| Hepatocellular carcinoma(HCC)is one of the most common cancers: the fifth most common cancer and the third leading cause of cancer related deaths worldwide.HCC is also a common digestive system malignant tumor in China.Newly found cases and death cases of liver cancer in China is account for almost 50% of the world.Due to the characteristics of insidious onset,rapid progress,easy metastasis at early stage,high case fatality rate and poor overall survival prognosis,the 5-year survival rate of HCC is still low and it causes serious damage to human health.The diagnosis and treatment principles of liver cancer are early imaging diagnosis and surgical R0 resection of carcinoma tissues.However,lack of effective early diagnostic indexes,high post-operation relapse and metastasis rate are still the main reasons of poor prognosis of HCC,and the key molecular mechanisms which influence the process are still not entirely clear.Therefore,searching for tumor molecular targets and mechanisms are critical for improving early diagnosis and increasing long-term survival rate of HCC patients.In recent years,with the rapid development of high throughput sequencing technology and declining costs,researchers both in China and abroad have made a lot of achievements in regulation of tumor transcription and post-transcription level,using high throughput technologies,including microarray,whole genome sequencing and tumor metabonomics.It was shown that microRNAs(miRNAs)and long non-coding RNAs(lncRNAs),which transcribed in non-coding protein area of more than 95% of cancer genome,played an critical role in tumorigenesis and progression.There exist a complex functional relation between non-coding RNAs,and coding protein RNA,which was of great research significance in occurrence and development of HCC.Studies related to HCC have shown that lncRNAs play an important role in proliferation,apoptosis and invasion,and are closely related to the occurrence,evolution and prognosis of HCC;some lncRNAs,specifically expressed in HCC cells and tissues influence cell proliferation,apoptosis,cell cycle,invasion and metastasis,tumor cell stemness and transdifferentiation,through various mechanisms in transcription and post-transcription level.Although sequencing technology and microarray technology are improving and are popular in liver cancer research,nc RNA(miRNAs and lncRNAs)related to liver,each research group selected and focused on,are quite different.There are not many long non-coding RNA,which are found by similar researches and spread out among researchers,and they are not completely same.There are still a lot of functional regulation molecular mechanisms,which differentially expressed lncRNA in HCC,remain unclear.By integrating analysis of Gene Expression Omnibus(GEO)datasets of HCC in this study,screening the function and molecular mechanisms of relevant lncRNA in occurrence and evolution of HCC will enrich our understanding about functions of lncRNA in occurrence and progress of HCC.Meanwhile,it will provide new theoretical evidence and experimental basis for the diagnosis,treatment and prognosis evaluation of HCC.Methods:Part ?: Screening of differentially expressed mRNAs,miRNAs and lncRNAs related to liver cancer progression based on GEO database.1.Define the GEO datasets search strategy to search and download datasets related to HCC,build an in and out criterion to filtrate GEO datasets,and conduct a variance analysis using R language.The criterion of different mRNAs,mi RNAs and lncRNAs is: P value<0.05 with |Fold change| >1.5.2.By integration analysis of differencially expressed genes,the miRNAs-mRNAs regulation network,lncRNAs-mRNAs co-expression network and lncRNAs-miRNAs relationship network were builded using cytoscape software.And executed functional annotation for differentially expressed miRNAs and lncRNAs through target mRNAs or co-expression mRNAs.3.The qRT-PCR detection was used to verify the regulation effect of miRNAs on lncRNAs in HepG2 cells after mi RNA mimics transfection,and the expression level of differencially expressed lncRNA were validated in 10 HCC tumor tissues and adjacent non-tumor tissues.The TCGA RNA sequencing data of HCC was downloaded to verify the expression level and correlation between miRNAs and lncRNAs.Part ?: Cell biology function and molecular biology mechanism of lncRNA LOC90784 in the process of HCC occurrence and evolution.1.Detect expression and distribution of long non-coding RNA LOC90784 in HegG2 and SMMC7721 cells and HCC tissues using fluorescence in situ hybridization(FISH).2.Detect expression level of lncRNA LOC90784 in primary liver cancer tissues and adjacent cancer tissues of 64 cases using qRT-PCR and compare the expression difference of lncRNA LOC90784 between HCC tumor tissues and adjacent non-tumor tissues.Analyze relationships between lncRNA LOC90784 expression and clinical characteristics,including clinical pathologic staging classification,pathological differentiation and tumor micrometastasis in HCC.3.In order to further identify the influence of lncRNA LOC90784 on liver cancer cell phenotype,three lncRNA LOC90784 siRNAs were designed and synthesed.After transfecting of lncRNA LOC90784 siRNA negative control(NC)and lncRNA LOC90784 siRNAs in HegG2 and SMMC7721 cells respectively,the cell biology function of lncRNA LOC90784 in liver cancer occurrence and development were proved respectively through CCK-8 proliferation,clone forming arrsay,Transwell,cell apoptosis and cell-cycle progression with flow cytometry.4.According to the predicted results in Part I,validate regulation function of miR-145 and miR-195 on lncRNA LOC90784 expression in histology and cytology level;further investigate the mechanisms of lncRNA LOC90784 in the aspects of invasion and migration;meanwhile,verify the prognosis assessment value of lncRNA LOC90784 in R0 postoperative patients with TCGA HCC data.Part ?: Regulatory mechanism of lncRNA OTUD7AP1 in the process of liver cancer evolution and relationship between lncRNA OTUD7AP1 expression and clinical characteristics in HCC patients.1.Detect expression and distribution of lncRNA OTUD7AP1 in HepG2 and SMMC7721 cells and HCC tissues using fluorescence in situ hybridization(FISH).2.Detect expression level of lncRNA-OTUD7AP1 in HCC tumor tissues and adjacent non-tumor tissues of 52 patients using qRT-PCR and compare the expression difference of lncRNA OTUD7AP1 between HCC tumor tissues and adjacent non-tumor tissues.Analyze relationship between lncRNA OTUD7AP1 expression and clinical characteristics,including clinical pathologic staging classification,pathological differentiation and tumor micrometastasis in HCC.3.In order to identify the influence of lncRNA OTUD7AP1 on liver cancer cell phenotype,three lncRNA OTUD7AP1 siRNAs were also designed and synthesed.After transfecting of lncRNA OTUD7AP1 siRNA negative control(NC)and lncRNA-OTUD7AP1 siRNAs in SMMC7721 cells respectively,the cell biology function of lncRNA-OTUD7AP1 in liver cancer occurrence and development were proved respectively through CCK-8 proliferation,clone forming arrsay,and analysis of cell apoptosis and cell-cycle progression with flow cytometry.4.According to the predicted results in Part I,validate regulation function of miRNAs on lncRNA OTUD7AP1 in histology and cytology level;further investigate the regulation between lncRNA OTUD7AP1 and OTUD7 A through sequence alignment analysis,and validate it on cytological and histological expression level.Results:Part ?: Screening of differentially expressed mRNAs,miRNAs and lncRNAs related to liver cancer progression based on GEO database.1.Obtained 9 GEO datasets through retrieving and further screening,and selected 251 upregulated mRNAs and 377 downregulated mRNAs within mRNA microarray after integration;selected 2 upregulated miRNAs and 13 downregulated miRNAs within miRNA microarray after integration;selected 174 upregulated mRNAs,78 downregulated mRNAs,42 upregulated lncRNAs and 7 downregulated lncRNAs within lncRNA microarray after integration(selection criteria: P<0.05 and |Fold change|>1.5).2.Expression network construction and function annotation: successfully constructed lncRNAs-miRNAs-mRNAs regulation or co-expression network by differentially expressed mRNAs,miRNAs and lncRNAs.Function and KEGG pathway analysis of differentially expressed miRNAs and lncRNAs were annotated by miRNA target mRNAs and lncRNA co-expression mRNAs.3.After screening,10 high expressed lncRNAs were selected and validate their expression level using qRT-PCR detection,and most lncRNA were highly expressed in tumor cell lines and tumor tissues(P<0.05).It was found that some lncRNAs were targeted-regulated by miRNAs after transfection of mi RNA mimics(P<0.05).Expression and relevance of partial differentially expressed miRNAs and lncRNAs were validated by TCGA HCC sequencing data(P<0.05).Part ?: The lncRNA LOC90784 expression level in HCC tissues was associated with clinical poor prognosis characteristics of HCC,knockdown expression of lncRNA LOC90784 could significantly inhibit proliferation,invasion and migration and survival of HCC cells.1.Compared with normal tissue adjacent liver cancer tissue or normal liver cell line,lncRNA LOC90784 was significantly highly expressed in HCC tumor tissues and cell lines,and mainly located in cytoplasm by FISH.Expression in HCC tissue was significantly associated with liver cancer tissue differentiation(P < 0.001),T stage of TNM stage system(P < 0.001),vascular invasion(P = 0.0237),serum AFP level(P = 0.0275),and HBV infection status(P = 0.0289).2.Result of CCK-8 proliferation detection,clone formation assay and Transwell experiment showed that,compared with siRNA NC group,lncRNA LOC90784 siRNAs significantly downregulated the expression of lncRNA LOC90784,and significantly inhibited the proliferation,invasion and metastasis,induced cell apoptosis and cell-cycle arrest in HepG2 and SMMC7721 cells(P<0.05).3.Consistent with the predicted results in the Part I,lncRNA LOC90784 expression was targeted-regulated by miR-145 and miR-195 through cytological and histological level detection.Knockdown lnc RNA LOC90784 expression could inhibit HCC cells invasion and migration by depressing mRNA and protein expression of MMP2 and MMP9.4.Expression level of mi R-145 was negatively correlated with lncRNA LOC90784 in HCC matched tissues(r=-0.6612,P=0.0015),and it was similar in 49 TCGA matched samples(r=-0.4549,P< 0.001).After survival analysis of 261 TCGA HCC patients(R0),it was found that high expression level of lncRNA LOC90784 was significantly correlated with poorer overall survival(P<0.05).Part ?: The lncRNA OTUD7AP1 level in liver cancer tissues was associated with clinical poor prognosis characteristics of HCC,knockdown expression of lncRNA-OTUD7AP1 could significantly inhibited the proliferation,and induced cell apoptosis and cell-cycle arrest in HCC cells.1.Compared with normal tissue adjacent HCC non-tumor tissues or normal liver cell line,lncRNA-OTUD7AP1 was significantly highly expressed in HCC tumor tissues and cell lines,and mainly located in cytoplasm by FISH.Eexpression of lncRNA OTUD7AP1 was significantly associated with clinical features,including liver cancer tissue differentiation(P < 0.001),T stage of TNM stage system(P < 0.001),serum AFP level(P = 0.0124)and number of tumor(P< 0.001)in HCC.2.Result of CCK-8 proliferation detection,clone formation assay and flow cytometry cycle analysis showed that,compared with siRNA NC group,lncRNA OTUD7AP1 siRNA significantly downregulated the expression of lncRNA OTUD7AP1,and significantly inhibited the proliferation and induced cell apoptosis and cell-cycle arrest in SMMC7721 cells(P<0.05).3.Consistent with the predicted results in the Part I,lncRNA OTUD7AP1 was targeted-regulated by miR-125 b,miR-101 and miR-130 a through cytological level detection.Tissue expression level correlation analysis showed expression level of miR-125 b was negatively correlated with lncRNA OTUD7AP1(n=20,r=-0.4645,P=0.0168).The OTUD7 A mRNA expression was lowly expressed in liver cancer tissues compared to normal tissues,and knockdown the lncRNA OTUD7AP1 expression with siRNAs could upregulated the expression of OTUD7 A mRNA,and there existed a negative correlation between tissue expression levels(n=17,r=-0.4005,P=0.0283).Conclusion:Part ?: Identification of HCC occurrence and evolution related mRNAs,miRNAs and lncRNAs based on GEO database.This part effectively improved the utilization rate of GEO data through integrated analysis,and systematically selected and identified HCC occurrence and evolution related mRNAs,mi RNAs and lncRNAs;selected potential regulating ncRNAs by building regulation and co-expression network;this finding effectively enriched our understanding of liver cancer development.It provided a new entry point of further investigation on occurrence and development of HCC.Meanwhile,it also provided a new thinking and new targets for early diagnosis and prognosis evaluation of HCC.Part ?: Cell biology function and mechanism of lncRNA LOC90784 in the process of liver cancer progression.This part systematically identified the cytological functional mechanism in promoting tumor of lncRNA LOC90784 in liver cancer and the molecular marking effect closely related to liver cancer prognosis.High expression of lncRNA LOC90784 was negatively regulated by mi R-145 and miR-195 in HCC.LncRNA LOC90784 played a critical role in occurrence and evolution of tumors by promoting cytological behaviors,including proliferation,invasion and migration,cell survival and cell-cycle progression,and acted as molecular markers in HCC prognosis.The study explained functions of lncRNA LOC90784 in proliferation,invasion and prognosis of liver cancer for the first time and would provide new thinking and theoretical basis for the diagnosis,treatment and prognosis assessment of liver cancer.Part ?: Cytological regulatory mechanism in promoting liver cancer proliferation of lncRNA OTUD7AP1 in the process of liver cancer evolution,and analysis of clinical characteristic.This part systematically identified the mechanism in promoting HCC of pseudogene transcript-lncRNA OTUD7AP1 and analyzed the relevance between lncRNA OTUD7AP1 expression and clinical features in HCC.Expression of lncRNA OTUD7AP1 was inhibited by mi R-125 b,mi R-101 and miR-130 a,and there is a negatively correlation between lncRNA OTUD7AP1 and miR-125 b in tissues;lncRNA OTUD7AP1 and parental gene OTUD7 A mRNA were negatively regulated.Knockdown expression of lncRNA OTUD7AP1 inhibited proliferation,induced apoptosis,and regulated cell-cycle in HCC,which played an important role in “tumor promoter”.This part would provide new thinking and mode for study of pseudogene in cancer research. |
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