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

【Background and Objective】Hepatic cysts are a group of heterogeneous diseases with different pathogeneses,of which simple hepatic cysts and polycystic liver disease（PLD）are two major types.Most patients with these two diseases have a benign course,but some suffer from symptoms and complications associated with the enlargement of cysts and the subsequent compression of abdominal organs,such as abdominal pain,early satiety,oesophageal reflux or intracystic haemorrhage,infection,and even rupture of the cyst.In severe cases,liver transplantation is required due to excessive size and number of liver cysts resulting in liver function damage or even liver failure.At present,the main treatment methods for hepatic cysts include aspiration-sclerotherapy,fenestration,partial liver resection and even liver transplantation.Partial hepatectomy and liver transplantation are effective treatments for hepatic cysts but still have many problems including postoperative complications,restrictions in clinical practice.Therefore,exploring the molecular mechanism of hepatic cystogenesis is helpful to find new therapeutic targets.PLD can occur as isolated autosomal dominant polycystic liver disease（ADPLD）or in combination with polycystic kidney diseases（PKD）.Genetic disorders of PLD accompanied by autosomal dominant polycystic kidney disease（PKD）are PKD1 or PKD2germline mutations,while ADPLD may arise from mutations of PRKCSH,SEC63,and LRP5.In contrast to PLD,simple hepatic cysts are sporadic,and their genetic background has been elusive so far.The pathophysiological changes of hepatic cystogenesis include ductal plate malformation（DPM）and abnormal proliferation of bile duct epithelial cells.DPM refers to the abnormal remodeling of the biliary plate during the development of the bile duct,which is associated with the disturbed polarization of bile duct plate cells and assembly and functional abnormalities of primary cilia.SEC63 is an endoplasmic reticulum-related gene involved in the biotransformation process of proteins,and mutations of SEC63 lead to the development of ADPLD.It has also been shown that knockout of SEC63 in human bile duct cells disrupts primary cilia formation.However,the transcriptional regulation of SEC63 has not been elucidated.Sex-determining region Y-box protein 9（SOX9）is a vital transcription factor which is expressed in many tissue types and organs,such as brain,lung,kidneys,intestine,liver,controlling the development of organs and differentiation of many cell types.SOX9 is known as a progenitor/stem cell marker in many tissues.In the liver,SOX9 is mainly expressed in bile duct epithelial cells,and thus regarded as commonly used biliary markers.During intrahepatic bile duct development,SOX9 determines the timing of bile duct morphogenesis and regulates the formation of symmetrical bile duct structure.A recent study also reported that SOX9 controls the differentiation,polarity,and cilium formation of biliary epithelial cells and regulates the expression of intermediates in several signaling pathways required for normal bile duct development,such as transforming growth factor-β（TGF-β）,Notch,and Hippo signaling pathways.These results suggest that SOX9 plays an important role in bile duct development and determining the fate of biliary cells.The previous work of our group proved that liver-specific knockout of Sox9 can promote the development of liver cancer.In this process,we observed that liver cysts were spontaneously formed in mice with liver-specific knockout of Sox9 without modeling,suggesting that deletion of Sox9 may be involved in hepatic cystogenesis.Based on the above research background and preliminary basis,this study aims to further clarify the role of SOX9 in hepatic cystogenesis and further explore its molecular mechanism.【Methods】Part1.The effect of liver-specific knockout of Sox9 on hepatic cystogenesis 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 establish Sox9^LKO(Sox9^f/f:Alb-Cre+)mice.Using PCR and agarose gel electrophoresis to detect the genotype of mice.The liver of the two groups was collected,and the expression of SOX9 protein in liver tissue was detected by Western blot,immunohistochemistry and immunofluorescence.2.To observe the effect of liver specific deletion of SOX9 on hepatic cystogenesis in mice.Sox9^f/f and Sox9^LKO mice were sacrificed at different ages after birth.After the mice were sacrificed,the cysts on the liver surface were observed,grossly counted.Using microcomputed tomography（micro-CT）scan to visualize the internal cysts in the livers of mice.The liver tissue sections of the two groups were analyzed histopathologically by H&E,sirius red staining and IHC.Meanwhile,serum liver biochemistry indexes including alanine aminotransferase（ALT）,alkaline phosphatase（ALP）,aspartate aminotransferase（AST）and total bilirubin（TBIL）were evaluated.Part2.The effect of depletion of SOX9 on proliferation,polarity and formation of primary cilia of biliary epithelial cells1.Knockdown of SOX9 by siRNAs in human intrahepatic bile duct epithelial cells in vitro,cell activity was detected by CCK-8 kit.2.To assess the proliferation of biliary epithelial cells in Sox9^LKO mice and Sox9^f/fmice,Brd U was injected intraperitoneally into mice（50 mg/kg,daily）for 5 days.The mice were sacrificed 2h after the last Brd U injection,and Brd U positive cells were visualized by immunofluorescence.3.Immunofluorescence co-localization analysis was used to assess the proliferation of biliary epithelial cells in Sox9^LKO mice and Sox9^f/f mice by detecting PCNA/CK19 double-positive cells.4.Immunofluorescence co-localization analysis was used to assess the polarity of biliary epithelial cells in Sox9^LKO mice and Sox9^f/f mice by detecting OPN/CK19 double-positive cells or Laminin/EpCAM double-positive cells.5.Immunofluorescence co-localization analysis was used to assess the formation of primary cilia of biliary epithelial cells in Sox9^LKO mice and Sox9^f/f mice by detecting Ac-tubulin-positive or ARL13B signals.6.The expression of primary ciliary markers（AC-tublin）was detected by immunofluorescence to observe the effect of SOX9 knockdown on the formation of primary cilia in biliary epithelial cells.Part3.The molecular mechanism of depletion of SOX9 on hepatic cystogenesis1.The primary biliary epithelial cells were isolated from 6-and 12-month-old Sox9^LKOmice and Sox9^f/f mice.EpCAM positivity determined by flow cytometry and biliary marker expression detected by RT-qPCR.2.Expression of hepatic cysts related genes（including PKD1,PKD2,SEC63,LRP5,PRKCSH）in primary biliary epithelial cells of Sox9^LKO mice and human intrahepatic biliary epithelial cells transfected with siSOX9 were detected by RT-qPCR.3.Western blot was used to detect the level of SEC63 protein in the siSOX9 group of HIBEpic.4.The binding sites of SOX9 and its possible downstream target genes（SEC63,PKD1,LRP5）were analyzed by using JASPAR and h TFtarget databases.5.The transactivation ability of SOX9 to downstream target genes SEC63,PKD1 and LRP5)was detected by the luciferase assay,and the binding sites were preliminatively explored.6.To observe whether overexpression of SEC63 in human primary biliary epithelial cells can reverse the effect of siSOX9 in biliary epithelial cells.Part4.Statistical analysisAll statistical analyses were performed by SPSS 22.0 software.Student’s T test was used for data of normal distribution.If the data did not obey the normal distribution,the nonparametric Mann-Whitney test was used.Animal-related data are expressed as mean±standard error,and other data are expressed as mean±standard deviation.Statistical test was two-sided,P<0.05 was considered statistically significant.【Results】Part1.Knockout of Sox9 in the liver leads to hepatic cystogenesis in mice1.The expression of SOX9 protein by Western blot,immunohistochemistry and immunofluorescence confirmed that SOX9 was almost not expressed in the liver tissue of Sox9^LKO mice.2.Through gross observation and micro-CT scanning,liver tissue of 3-month-old Sox9^LKO mice showed no obvious changes,while liver cysts appeared from the 6-month-old Sox9^LKO mice.The incidence,number and size of liver cysts in Sox9^LKO mice increased with age.No cysts were observed in Sox9^f/f mice even up to 12 months old.3.H&E staining and immunohistochemical staining for biliary tract marker CK19showed that the mice in Sox9^LKO group showed progressive abnormal bile duct dilation in liver from 3 months of age.Sirius red staining was performed and showed increased collagen deposition in Sox9^LKO mice compared with Sox9^f/f mice。4.Compared with Sox9^f/f mice,serum test of Sox9^LKO mice showed an increase in alkaline phosphatase ALP,while other liver biochemistry indexes,including ALT,AST and TBIL,did not change.Part2.Depletion of SOX9 resulted in the hyperproliferation and reduced the formation of the primary cilium and disordered polarity in biliary epithelial cells1.In vitro experiments showed that SOX9 knockdown promoted the proliferation of human primary bile duct epithelial cells（HIBEpic）.2.The Brd U incorporation assays showed that the increase of Brd U/CK19 double-positive cells was detected in liver of 3-and 6-month-old Sox9^LKO mice,but not in Sox9^f/fmice at the same age.3.Immunofluorescence staining of liver tissues of 6-month-old and 12-month-old Sox9^LKO mice showed increased PCNA/CK19 double-positive cells,while almost no PCNA staining was detected in Sox9^f/f mice.4.Immunofluorescence staining of liver tissues of 12-month-old Sox9^LKO mice showed obvious PCNA staining in the endothelial cells of the cyst wall.5.In the liver tissues of Sox9^f/f mice,immunofluorescence staining showed that the apical marker OPN and the basal marker Laminin were normally expressed at the apical and basal poles of biliary epithelial cells,while in the liver tissues of 6-month-old Sox9^LKOmice,some biliary epithelial cells showed no OPN expression at the apical pole,while the basal marker Laminin was thickened.The apical marker OPN of the biliary epithelial cells of 12-month-old Sox9^LKO mice displayed mislocalization,and the basal marker Laminin was fragmented and thickened.6.In 6-month-old mice,Ac-tubulin positive or ARL13B positive primary cilia were significantly reduced in biliary epithelial cells of Sox9^LKO mice compared with Sox9^f/f mice.In 12-month-old Sox9^LKO mice,primary cilia were still absent in biliary epithelial cells even though the bile ducts were in a state of hyperplasia.7.In vitro,immunofluorescence staining showed that the positive signal of Ac-tubulin of human primary bile duct epithelial cells（HIBEpic）with Sox9 knockdown decreased,indicating that the formation of primary cilium was reduced.Part3.Depletion of SOX9 leads to hepatic cystogenesis by transcriptionally downregulating SEC631.EpCAM positivity was detected by flow cytometry,and the mRNA expression of biliary tract marker was detected by RT-qPCR,confirming that EpCAM-positive primary biliary epithelial cells of mice were successfully isolated.2.RT-qPCR results showed that compared with Sox9^f/f mice,mRNA levels of PKD-related genes,including Pkd1 and Pkd2,and ADPLD-related genes,including Sec63,Lrp5,and Prkcsh,were significantly reduced in primary biliary epithelial cells of Sox9^LKO mice.3.RT-qPCR results showed that in vitro,the knockdown of SOX9 in HIBEpic only led to the downregulation of SEC63,while PKD1,PKD2 and LRP5 mRNA were not reduced.4.Western blot also confirmed that the knockdown of SOX9 led to a decrease in SEC63 protein level in HIBEpic.5.JASPAR and h TFtarget databases were used to analyze and speculate that the binding sites of SOX9 and downstream target genes might be located in the 2kb region upstream of PKD1,SEC63 and LRP5 genes.6.Luciferase assay showed that overexpression of SOX9 only increased the activity of the SEC63 promoter reporter,and two possible SOX9 binding sites were found in the SEC63 promoter.When SEC63 site 1 was mutated,the activity of SEC63 promoter reporter was significantly impaired.7.Cell immunofluorescence showed that overexpression of SEC63 could reverse the effect of the depletion of SOX9 on the formation of primary cilia in HIBEpic.8.In vitro CCK8 detection showed that overexpression of SEC63 partially reversed the promoting effect of the knockdown of SOX9 on HIBEpic proliferation.【Conclusions】1.Knockout of Sox9 in the liver led to hepatic cystogenesis in mice.2.Depletion of SOX9 resulted in the hyperproliferation and reduced the formation of the primary cilium and disordered polarity in biliary epithelial cells.3.Depletion of SOX9 led to hepatic cystogenesis by transcriptionally downregulating SEC63.