 46-week-old mice were injected with AAV8-TBG-Cre and were sacrificed two weeks later.Then,the expression of Td Tom was detected and quantified by image-protein plus.?3?Sox9-Flp/Ai65 D mice tracing the origin of the primary HCC We established primary HCC models by DEN and STZ-HFD in the AAV8-TBG-Cre-injected Sox9-Flp/Ai65 D mice.And the mice were sacrificed when HCCs were induced.The expression of Td Tom in tumors was assessed by Immunofluorescence.3.The effect of specific knockout of SOX9 in liver on hepatocarcinogenesis in mice.?1?To verify the expression of SOX9 in the liver of Sox9^LKO mice Sox9^f/f mice were crossed with Alb-Cre mice to obtain Sox9^LKO mice,which was specifically knock out the SOX9 in hepatocytes and biliary epithelial cells?BECs?.All the 8-week-old mice were sacrificed,and the tail DNA was extracted and the genotype was detected by PCR.The expression of SOX9 in liver tissue was analyzed by Western blotting and immunohistochemistry.?2?The effect of liver-specific deletion of SOX9 on hepatocarcinogenesis Mice were divided into two groups: Sox9^LKO and Sox9^f/f group according to the genotype.Three HCC models were established according to the protocols above.The mice were sacrificed at several time points and the liver morphology,tumor number,tumor size were recorded.The liver tissue would be stained by H&E and Sirius red.SOX9,Ki67 and CK19 were detected by IHC and the serous liver function indicators were tested.4.The effect of hepatocyte-specific deletion of SOX9 on liver tumorigenesis in mice?1?To assess the expression of SOX9 in the liver of Sox9^HKO mice Hepatocytes were isolated from the Sox9^HKO and Sox9^f/f mice of the STZ-HFD model after chowing HFD for 8 weeks.The expression of SOX9 in hepatocytes was detected by RT-PCR and western blotting.The liver tissue was analyzed by IHC.?2?The effect of hepatocyte-specific deletion of SOX9 on hepatocarcinogenesis Sox9^f/f mice were injected i.p.with AAV8-TBG-Cre/AAV8-TBG to obtain the Sox9^HKOmice and control mice.Then the STZ-HFD and DEN×1 model were induced as described above.Mice were sacrificed at several time points and the liver morphology,tumor number,tumor size were recorded.The liver tissue would be analysed by H&E and Sirius red.SOX9,Ki67 and CK19 were detected by IHC and the serous liver function indicators were tested.5.The molecular mechanism of SOX9 inhibit heaptocarcinogenesis?1?To detecte the differential gene expression in the Sox9^LKO mice The m RNA was extracted from the total liver of Sox9^f/f mice and Sox9^LKO mice treated with DEN for 13 times?DEN×25 model?.The quality RNA was evaluated and the expression of Sox9 was confirmed.Then RNA-Seq assay was performed to seek for the differential genes.?2?To verify the accuracy of RNA-seq According to the analysis of RNA-seq,the differential genes in Wnt signaling pathway were tested in the samples used for RNA-Seq.?3?To validate the activation of Wnt signaling in another HCC model The hepatocytes and liver were collected from Sox9^HKOmice in the HFD-STZ model chowing HFD for 8 weeks.m RNA was extracted and the genes were detected by RT-PCR.?4?Tumorigenesis related signal pathways in Sox9 KO livers of HCC models Western blotting was used to detect the signal pathways in liver tissue of Sox9^HKOand Sox9^LKO mice in the HCC models.5.Statistical analysis Statistical analyses were performed by SPSS software?21.0 version?.Two-tailed P value <0.05 was considered statistically significant.Student's t test was used to analyze the data of experiments involving only two groups of equal variances.The unequal variances pairing using the Wilcoxon signed rank test or Mann-Whitney U test.?Results? 1.Expression of SOX9 in hepatocytes isn't indispensable in hepatocarcinogenesis?1?The Td Tom + cells in the liver tusse also expressed the hepatocytes marker HNF4?,but didn't express the maker of biliary epithelial cells?CK19?and hepatic stellate cells?Desmin?.These results indicated after the injection of AAV8-TBG-Cre,only hepatocytes which expressed SOX9 could be marked with Td Tom.?2?The quantity of the Td Tom+ cells in liver tissue showed about 10% of hepatocytes could be maked with Td Tom without any injuries and these can be regarded as “background”.The amount of the Td Tom+ heaptocytes had no significant differences among 16-,32-and 48-week-old mice.?3?In the STZ-HFD-and DEN-induced mouse HCC models,we could see the liver became red and most of hepatocytes expressed Td Tom.Furthermore,the Td Tom+ and Td Tom-tumors could be observed in the livers and sometimes existed in one liver.2.Liver-specific SOX9 deletion promotes hepatocarcinogenesis?1?To verify the expression of SOX9 in the liver of Sox9^LKO mice Western blotting showed the expression of SOX9 significantly decreased in the liver of Sox9^LKO mice.The expression of SOX9 in BECs and hepatocytes decreased significantly in Sox9^LKO mice by IHC analysis.?2?Deletion of SOX9 in liver promotes hepatocarcinogenesis in HCC model DEN×25 model We regularly sacrificed one cohort of mice after DEN injection for 13,17,21 and 24 weeks to evaluate the development of HCC.Our results showed that liver tumor incidence was significantly higher in Sox9^LKO mice compared with Sox9^f/f group at every time point,especially after 21 injection of DEN.At the terminal point,macroscopic tumors of liver were detected 100%?6/6?in Sox9^LKO mice,while only 28.6%?2/7?could be seen in Sox9^f/f counterpart.Consistently,the number and sizes of liver tumors of Sox9^LKO mice dramatically increased compared to that of control mice.Interestingly,the ductular reaction?DR?and liver cirhhosis were significantly enhanced in the SOX9-deletion group via IHC and Sirius red staining.STZ-HFD model Mice from two groups were sacrificed after chowing HFD for 4,8,12 and 18 weeks to assess the occurance of HCC.The results displayed that HCC incidence increased in Sox9^LKO mice at any time point,especially after 18-week HFD.At the last time point,tumors could be seen 100%?7/7?and 28.6%?2/7?in Sox9^LKO mice and Sox9^f/f counterpart,respectively.As is consistent with the DEN×25 model,the tumor number and size of liver tumors in Sox9^LKO mice dramatically elevated.Besides,the ductular reaction and liver cirhhosis are more severe in Sox9^LKO mice.3.Hepatocyte-specific deletion of SOX9 promotes hepatocarcinogenesis?1?To verify the expression of SOX9 in the hepatocetes of Sox9^HKO mice Western and RT-PCR showed the expression of SOX9 dramatically decreased in the hepatocytes isolated from Sox9^HKO mice treated with HFD for 8 weeks in STZ-HFD model.IHC staining displayed that BECs still expressed SOX9,while the SOX9 positive hepatocytes rarely existed.?2?Hepatocytes-specific deletion of SOX9 promoting HCC initiation.STZ-HFD model Mice of Sox9^HKO and Sox9^f/f groups were fed with HFD for 8 and 12 weeks prior to being sacrificed.Interestingly,the results showed to us were consistent with that of Sox9^LKO and Sox9^f/f mice.After chowing HFD for 12 weeks,HCC could be seen 100%?5/5?and 25%?1/4?in Sox9^HKO and Sox9^f/f mice,respectively.Compared Sox9^HKO mice with Sox9^LKO mice,we could see more HCC and larger tumor zise in Sox9^LKO mice.Pathological tissue analysis showed enhanced ductular reaction and cirhhosis in Sox9^LKO mice.DEN×1 model As was consistent with the STZ-HFD model,the tumor number and tumor sizes of liver tumors between groups were in that order :Sox9^LKO > Sox9^HKO> Sox9f/ f.Besides,the relatively severe DR,cirhhosis and elecated liver function indicators could also be detected in Sox9^LKO mice rather than in Sox9^HKO mice and Sox9^f/f mice.Collectively,these results vastly demonstrated that SOX9 acted as a tumor suppressor in hepatocarcinogenesis and may have opposite functions in tumor initiation and development.4.The activation of Wnt,AKT and ERK signaling pathways contribute to the accelerated hepatocarcinogenesis in Sox9 KO mice.?1?RNA-Seq showed 1397?89%?upregulated genes in Sox9^LKO mice and KEGG analysis indicated the activation of pathways in cancer among the upregulated genes.Furthermore,GSEA analysis showed the activation of Wnt,Hedgehog and chemokine signaling in Sox9^LKO mice.The activation of Wnt signaling was confirmed in Sox9^LKO and Sox9^HKOmice.?2?The consistent results of RT-PCR and RNA-seq of the same samples indicating the accurancy of RNA-seq.?3?Consistent with the result of RNA-seq,the genes changed in Wnt signaling pathway?wnt5a?wnt7a?wnt7b?FZD2?FZD6 et al.?also elevated in the liver and hepatocytes of Sox9^HKO mice of STZ-HFD model.?4?Western blotting analysis displayed that the elevated phosphorylation of AKT and ERK in Sox9^LKO mice and Sox9^HKO mice of two models.?Conclusions? 1.Expression of SOX9 in hepatocytes isn't indispensable in hepatocarcinogenesis.2.Liver-Specific deletion of SOX9 induces ductular reaction and liver cirrhosis and could promote carcinogenesis.3.Hepatocyte-Specific deletion of SOX9 could also promote hepatocarcinogenesis without giving rise to ductular reaction and liver cirrhosis.4.Deletion of SOX9 in BECs could induce ductular reaction and plays a role in accelerating HCC initiation.5.The activation of Wnt,AKT and ERK signaling pathways contributes to the promoted hepatocarcinogenesis in Sox9 KO mice.SOX9 may have dual roles in liver tumor initiation and development.