The Exploration Of Regulatory Mechanisms Of PTTG3P And Associated Genes In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the most frequently occured liver cancer with the largest number of new patients in the world.The treatment strategies for HCC include surgical resection and liver transplantation,transarterial chemotherapy and embolization(TACE),radiotherapy,targeted therapy and immunotherapy.Notwithstanding tremendous advances have been achieved in radiation therapy,surgery and other treatments,the standardized mortality rate of HCC patients is 15.9/100,000,second only to lung cancer.The 5-year survival rate of early stage and end stage HCC patients is more than 50%and less than 10%respectively,most patients have lost the opportunity of surgery when they are diagnosed with HCC.Therefore,more accurate and diversified biomarkers are needed in the clinical diagnosis and treatment of HCC.As a novel oncogene,the long non-coding RNA PTTG3P has shown its potential in becoming a new biomarker and target in many malignant tumors including HCC.Recent studies have proved that PTTG3P can be used as an independent prognostic factor in HCC patients with a limited sample size and survival data,which render it unconvincing.Meanwhile,the mechanism of PTTG3P and its downstream genes has not been clarified.In light of the above reasons,this dissertation first verified the clinical value of PTTG3P via bioinformatic analysis.Secondly,we built the over-expression and knock down HCC cell model to clarify the relationship between PTTG3P and malignant phenotypes.Finally,the functional mechanism of PTTG3P was explored through molecular and animal experiments as well as clinical specimen analysis.1.Bioinformatics analysis of LncRNA PTTG3P in liver cancerIn order to verify the prognostic value and reliability of the previous study,we included clinical samples from more than 300 HCC patients in the TCGA-LIHC database and follow-up data of more than 5 years to comprehensively evaluate the prognostic value and functional mechanism of PTTG3P.First of all,we downloaded the level-3 data of HCC patients in TCGA-LIHC from the UCSC Xena browser After screening out the patients who did not meet the inclusion criteria,353 HCC tissues and 49 matched normal tissues were obtained.According to RNA-seq data,the expression of PTTG3P in HCC samples was enormously higher than adjacent liver samples,and the expression level was proportional to the clinical stage and Grade of the patients.Secondly,Kaplan-Meier survival curve showed that PTTG3P,which is negatively correlated with the OS and RFS of the HCC patients,could be an unfavorable independent biomarker.Finally,we used String 10.5 and KEGG/GO to explore the downstream regulatory network of PTTG3P and found 130 co-expressed genes with PTTG3P.These genes are mainly correlated with cell division,cytokines,cell cycle.2.PTTG3P knockdown and overexpression cell model construction and phenotype explorationBased on the in silico analysis in the first part,we further explored the internal relationship between PTTG3P and HCC phenotypes.First,we selected Hep3B and HepG2 cell lines with the highest and lowest constitutive expression level of PTTG3P respectively to establish the stable PTTG3P over-expressed and knockdown cell models.Secondly,the CCK8 assay showed that the expression of PTTG3P in HCC was directly proportional to cell proliferation.Flow apoptosis assay showed that the expression of PTTG3P was inversely proportional to the apoptosis of HCC cells.Flow cytometry showed that PTTG3P was positively related to S phase ratio.In this part of the dissertation,a stable transformation model of overexpression and knockdown of PTTG3P was established to verify the correlation between PTTG3P and HCC cell proliferation and migration,and to provide a consistent foundation for subsequent mechanism experiments.3.Exploration of the regulatory mechanism of PTTG3P in HCCIn order to investigate its hidden molecular mechanism,fluorescence in situ hybridization(FISH)experiment was first performed to clarify its subcellular localization,and the results showed that LncRNA PTTG3P was mainly localized in the cytoplasm.Then we use RNA pulldown,mass spectrometry and RIP experiment to discover the ILF2/ILF3 complex as the binding protein of PTTG3P.At the same time,we ues the PTTG3P overexpression,knockdown Hep3B cells and their respective control groups for transcriptome sequencing.Then we integratively analyze our data and in silico analysis,together with the previous study to screen the potential downstream genes of PTTG3P and ILF2/ILF3 complex.TOP2A was eventualy confirmed by ChIP.According to IF and WB results,ILF2 has a significant tendency to translocate from cytoplasm to nucleus and bind to the TOP2A promoter region.The dual luciferase reporter gene experiment also confirmed that ILF2 can promote the transcription of TOP2A.According to the previous studies,ILF2/ILF3 usually enhance the stability of mRNA in cytoplasm.We further confirmed PTTG3P and ILF2 could enhance the stability of TOP2A mRNA by the degradation experiment of actinomycin D.Subsequently,we constructed a nude mouse tumor-bearing model with PTTG3P overexpression,PTTG3P knockdown and TOP2A knockdown in animal experiments and obtained tumor tissues for immunohistochemistry.It was found that PTTG3P overexpression significantly promoted the growth of tumor in nude mice,while PTTG3P and TOP2A knockdown had the opposite effect.HE and TUNEL staining results showed that PTTG3P and TOP2A knockdown significantly inhibited tumor necrosis and apoptosis.Ki67 staining showed that PTTG3P knockdown group and TOP2 A knockdown group tremendously reduced the proliferation of HCC cells.Finally,qPCR assay was uesed to examine the level of PTTG3P,TOP2A and ILF2 in liver cancer and adjacent normal tissue samples of 40 HCC patients.Meanwhile,HE staining and TUNEL fluorescence staining were performed on 15 samples of patients,and the basic and clinical information of all patients were collected.The results showed that the expression level of PTTG3P is positively correlated with ILF2 and TOP2A,all of them were highly expressed in liver cancer tissues.The expression level of PTTG3P was positively correlated with AFP and HBsAg levels,the expression level of TOP2A was positively correlated with AFP level,and the expression level of ILF2 was positively correlated with CNCL staging.Finally,HE and TUNEL staining were performed on HCC specimens and matched normal liver tissues of 15 patients.It was found that tumor necrosis and apoptosis were significantly decreased when PTTG3P and TOP2A expression levels were higher in HCC tissues,while the ratio of necrosis and apoptosis was significantly increased when PTTG3P and TOP2A expression levels were low.