Study On The Role And Mechanism Of Long Non-coding RNA LINC00324 In Non-small Cell Lung Cancer

Up to now,the morbidity and mortality of lung cancer are still high worldwide.Non-small cell lung cancer is the main type,accounting for about 85%.Although great progress has been made in the diagnosis and treatment of lung cancer,the 5-year survival rate is still limited,less than 20%^[2].The main reason is that the molecular mechanism of lung cancer is still unclear.In recent years,studies have shown that long non-coding RNA has been found to be closely related to the occurrence and progression of various tumors,including lung cancer.In this study,we focused our research on a new,downregulated long non-coding RNA（LINC00324）in non-small cell lung cancer.To verify its expression and clinical significance in the organizations of non-small cell lung cancer（NSCLC）,and to explore the effects on the biological function of non-small cell lung cancer cells and the possible mechanism.To provides the theory basis for seeking new diagnostic or prognostic molecular markers and potential therapeutic targets.Chapter 2 Expression and significance of lncRNA LINC00324 in non-small cell lung cancer and paracancer tissuesObjective:The biomolecular informatics characteristics of LINC00324 were analyzed.We then detected the expression of LINC00324 in lung cancer organizations and adjacent normal tissues of patients with non-small cell lung cancer,and analyzed its association with prognosis,so as to provide clues for further cell biology experiments.Methods:NCBI and UCSC gene browsers were used to analyze the features and coding capabilities of LINC00324.The expression level of long non-coding RNA LINC00324 in non-small cell lung cancer organizations and adjacent normal tissues was detected by qRT-PCR.Kaplan-meier was used to analyze the lung cancer information in the TCGA database to verify the expression of LINC00324 in non-small cell lung cancer and its relationship with the prognosis of patients.Results:1.LINC00324 belongs to intergenic long non-coding RNA,which is located on chromosome 17p13.1 and contains three exons.The total length of LINC00324 is 2115nt.Moreover,LINC00324 has a low coding potential,which conforms to the characteristics of long non-coding RNA.2.The qRT-PCR results of 65 non-small cell lung cancer and adjacent normal tissues showed that LINC00324 was significantly down-regulated in lung cancer tissues（P<0.05）.3.Kaplan-meier survival analysis was used to analyze the database information and showed that the low expression of LINC00324 in NSCLC was significantly correlated with poor overall survival（HR=0.44,CI=0.37-0.52,P<0.01）.Multivariate COX regression analysis also showed that low expression of LINC00324 was significantly associated with poor overall survival of NSCLC（HR=0.742,CI=0.577-0.954,P=0.020）.Conclusion:LINC00324,as an intergenic long non-coding RNA,is located on chromosome 17p13.1.The expression of LINC00324 was significantly decreased in NSCLC tissues,and the poor prognosis was associated with its low expression.Chapter 3 The role of lncRNA LINC00324 in the cellular biological behavior of non-small cell lung cancerObjective:To observe the effect of LINC00324 on the biological behavior of non-small cell lung cancer cell.Methods:The siRNA and overexpression plasmid of LINC00324 were designed and constructed,and the interference and overexpression model of LINC00324 were established by transfecting the non-small cell lung cancer cells.Cell counting kit-8（CCK-8）assay was used to observe the effect of LINC00324 on proliferation of non-small Cell lung cancer cells.The effect of LINC00324 on the migration and invasion ability of non-small cell lung cancer（NSCLC）cells was observed in Transwell Chambers with and without matrix gels.Flow cytometry was used to detect the effect of LINC00324 on apoptosis.The effect of LINC00324on oncogenesis and metastasis of non-small cell lung cancer cells was observed in tumorigenesis test and Metastatic tumor experiment of Balb/c mice.Results:1.LINC00324 gene expression levels were different in different non-small cell lung cancer cell lines,which were high in SPCA1 and H1299 cell lines,and low in A549 and H460cell lines relatively.2.Cell biological behavior experiments showed that interference of intracellular LINC00324 expression can promote proliferation,migration,invasion and inhibit apoptosis of SPCA1 and H1299 cells.On the contrary,the overexpression of LINC00324 can inhibite proliferation,migration,invasion and promote apoptosis of A549 and H460 cells.3.Subcutaneous tumorigenesis showed that LINC00324 inhibited the growth of non-small cell lung cancer cells in nude mice;Metastatic tumor experiment showed that LINC00324 could inhibit the metastasis of non-small cell lung cancer cells in Balb/c mice.Conclusion:Long non-coding RNA LINC00324 plays a role of tumor suppressor in NSCLC and can inhibit the occurrence and development of NSCLC.Chapter 4 Molecular mechanism of lncRNA LINC00324 in non-small cell lung cancerObjective:To investigate the molecular mechanism of how LINC00324 regulating the biological behavior of lung cancer cells.Methods:Nucleocytoplasmic separation and RNA fluorescence in situ hybridization（RNA-FISH）were used to observe the subcellular localization of LINC00324 in non-small cell lung cancer cells.The interaction between LINC00324 and HuR Protein was verified by RNA Binding Protein Immunoprecipitation（RIP）assay.To study whether LINC00324 can regulate the biological function of non-small cell lung cancer cells by combining with HuR through the biological function recovery experiment.Western blot was used to verify the relationship between LINC00324 and HuR protein expression.Results:1.Nucleocytoplasmic separation and rna-fish experiments showed that LINC00324 was mainly distributed in the cytoplasm of non-small cell lung cancer cells.2.Western blot showed that interference of LINC00324 expression in non-small cell lung cancer cells could increase the expression of HuR protein.In contrast,overexpression of LINC00324 in NSCLC cells decreased the expression of HuR protein.3.RIP experiment showed that LINC00324 could bind HuR protein.4.Interference with HuR expression can reverse the effect of interference with LINC00324 expression on promoting proliferation of non-small cell lung cancer cells H1299,and also reverse the effect of interference with LINC00324 on promoting migration and invasion of non-small cell lung cancer cells.Conclusion:LINC00324 can bind with HuR protein and play its anti-cancer role in the occurrence and development of NSCLC through the regulatory effects of HuR protein.