| Objectives: Primary hepatocellular carcinoma(HCC)is one of the malignant tumors threatening human health.The incidence and mortality of PHC accounted for the sixth and fourth place of all malignant tumors,respectively.China is a big country of liver cancer,and its new cases and deaths account for more than a half.However,HCC has poor sensitivity to chemotherapeutic drugs and is prone to drug resistance,which greatly limits the application of chemotherapy.The mechanisms of drug resistance in HCC are sophisticated.A series of signal transduction pathways occur from receiving stimulating signals of chemotherapeutic drugs to tumour cells,up-regulating the expression of some genes that promote survival and promote drug pumping,resulting in tolerance to chemotherapeutic drugs.In this process,the anti-apoptotic mechanism play an important role in tumor cells stimulated by chemotherapeutic drugs.SND1(Staphylococcal Nuclease and Tudor Domain Containing 1)protein is an evolutionarily conserved multifunctional protein,which can be used as transcriptional co-activator and RISC(RNA-induced Silencing Complex)complex member to promote the occurrence and development of HCC.In recent years,several studies have shown that SND1 protein is involved in the anti-apoptotic mechanism of tumors,and plays an important role in chemotherapy resistance of non-small cell lung cancer.However,the role of SND1 protein in chemotherapeutic resistance of HCC has not been reported.Therefore,we aimed to investigate the correlation between SND1 protein and HCC,and the anti-apoptotic mechanism of HCC cells stimulated by chemotherapeutic drugs.Methods: This study were divided into three parts.Part 1:(1)The HCC histochemical samples were retrievd from the Human Protein Atlas(HPA)network database and analyzed the difference of SND1 protein levels between hepatocellular carcinoma(HCC)and adjacent tissues.(2)The clinical information and expression profile information datasets of HCC patients were download from the Cancer Genome Atlas(TCGA)database and the differences of SND1 gene expression in HCC and adjacent cancers were analyzed.(3)The downloaded HCC datasets were subgrouped according to general clinical data(gender,age,race,race)and TNM classification of malignant tumors,histopathological grading and Barcelona staging of HCC,and then the correlations between SND1 expression level and overall survival rate were analyzed.Part 2:(1)The flow cytometry and Western Blot(WB)were used to study the apoptotic status of HCC cell lines interfering with SND1 expression in normal condition(normal growth of tumors)and under the stimulation of 5-Fluorouracil(5-Fu)or cisplatin(CPT)(Chemotherapy against tumors)to clarify the role of SND1 in HCC apoptosis.(2)The downstream target genes of SND1 protein in HCC treated with chemotherapeutic drugs were identified by RNA microarray and fluorescence quantitative polymerase chain reaction(qPCR).(3)The apoptotic mechanisms of SND1-LncRNA UCA1(Urothelial Cancer Associated 1)axis in HCC cells stimulated by 5-Fu was investigated by flow cytometry.(4)The anti-apoptotic function of SND1-UCA1 axis at the animal level were explored by xenografts in nude mice combined with intraperitoneal administration.Part 3:(1)The specific regions of SND1 effected on LncRNA UCA1 promoter were determined by Secreted and Robust Gaussia Luciferase Gene Reporter System(GLuc).(2)The transcription factors of LncRNA UCA1 promoter were predicted by JASPAR online prediction website,and then analyzed by STRING protein network database and immunoprecipitation(Co-immunoprecipitation,Co-IP)to identify transcription factors that activate UCA1 in combination with SND1 protein.(3)The specific regions of MYB in LncRNA UCA1 promoter were analyzed by GLuc and Electrophoretic Mobility Shift Assay(EMSA)experiments.(4)Chromatin Immunoprecipitation Assay(ChIP)assay was used to determine the binding of SND1 and MYB in UCA1 promoter regions.Results: Part 1:(1)Immunohistochemical results of HPA database showed that SND1 protein was scarcely stained in normal liver tissues,but deeply stained in HCC.(2)Analysis of HCC datas collected in TCGA database showed that the SND1 gene was in high-expression region,and there were significant differences compared the expression of SND1 gene in tumor data with that in tumor-adjacent data.(3)There was no significant correlation between SND1 and overal survival rate of HCC patients in general clinical data(gender,age,race,ethnicity),TNM stage,histopathological grade and Barcelona stage.Part 2:(1)Simply interfering with the expression of SND1 did not affect the apoptotic level of HCC cells.But compared with the normal expression of SND1,the apoptotic level of HCC cells interfering with the expression of SND1 increased significantly after being treated with 5-Fu or CPT,and the proportion of BAX/Bcl-2 increased significantly.(2)Treating cells with 6 μg/mL 5-Fu,it was found that compared with the normal expression of SND1 cells,a series of genes were up-regulated or down-regulated in HCC cells intefering with SND1 and LncRNA UCA1 was selected as the downstream of SND1.(3)When treated with 6 μg/mL 5-Fu,compared with normal cells,the apoptotic rate of HCC cells increased after interfering with UCA1,but the apoptotic level of HCC cells was decreased after restoring LncRNA UCA1 expression on the basis of interfering with SND1(compared with cells interfering with SND1).(4)Compared with the tumors of HepG2-shNC control cells,the tumors of HepG2-shSND1-#1 and HepG2-shUCA1-#1 were significantly reduced,while the tumors of HepG2-shSND1-#1+UCA1 could recover to tumors similar to those of control cells.Part 3:(1)SND1 could activate the UCA1 promoter region of-2000 bp to transcription initiation site(TSS)and the main activation sites were found in the core promoter region of-500 bp~TSS.(2)JASPAR predicted nearly 200 transcription factors,but the results of STRING database showed that only the transcription factor MYB has a potential binding relationship with SND1.SND1 protein enriched by anti-SND1 antibody could catch MYB.(3)Similarly,MYB could activate each part of UCA1 promoter region of-2000 bp to TSS and mainly focused on the core promoter region of-500 bp~TSS.EMSA experiments confirmed that MYB could bind to the UCA1 promoter region of-231~-222 bp.(4)ChIP results showed that SND1 could bind to the UCA1 promoter region of-231~-222 bp,and SND1 knockdown could affect the binding of MYB at the UCA1 promoter region of-231~-222 bp.Conclusions:(1)SND1 was highly expressed in HCC,but there is no significant difference between the expression level of SND1 and the survival rate of patients.(2)Interference with the expression of SND1 or LncRNA UCA1 could enhance the sensitivity of HCC to 5-Fu,and increase the apoptotic level of HCC cells.Interference with SND1 while restoring the expression of LncRNA UCA1 could counteract the increase of apoptotic level of HCC interfering with SND1 induced by 5-Fu.(3)SND1 activated the expression of LncRNA UCA1 by binding to transcription factor MYB,and plays a role in resistance to apoptosis induced by chemotherapeutic drugs in HCC. |
