The Role And Mechanism Of Small Nuclear Ribonucleoprotein E In Hepatocellular Carcinoma

BACKGROUNDHepatocellular carcinoma(HCC)is a type of primary liver cancer in which the proportion of HCC is almost 90%,higher than all the other types of liver cancer..In2018,the number of deaths due to liver cancer in the world ranked fourth in the total number of cancer-related deaths.The prognosis of liver cancer is not optimistic,the5-year survival rate of liver cancer is less than 18%.This low survival rate is mainly caused by the biological behavior of liver cancer cells.Therefore,it is necessary to find new markers and targets for early diagnosis and therapeutic intervention of liver cancer.Human small nuclear ribonucleoprotein polypeptide E(SNRPE)is a protein that encodes a spliceosome,which mainly acts on the regulation of pre-m RNA.SNRPE is a type of abnormally expressed in a variety of human malignancies.However,the functional role of SNRPE in hepatocellular carcinoma remains unclear.AIMDetect the expression level of SNRPE in HCC tissues and liver cancer cells;analyze general information on clinical samples of SNRPE and surgical resection,such as patient age,gender,tumor size,tumor differentiation,TNM stage,presence or absence of vascular tumor thrombus and other clinicopathological parameters Correlation between;observe the effect of SNRPE on the biological phenotype of HCC cell lines,screen out the more obvious phenotypes;verify the effect of SNRPE on HCC in vivo experiments;and further explore the specific molecular signaling mechanism of SNRPE affecting the biological phenotype of HCC cells.METHODSReal-time fluorescent quantitative PCR(q RT-PCR)is used to detect the expression levels of SNRPE in 80 HCC patient tissues,corresponding normal liver tissues,and human liver cancer cell lines(Hu H7,SK-HEP-1,Hep G2);analyze SNRPE and the age of liver cancer patients,Gender,tumor size,tumor differentiation degree,TNM staging and the correlation with vascular tumor thrombus;Western blot(WB)is used to test the SNRPE content in HCC tissues and liver cancer cell lines,and analyzes the expression level of SNRPE in liver cancer and the impact of SNRPE on the prognosis of patients through TCGA and GEPIA database;short hairpin RNA(Short hairpin RNA,sh RNA)is constructed to knock down SNRPE,and after sh RNA is transfected into liver cancer cell lines,Western blot is used to verify sh RNA knockdown Expression after low SNRPE;CCK-8 test is used to test the changes of Hu H7 and Hep G2 proliferation function;the cell colony formation test is used to test the changes of Hu H7 and Hep G2 clonality;the scratch width healing test is used to test the changes of Hu H7 and Hep G2 Migration ability;flow cytometry is used to test the changes of Hu H7 and Hep G2 cell apoptosis rate;Knockdown SNRPE expression hep3 B cells are inoculated into mice subcutaneously,the tumor formation and growth of mice after knocking down SNRPE are observed;the co-expressed genes are predicted by Oncomine database data which are RAS,RMB34,CCT3,FBL,JTB,NENF,GAPDH,respectively.After knocking down SNRPE,q PCR detects the expression levels of the above genes.Finally,Western blotting was used to determine that SNRPE affects the biological phenotype of liver cancer cells through the RAS/RAF/MEK/ERK signaling pathway.RESULTSThe results of q RT-PCR experiments showed that the expression level of SNRPE in liver cancer tissues was significantly higher than that of the corresponding normal liver tissues(P<0.05).Immunofluorescence showed that SNRPE was mainly expressed in the cytoplasm,and partly in the nucleus.Combined with the analysis of general clinical data of patients,the expression of SNRPE in liver cancer is increased,and the content of SNRPE in liver cancer tissue is significantly correlated with the patient’s tumor size,TNM stage,tumor differentiation and the presence or absence of vascular tumor thrombi(P<0.05).WB results showed that the content of SNRPE in Hu H7,SK-HEP-1 and Hep G2 was higher than that of HL-7702.Among them,the expression level of SNRPE in Hu H7 and Hep G2 cells was higher than that of SK-HEP-1.The short hairpin SNRPE plasmid was introduced into Hu H7 and Hep G2 HCC cells to construct a cell line with targeted knockdown expression of SNRPE.WB was used to test the changes in SNRPE content after transfection.Compared with the control groups,the expression of SNRPE in Hu H7 and Hep-G2 cell lines decreased significantly after transferring the short hairpin SNRPE plasmid(P<0.05).The CCK-8 experiment showed that the absorbance value(ie proliferation ability)of the liver cancer cell lines Hu H7 and Hep G2 after reducing the expression of SNRPE was lower than that of the respective control groups at the same time(P<0.05).In the cell colony formation experiment,knockdown expression of SNRPE,the number of Hu H7 and Hep G2 hepatocarcinoma cell line colonies formed after 2 weeks was reduced compared with the respective control groups(P<0.05).In the scratch experiment,the edge migration distance of Hu H7 and Hep G2 liver cancer cells was shorter than that of the control group at the same time(P<0.001).Flow cytometry showed that after SNRPE silence,the early and late apoptosis rates of Hu H7 and Hep G2 were significantly increased compared with the respective control groups(P<0.001).In vivo tumor formation experiments of mice showed that the weight of tumors in the experimental group was significantly lower than that of the control group after knocking down SNRPE(P<0.001).RAS was knocked down most significantly after reducing the expression of SNRPE.RAS,p-RAF,p-MEK,and p-ERK were all knocked down by detecting the RAS pathway signaling protein.CONCLUSIONS1.The content of SNRPE is elevated in HCC and HCC cells.SNRPE is mostly distributed in the cytoplasm.2.The content of SNRPE is relate to the clinicopathological parameters of HCC patients in tumor diameter,TNM stage,differentiation presence and vascular tumor thrombus.3.If the expression of SNRPE is Knocked down,the proliferation and migration ability of HCC cells will decrease,and meantime the apoptosis of HCC cells rate will increase.4.Mouse tumor-bearing experiments show that reducing the expression of SNRPE can inhibit the growth of HCC.5.SNRPE affects the biological phenotype of liver cancer cells that through phosphorylation pathways related to RAS.Accelerates the pathology of HCC.