Exploring The Meehanism Of Lycorine Hydrochloride In Surpressing ICCA

ObjectiveCholangiocarcinoma is a malignant tumor originated from the bile duct epithelial cells.According to the location of the pathological structure,it is divided into two types clinically:extrahepatic cholangiocarcinoma(ECCA)and intrahepatic cholangiocarcinoma(ICCA).In recent years,the ICCA has achieved growing attention due to its increasing incidence.Generally,it is difficult to diagnose ICCA at its early stage and lack of efficient drug therapy also constrains its prognosis,causing high mortality rate,which hinders its therapy development of tumor.Therefore,it is urgent to find new therapeutic targets and effective drugs for the treatment of ICCA.In our study,we used high-throughput screening of natural compounds to identify candidate drugs for ICCA.And we selected lycorine hydrochloride as the candidate.RNA-Seq bioinformatics analysis and in vitro experiments were proposed to investigate the potential mechanisms of the candidate of lycorine hydrochloride on intrahepatic cholangiocarcinoma cells.The purpose of this study is to decipher the mechanisms of actions of lycorine hydrochloride on ICCA and provide theoretical practice for further clinic treatment of lycorine hydrochloride on ICCA.Methods:1.Natural compounds screening for targeting RBE cellsThe inhibitory effect of 640 natural compounds on intrahepatie cholangiocarcinoma cell line RBE was screened by CCK-8 kits.First,the RBE cells were treated with natural compounds at a concentration of 30 ? M,screening the candidate natural compounds with inhibition rate greater than 50%.Subsequently,the RBE cells were treated with the candidate natural compounds at a concentration of 10 ?M,and obtained the candidate natural compounds with inhibition rate greater than 50%.Furthemore,the RBE cells were treated with the candidate natural compounds at a concentration of 5? M,and the candidate compounds were reserved,which satisfied the condition of inhibition rate greater than 5006.Finally,we selected Lycorine hydrochloride for further research on the treatment of ICCA by combining literature evidence.2.The effect of Lycorine hydrochloride on RBE cellsTo explore the potential mechanism of Lycorine hydrochloride against ICCA,we design experiments such as cell proliferation assay by colony formation and CCK-8 kits,cell cycle and cell apoptosis by flow cytometry and immunofluorescence assay,cell migration and cell invasion by transwell assay.3.RNA-Seq revealed the potential molecular targets and pathways of Lycorine hydrochloride on RBE cellsThe RBE cells were treated with lycorine hydrochloride at a concentration·of 5 uM for 12hours.Total RNA was extracted by TRIzol for RNA-Seq.RNA-Seq revealed that the anti-tumor effects of lycorine hydrochloride on RBE cells were associated with cholesterol biosynthesis.The expression level of related targets such as SQLE,FDFT1,HMGCS1,DHCR24,DHCR7,MVK,IDI2,and IDI1 in the cholesterol biosynthesis were validated by Western Blot.The results showed that SQLE and FDFT1 were potential targets of lycorine hydrochloride which suppressed the growth of RBE cells.4.The role of SQLE and FDFT1 in the treatment of intrahepatie cholangiocarcinomaThe lentivirus shRNA vector of SQLE and FDFT1 were constructed and transfected into 293T cells.Then,the obtained virus solution were transfected into RBE cells and the transfected RBE cells were screened by antibiotics.The cells were collected to ensure silencing SQLE and FDFT1 by Western Blot.The effects of SQLE and FDFT1 knockdown on RBE cells were investigated by CCK-8 and clony formation assay.Our results indicated that lycorine hydrochloride can inhibit the growth of RBE cells by decreasing the expression of SQLE and FDFTI.5.Combined effect of Lycorine hydrochloride and gemcitabine on RBE cellsThe effect of lycorine hydrochloride combined with gemcitabine on RBE cells was detected by CCK-8 assay.Then,the expression of SQLE,FDFTI,HMGCSI,DHCR24,DHCR7,MVK,IDI2 and IDI1were detected with Western Blot,which revealed the effect of lycorine hydrochloride combined with gemcitabine on intrahepatic cholangiocarcinoma cell line RBE.Results:1.With natural compounds screening,we found the growth of RBE cells was inhibited by lycorine hydrochloride effectively.Lycorine hydrochloride was selected as a potential candidate for further mechanism analysis on RBE cells.We expected to identifying new targets for the treatment of ICCA.2.According to cell proliferation assay and clony formation assay,we found that the growth of RBE cells was inhibited by lycorine hydrochloride.The cell cycle and apoptosis were tested by flow cytometry.Lycorine hydrochloride induced G2/M phase arrested and apoptosis in RBE cells.Immunofluorescence assay which stained RBE cells nuclear with DAPI proved that lycorine hydrochloride promoted cell apoptosis in RBE cells.Lycorine hydrochloride which inhibited cell migration and invasiveness of RBE cells were verified by cell migration assay and invasion assay.Overall,we concluded that lycorine hydrochloride surpressed the growth of intrahepatic cholangiocarcinoma cells based on the results of in vitro experiments.3.The total RNA was extracted from the RBE cells which were treated by lycorine hydrochloride for 12hours for RNA-seq.The RNA-seq data suggested that SQLE and FDFT1 were new potential targets of lycorine hydrochloride for treatment on intrahepatic cholangiocarcinoma cell line RBE.We conducted experiments to investigate the role of SQLE and FDFT1 as new targets in inhibiting the growth of RBE cells.Western Blot was utilized to confirm that lycorine hydrochloride inhibited the expression of SQLE and FDFT1 in RBE cells.4.Our results showed that lycorine hydrochloride could suppress the growth of RBE cells.The growth of RBE cells was inhibited when the expression of SQLE and FDFT1 were decreased.In addition,lycorine hydrochloride suppressed the growth of RBE cells by decreasing the expression level of SQLE and FDFT1.We concluded that knockdown of SQLE and FDFT1 can inhibit the growth of RBE,mimicking the effect of Lycorine hydrochloride.On the basis of the results of CCK-8 assay,we found that lycorine hydrochloride enhanced the sensitivity of gemcitabine to intrahepatic cholangiocarcinoma cells.Moreover,we confirmed that lycorine hydrochloride promoted the effects of gemcitabine on RBE cells by redueing the expression of SQLE and FDFT1 according to the outcome of Western Blot.Conclusion:1.The growth of intrahepatic cholangiocarcinoma cell line RBE was surpressed by lycorine hydrochloride.2.Lycorine hydrochloride can downregulate the expression of SQLE and FDFT1 in RBE cells.3.The growth of RBE cells was inhibited by lycorine hydrochloride via decreasing the expression of SQLE and FDFT1.