Linc02154 Promotes The Proliferation And Metastasis Of The Hepatocellular Carcinoma By Enhancing The Activity Of The SPC24 Promoter And Activating The PI3K-AKT Signaling Pathway

According to the latest distribution of the morbidity and mortality of the 10 most common cancers in the world,primary liver cancer is the sixth most common cancer in the world and the fourth leading cause of cancer death.There are approximately 8.410 million new cases and 782,000 people each year.The incidence of death in China is also increasing year by year.The main type of primary liver cancer is Hepatocellular Carcinoma(HCC).HCC has an insidious onset,rapid progress,prone to metastasis,and extremely high mortality and malignancy.The pathogenesis of HCC is still unclear.The non-coding RNA in the human genome sequence is related to the occurrence and metastasis of many tumors.In-depth exploration of the pathogenesis of HCC in order to find early diagnosis methods and effective treatment measures,and to find new targets for early liver cancer molecular therapy intervention is of great significance for improving the overall survival rate of early liver cancer patients.Long non-coding RNAs(Lnc RNAs)are widely involved in the occurrence and progression of tumors in tumor diseases.The mechanism of action of most Lnc RNAs in HCC is still unclear.Lnc RNA has the characteristics of multiple types,numbers,and modes of action.It can participate in DNA methylation,histone modification,or chromatin remodeling through mechanisms such as transcription,post-transcription,and epigenetic regulation,and mediate gene activation or silencing.Lnc RNA is related to the metastasis and invasion of hepatocellular carcinoma,and is of great significance to the diagnosis,treatment and prognosis of hepatocellular carcinoma.At present,high-throughput sequencing has found that Linc02154 is highly expressed in HCC tissues,but whether it has biological functions in HCC cells remains to be studied.Therefore,this study will explore the role and mechanism of Linc02154 in the development,metastasis,invasion and other biological behaviors of hepatocellular carcinoma from in vivo and in vitro functions and effects,and try to find effective hepatocellular carcinoma gene therapy targets and enrich the liver.The mechanism of the occurrence and development of cell carcinoma provides a strong theoretical and experimental basis for clinical diagnosis,treatment and prognosis.Objective:1.Explore the expression of Linc02154 in HCC tissues and itsrelationship with survival prognosis,and study the expression ofLinc02154 in HCC cell lines and its distribution in cells.2.Using function gain and loss experiments,determine the biologicalfunction of Linc02154 in HCC in vivo and in vitro.3.Explain the effect of Linc02154 on the biological functions of HCCcells and the molecular mechanism of the effect.Method:1.Download the high-throughput sequencing results of liver cancer inthe TCGA database for bioinformatics analysis to obtain differentiallyexpressed candidate Lnc RNAs related to survival and prognosis.Real-time fluorescent quantitative PCR was used to detect the expressionof candidate Lnc RNA in HCC cell lines MHCC-97 H,Hep G2,SMMC7721,Hep3 B,Huh7.The tissues of 20 clinical patients with HCCwere collected to detect the expression of Linc02154.Two kinds ofcoding ability prediction software were used to analyze the codingpotential of Linc02154 to confirm the non-coding ability.Thelocalization of Linc02154 in HCC cells was detected by fluorescence insitu hybridization experiment and nuclear-plasma separation experiment.2.Construct a plasmid that interferes with and overexpress Linc02154through molecular cloning,and package the lentivirus to infect HCCcells to form an HCC cell line with stable interference and overexpressLinc02154.The q RT-PCR experiment verifies the efficiency ofinterference and overexpression.Cell proliferation and toxicity detection MTS test,Ed U test,and clone formation test were used to detect the effect of Linc02154 on the proliferation ability of HCC cells.Flow cytometry was used to detect the effect of Linc02154 on HCC cell cycle and apoptosis.Wound healing experiment,Transwell cell migration test without glue coating,and Transwell cell coating invasion test was used to detect the effect of Linc02154 on the migration and invasion of HCC cells.Construct an in vivo nude mouse subcutaneous tumor model,observe the effect of Linc02154 on the growth and proliferation of HCC cells in vivo,and detect the expression of Ki67 protein by immunohistochemistry to detect the effect of Linc02154 on the proliferation of cells.3.After the interference of Linc02154 highly expressed SK-Hep1 cells,the stable cell line was subjected to RNA-seq transcriptome sequencing to screen for differentially expressed genes,and the expression level of candidate target genes was confirmed in HCC cells by q RT-PCR,and the screening expression was significant The differential target gene SPC24,q RT-PCR and Western blot were used to detect the changes in m RNA and protein levels of SPC24 in HCC cells that interfered with and overexpressed Linc02154.The TCGA database was used to analyze the correlation between the co-expression of SPC24 and Linc02154.A dual luciferase reporter gene plasmid with the full length of the SPC24 promoter and a truncated fragment(with 500 bp as the truncation unit)was constructed,and the effect of Linc02154 on the activity of theSPC24 promoter was detected by the dual luciferase reporter geneexperiment.Using KOBAS and KEGG to enrich the signal pathwayrelated to the expression of SPC24 by Linc02154,and detect theinfluence of Linc02154 on the regulation of the signal pathway.According to the biological function of Linc02154 to promote migrationand invasion,the influence of Linc02154 on the process ofepithelial-mesenchymal transition was detected.Result:1.Linc02154 highly expressed log FC=4.033143521 in the TCGAdatabase and was related to survival prognosis P=0.034747372(P<0.05),which was significantly higher in different HCC cell lines than in thecontrol group.Linc02154 does not have the ability to encode proteinsand is less conservative.q RT-PCR detected that Linc02154 was highlyexpressed in 20 pairs of HCC tissues.Fluorescence in situ hybridizationFISH experiment and nucleoplasm separation experiment showed thatLinc02154 was localized in the nucleus and cytoplasm of HCC cells.2.Overexpression of Linc02154 promotes the proliferation,migrationand invasion of HCC cells,while interference with Linc02154 has aninhibitory effect on the proliferation,migration and invasion of HCCcells.Flow cytometry detection of cell cycle found that interference withLinc02154 makes HCC cells G1/S Arrest,overexpression of Linc02154 promotes G1/S transformation of HCC cells.Flow cytometry detected cell apoptosis and found that interference and overexpression of Linc02154 had no significant effect on HCC cell apoptosis.In the nude mouse subcutaneous tumor model,it was found that the overexpression Linc02154 group increased the volume and weight of subcutaneous tumors compared with the control group.The immunohistochemical detection of overexpression Linc02154 group Ki67 protein expression level was significantly up-regulated,while the interference Linc02154 group obtained the opposite experimental results.3.After the interference of Linc02154 highly expressed SK-Hep1 cells,RNA-seq sequencing analysis was performed to review the literature and select the first 11 low-expressed proteins after interference.Validation by q RT-PCR found that SPC24 was the most significant gene down-regulated after interfering with Linc02154 in HCC cells.In SK-Hep1 and MHCC-97 H cells,the m RNA and protein expression levels of SPC24 were significantly reduced after Linc02154 was interfered with.The m RNA and protein expression levels of SPC24 were significantly increased after Linc02154 was overexpressed,and the expression trends of the two were consistent.SPC24 is highly expressed in hepatocellular carcinoma in the TCGA database,and is related to survival p<0.05,and its expression is different in different clinical stages.The double-luciferase experiment co-transfected the plasmid and foundthat Linc02154 can enhance the activity of the promoter in the-500 bp～-1000 region of the SPC24 promoter.The KEGG pathway enriched byLinc02154 and SPC24 is related to the cell cycle,and the PI3K-Aktsignaling pathway is significantly different.Western Blot detectedchanges in the protein levels of PI3 K and Akt in SK-Hep1 andMHCC-97 H cells.The results showed that compared with the controlgroup,the phosphorylation levels of PI3 K and Akt in SK-Hep1 andMHCC-97 H overexpressing Linc02154 increased,while after interferingwith Linc02154,the opposite result was obtained.The study of themechanism of Linc02154 promoting HCC migration and invasion foundthat overexpression of Linc02154 significantly reduced the expression ofepithelial marker E-cadherin,and promoted the expression ofmesenchymal cell markers N-cadherin,Vimentin,Snail and Slug.Interfering with Linc02154 has the opposite result.Linc02154 maypromote the occurrence of EMT in HCC and promote the migration andinvasion of HCC cells.Conclusion:1.Linc02154 is a long non-coding RNA that exists in both the cytoplasmand nucleus of HCC.It is highly expressed in HCC tissues and cells andis related to the survival and prognosis of HCC patients.It promotes theoccurrence and progression of HCC in clinical features.2.In vitro experiments of HCC cells show that Linc02154 promotes cell proliferation,migration and invasion,and promotes the transformation of HCC cells in the G1/S phase.3.Molecular mechanism studies confirmed that Linc02154 can enhance the activity of the SPC24 promoter,up-regulate the expression of SPC24,and activate the activity of the PI3K-Akt signaling pathway to promote the proliferation and cycle changes of HCC.In addition,the activation of PI3K-Akt signaling pathway promotes the expression of mesenchymal cell markers N-cadherin,Vimentin,Snail and Slug,reduces the expression of epithelial cell marker E-cadherin,and induces EMT to promote the migration and invasion of HCC cells.