The Role Of LINC00311 In The LPS Induced Increase Expression Of Endogenous β-glucuronidase Via TLR4/NF-κB/c-myc Signaling Pathway In Human Intrahepatic Biliary Epithelial Cells

Objective: Hepatolithiasis is a rare disease in western countries,but it’s very common in China.Due to the complicated course of this disease and the high recurrence rate,it becomes a benign biliary disease that is difficult to cure.Treatments such as bile duct exploration,removing stones by choledochoscopy or liver resection,etc.can only remove stones,but can not effectively prevent stone recurrence.Patients with recurrence of stones have to undergo surgery or endoscopic stone removal treatment,with increased pain,risk and difficulty.Therefore,it is of great clinical significance to study the mechanism of stone formation and explore effective methods for preventing or intervening the formation of stones.Most intrahepatic bile duct stones are pigment stones with bilirubin calcium as the main component.The exogenous β-glucuronidase(β-GD)produced by bacteria can decompose conjugated bilirubin to free bilirubin,which may cause the latter to combine with calcium to form calcium bilirubinate and deposit to form pigment stone.At present,it is found that after biliary infection,in addition to exogenous β-GD,endogenous β-GD is also significantly increased,which can also promote the decomposition of conjugated bilirubin to free bilirubin,and then participate in the formation of pigment stones.Therefore,the study of the regulation mechanism of endogenous β-GD activity will help us to explore effective measures to interfere with the formation or recurrence of hepatolithiasis.This study aims to determine whether LPS stimulates the expression of β-GD in human intrahepatic biliary epithelial cells(HiBEpiC)via TLR4/NF-κB/c-myc signaling pathway,thereby promoting the formation of hepatolithiasis.We also aim to identify long non-coding RNAs that play a key role in this disease process and explore whether curcumin could exert an anti-inflammatory effect on HiBEpiC by regulating this essential long non-coding RNA(lncRNA).Methods: Immunohistochemical staining analysis of NF-κB/c-myc signaling pathway molecules and β-GD was performed on paraffin sections of both normal liver tissues and hepatolithiasis liver tissues.Inflammatory injury model of HiBEpiC induced by LPS was used for Western blot analysis of β-GD and TLR4/NF-κB/c-myc signaling pathway molecules,and to verify whether intrahepatic bile duct stone formation is related to the increased expression of β-GD and activation of the TLR4/NF-κB/c-myc signaling pathway.Next,the lncRNAs associated with the three inflammatory molecules of the TLR4/NF-κB/c-myc inflammatory pathway were predicted online by using GCBI biological gene radar,to screen out key lncRNAs associated with LPS stimulation.After that,we validated the predicted lncRNA in the Protein-RNA Interaction Database(PRIDB).Quantitative real-time polymerase chain reaction(qPCR)was used to verify whether the predicted lncRNA was up-regulated in the LPS-induced inflammatory injury model of HiBEpiC,and also to detect the predicted lncRNA in both the normal liver tissues and hepatolithiasis liver tissues.After LINC00311 was identified as the candidate lncRNA,siRNA transfection was used to knock down the target lncRNA in HiBEpiC to explore whether LPS-induced inflammatory response could be alleviated.LINC00311 was also stably overexpressed in HiBEpiC by lentiviral infection.Western blot analysis was used to show the protein expression level of TLR4,NF-κB,c-myc,and β-GD in LINC00311 overexpressing biliary epithelial cells and the proliferation rate of HiBEpiC overexpressing LINC00311 was counted by CCK8 assay.The protein levels of epithelial cell marker Ecadherin and interstitial cell markers α-SMA and vimentin in LINC00311 overexpressing biliary epithelial cells were also detected by Western blot,to determine whether the epithelial-mesenchymal transition occurred.Then,we added NF-κB inhibitor JSH-23(Selleck)to the HiBEpiC overexpressing LINC00311 to block NF-κB/c-myc signaling pathway and detected β-GD expression level by Western blot,by means to explore whether LINC00311 could promote inflammatory response through the NF-κB/c-myc signaling pathway.Finally,curcumin was used to inhibit the inflammatory response caused by LPS in HiBEpiC.We used qPCR to detect the expression level of the LINC00311 in the control group,LPS-treated group and curcumin combined with LPS-treated group,to explore whether curcumin could inhibit LINC00311.Western blot was used to detect the expression of TLR4,NF-κB,c-myc,and β-GD in the same three groups,to explore whether curcumin could down-regulate β-GD by inhibiting the activation of the aforementioned inflammatory pathway.Results: In the liver tissue of patients with hepatolithiasis,the TLR4/NF-κB/c-myc signaling pathway is activated,and β-GD is highly expressed.In the model of LPS-induced inflammatory injury of HiBEpiC,the same result is obtained(P<0.05).By using the biological gene radar,we predicted that the two lncRNAs LINC00311 and LINC00467 are related to the three molecules of the TLR4/NF-κB/c-myc signaling pathway.After that,we predicted that LINC00311 could interact with NF-κB by using the Protein-RNA Interaction Database.It was validated by qPCR that LINC00311 was positively associated with the inflammatory response in LPS treated HiBEpiC,and the same results were also obtained in liver tissues of hepatolithiasis patients(P<0.05).Transfection of LINC00311 siRNA inhibited the inflammatory response in HiBEpiC caused by LPS(P<0.05).Overexpression of LINC00311 by lentiviral Infection in HiBEpiC could directly up-regulate β-GD and activate the TLR4/NF-κB/c-myc signaling pathway and also promote epithelialmesenchymal transition(P<0.05).Overexpression of LINC00311 could not promote HIBEpiC cell proliferation(P>0.05).When the NF-κB/c-myc signaling pathway was blocked by NF-κB inhibitor JSH-23 in LINC00311 overexpressing biliary epithelial cells,β-GD was down-regulated(P<0.05).Finally,we treated HiBEpiC with both LPS and curcumin at the same time.We found that curcumin can inhibit the high expression of LINC00311 caused by LPS,thus inhibiting the expression of endogenous β-GD and the corresponding TLR4/NF-κB/ c-myc signaling pathway(P<0.05).Conclusions: These results for the first time revealed a long non-coding RNA-LINC00311 which is closely related to hepatolithiasis could up-regulate the expression of β-GD by activating TLR4/NF-κB/c-myc signaling pathway,thus plays an important role in promoting the formation of intrahepatic bile duct stones.This provides a novel biomarker that can be used as a potential therapeutic target for hepatolithiasis.The traditional Chinese medicine curcumin can inhibit the activation of TLR4/NF-κB/c-myc signaling pathway by down-regulating LINC00311,resulting in decreased expression of β-glucuronidase and could inhibit the formation of intrahepatic bile duct stones.