Effect Of LncRNA BC040175 On The Proliferation Of Hepatocellular Carcinoma And Its Mechanism

This study is composed of three parts: 1.The differential expression of lnc RNA BC040175 in hepatocellular carcinoma and adjacent tissues and its clinical value in HCC.2.The effect of lnc RNA BC040175 on the biological function of hepatoma cells.3.The molecular mechanism of the effect of lnc RNA BC040175 on the proliferation of hepatoma cells.PART I Differential Expression of Lnc RNA BC040175 in Hepatocellular Carcinoma and Paracancerous Tissues and Its Clinical Value in HCCObjective To study the coding ability of lnc RNA BC040175 and its clinical value in hepatocellular carcinoma.Methods(1)We obtained the complete sequence of lnc RNA BC040175 from the NCBI website.The coding potential of lnc RNA BC040175 was predicted by CPAT,ORFfinder,and Phylo CSF.Conservation of lnc RNA BC040175 was predicted by UCSC.(2)We included patients hospitalized in the First Affiliated Hospital of Guangxi Medical University from January 2020 to October 2021 with a postoperative pathological diagnosis of hepatocellular carcinoma.Hepatocellular carcinoma and paired adjacent tissues,complete medical records were also collected,which included hospitalization number,gender,name,age,viral hepatitis history,liver function grade at admission,AFP,blood routine examination,liver function,tumor postoperative CNLC stage,postoperative pathological grade,postoperative immunohistochemical results and lymph node metastasis.Results(1)The genomic location of lnc RNA BC040175 is(Ch R2: 208104280-208106940).CPAT analysis showed that the coding probability of lnc RNA BC040175 is 0.014792140336423 and the coding label is no.ORFfinder showed that the length of its open reading frame was less than 300 NT with no homologous protein in the short peptide encoded by the sense chain.Phylo CSF output values are all negative,and they have no ability to encode proteins.lnc RNA BC040175 has some homologous regions with dogs but no homologous fragments with mice,zebrafish,chickens,elephants,rhesus monkeys,and other animals.(2)The expression of lnc RNA BC040175 in hepatocellular carcinoma was significantly lower than that in adjacent tissues(P < 0.005).The ROC curve showed that the area under the curve was 0.6593(95% IC 0.58-0.7385).The expression of lnc RNA BC040175 in tumor tissue of patients with CNLC stage I was significantly higher than that of patients with stages II and III(P < 0.05).Patients in the lnc RNA BC040175 high expression group have a significantly better CNLC stage than patients in lnc RNA BC040175 low expression group(P = 0.008).Conclusion lnc RNA BC040175 has no ability to encode protein and is a long noncoding RNA with poor conservation.The expression of lnc RNA BC040175 in hepatocellular carcinoma was lower than in adjacent tissues and was negatively related to the CNLC stage.Lnc RNA BC040175 is a potential diagnostic marker for HCC.PART II Effect of lnc RNA BC040175 on the biological function of hepatoma cellsObjective To investigate the effect of lnc RNA BC040175 on the biological function of hepatoma cells.Methods(1)The subcellular localization of lnc RNA BC040175 was detected by cell nuclear-cytoplasmic separation and fluorescence in situ hybridization.(2)The stable transfection cell line of lnc RNA BC040175 interfering lentivirus was constructed in hepatoma cells.The effect of lnc RNA BC040175 on the proliferation of hepatoma cells was studied by CCK-8 assay,Ed U assay,cell clone formation assay,and tumor formation in nude mice.TUNEL assay was used to detect the effect of lnc RNA BC040175 on the apoptosis of hepatoma cells.The impacts of lnc RNA BC040175 on the migration and invasion of hepatoma cells were explored by scratch assay,migration,and invasion test of Transwell assay.Results(1)Nucleocytoplasmic isolation and FISH experiments indicated that lnc RNA BC040175 was mainly distributed in the nucleus and partly in the cytoplasm in human hepatoma cells.(2)Overexpression of lnc RNA BC040175 inhibited the proliferation,migration,and invasion of hepatoma cells,while silencing lnc RNA BC040175 promoted the proliferation,migration,and invasion of hepatoma cells.Conclusion lnc RNA BC040175 plays an inhibitory role in the proliferation,migration,and invasion of liver cancer cells and promotes the apoptosis of liver cancer cells.Our results suggested that lnc RNA BC040175 may have biological functions in the occurrence and development of liver cancer.PART III Molecular Biological Mechanism of Lncrna BC040175 on the Proliferation of Hepatoma CellsObjective To investigate the molecular biological mechanism of lnc RNA BC040175 on the proliferation of hepatoma cells.Methods(1)We used the RNA pull-down binding mass spectrometry and RIP experiment to study the direct binding proteins of lnc RNA BC040175.The expression of the potential binding protein was detected by Western blot.(2)Hep 3B cells overexpressing lnc RNA BC040175 were sequenced to understand the changes in the genomic transcription level of HCC cells and analyze the changes of signal pathways that may be involved,which were further verified by Western blotting.Results(1)RNA pull-down showed that 365 proteins directly bind to lnc RNA BC040175(-10 lgp >20),of which 134 proteins were significantly associated with HCC(P < 0.05).There was no significant difference in the expression of ZC3H14 between control group and overexpression group in Hep 3B hepatoma cells overexpressing lnc RNA BC040175(P > 0.05).The expression of ZC3H14 in the Huh 7 cells interfering with lnc RNA BC040175 decreased significantly than controls(P < 0.01).(2)RIP assay showed that the expression of ZC3H14 in anti-ZC3H14 group was significantly higher than the Ig G group(P < 0.01).(3)KEGG analysis showed that the pathway changes were mainly related to cell growth and death,cell viability,cell community.MAPK-ERK signal pathway was selected for verification.In Hep 3B cells overexpressing lnc RNA BC040175,phosphorylated c-Raf,MEK and ERK decreased in Hep 3B cells overexpressing lnc RNA BC040175 while increasing significantly in Huh 7 cells interfering with lnc RNA BC040175.Conclusion lnc RNA BC040175 may regulate ZC3H14 through direct combination.lnc RNA BC040175 inhibits HCC by inhibiting the activation of the MAPK / ERK signaling pathway.