Function And Mechanism Of LncRNA H19 And MiR-21 In Non-small Cell Lung Cancer

With the increasing morbidity and mortality,lung cancer has become the leading cause of cancer death worldwide and is the main cause of tumor-related deaths worldwide.The main reasons for poor prognosis are lack of effective diagnostic tools in the early stage and effective treatment in the late stage.The main challenges associated with lung cancer treatment are recurrence and metastasis,and these are also the primary causes of death among patients.Although molecular networks for the development of lung cancer have been partially elucidated at the genetic and protein levels,and individualized therapies based on genetic changes and targeted drugs have made some progress in the past decade,the overall survival rate of lung cancer is still not encouraging.In recent years,domestic and foreign scholars had discussed the occurrence and development mechanism of lung cancer in different perspectives.The differences between normal cells and cancer cells lie in the enhancement of proliferation ability,the decrease of apoptosis,the high rate of gene mutation and the increase of multi-drug resistance.However,there are numerous regulatory molecules and the regulation of intracellular signaling pathways is comple.The specific mechanism of the synergistic regulation of non-coding RNA is still unclear.Non-coding RNA do not encode proteins,which generally include lncRNA,tRNA,snRNA,microRNA and other RNA with unknown functions.Those Non-coding RNA could be transcribed from the genome,however they will not be translated into proteins.They came into play biological functions at the RNA level.In recent years,non coding-RNA has attracted more and more attention in the aspect of transcriptional regulation of lung cancer.In addition,it was reported that miR-21 is closely related to lung cancer,which regulates the occurrence and development of lung cancer.MiRNA-21 regulates tumors through downstream target genes,such as PDCD4(programmed cell death 4)gene,PTEN(phosphatase-tensin homolog)gene and NFIB(nuclear factor I/B gene).LncRNA is a kind of non-coding RNA,with a length generally greater than 200 bases.It mainly plays a role in epigenetic regulation,transcriptional regulation and post-transcriptional regulation in the form of RNA.Many studies have reported that lncRNA is closely related to lung cancer,and may play a key regulatory role in promoting the invasion and metastasis of lung cancer,promoting the growth and colony formation of non-small cell lung cancer cells,resulting the occurrence and development of lung cancer,regulating the activation of oncogenes,enhancing the stability of lung cancer cells,and causing chemotherapy resistance.We used imported lncRNA chips and bioinformatics technology(TCGA database and Pubmed)to screen the target lncRNA-H19.However,there are numerous regulatory molecules(proto-oncogenes,tumor suppressor genes,etc.),complex signal pathway regulations(PI3K/AKT,Wnt signal,etc.),and regulatory networks are crisscrossed.Single non-coding RNA regulation may not play a key role,and the specific mechanism of non-coding RNA coordinated regulation is still unclear.Therefore,this study mainly focused on the expression of H19 and mir-21 in NSCLC and the correlation between them and the biological characteristics and prognosis of NSCLC.Then we further clarified the specific molecular regulatory mechanism of H19 on miR-21 and its downstream target genes in NSCLC through in vitro and in vivo experiments,which is conducive to assisting the early diagnosis of lung cancer,revealing the prognosis of lung cancer and providing more targets for the treatment of lung cancer.Part I:Association of long non-coding RNA H19 and microRNA-21 with the biological features and prognosis of non-small cell lung cancerObjective The aim of this study was to investigate the expression,diagnostic and prognostic value of lncRNA H19 and miR-21 expression in non-small-cell lung cancer(NSCLC).Materials and Methods H19 and miR-21 expression was measured in tumor tissues and corresponding non-tumor lung tissues from 200 patients by quantitative reverse transcription polymerase chain reaction.The relationship between the expression level of the H19 and miR-21 and the biological characteristics of lung cancer was analyzed.According to the follow-up data,the prognostic relationship between H19 and mir-21and non-small cell lung cancer was determined.Results Expression of both H19 and miR-21 was significantly higher in lung tissues from patients with NSCLC than in normal lung tissues.Increased expression of H19and miR-21 was positively correlated with advanced tumor-node-metastasis(TNM)stage and tumor size.MiR-21 expression was highest in stage I and II NSCLC,whereas H19 expression was highest in stage III and IV NSCLC.The high expression of H19 and miR-21 in lung cancer is negatively correlated with the prognosis of NSCLC.With the higher the expression of H19 and miR-21,the overally survival(OS)is lower.Conclusion H19 may mainly contributes to the progression of NSCLC,and its expression levels can reflect the invasive and metastatic status to some extent.Expression of miR-21 more likely plays an important role in early stage NSCLC.The high expression of H19 and mir-21 is negatively correlated with the prognosis of lung cancer.The combination of H19 and miR-21 may have diagnostic and prognosis value in NSCLC.Part II:Regulation of lncRNA H19 expression on lung cancer cell proliferation and mir-21 expression(in vitro)Objective The aim of this study was to investigate the effect of lung cancer cell proliferation and miR-21 expression inn vitro after overexpression/inhibition of H19.Materials and Methods Lung cancer cell lines A549 and H1299 were cultured in vitro.The over-expression of H19 was transfected with plasmids and the expression of H19 was inhibited by siRNA-H19 transfection.Then the proliferation of lung cancer cells was detected by MTT assay and flow cytometry.Changes in the expression of miR-21 were detected according to the overexpression/inhibition expression of H19.Results Lung cancer cell proliferation is active after H19 was overexpressed.The growth of lung cancer cells was significantly inhibited after H19 expression was inhibited.Overexpression of H19 increased the level of miR-21.If H19 was knocked down,the expression of miR-21 decreased.Conclusion In vitro experiments,H19 can regulate the proliferation of lung cancer cells.The expression of H19 is positively correlated with the expression of miR-21,and there may be a regulatory relationship.Part III:The mechanism of LncRNA H19 on microRNA-21 and its downstream target genes in animal models of non-small cell lung cancer(in vivo)Objective The aim of this study was to evaluate the effect of H19 gene-silencing on animal models of non-small cell lung cancer.Materials and Methods The A549 cells cultured in vitro were injected into the left back of nude mice,and the subcutaneous tumor volume was 50~100 mm~3.The experiment was divided into several groups,such as control group,H19 gene knockdown group(shH19),antagomir-21 group,shH19+pemetrexed+cisplatin group,H19-gene-knockdown+Gefitinib group,pemetrexed+cisplatin group and Gefitinib intervention group.After the mose was executed,HE staining,quantitative PCR and Western Blot were performed to identify the expression of the target gene and detect the expression level.Results All the experiment groups showed slower tumor growth than the control group,and the H19-gene-silence+gefitinib group showed the slowest tumor growth.H19-gene-silence+gefitinib had the most obvious tumor inhibition effect.QRT-PCR and western-blot experiments indicated that the expression of miR-21 was significantly lower after H19 gene was knocked down,and the expression of PTEN,PDCD4 which are the downstream target genes of miR-21,was significantly increased at the same time.However,the expression of NFIB was lower than control.Conclusion In vivo experiment,H19-gene-slience had a significant inhibitory effect on tumor.H19 may play regulatory role in miR-21,including its downstream target genes in lung cancer.H19-gene-silencing can enhance the efficacy of chemotherapeutic drugs and targeted drugs.H19 could be as a new target in NSCLC.