| Backgrounds:Human lung cancer is one of the most common aggressive malignancies.Even though there has been great improvement on traditional treatments,a considerable number of patients with NSCLC are diagnosed at the advanced stages and the prognosis of these patients is still poor.Therefore,it is extremely necessary to identify novel biomarkers used as therapeutic targets for human NSCLC,98%of the genome do not code for protein function,the lack of protein coding functions of RNA are known as non-coding RNAs(non-coding RNAs,ncRNAs),it is divided into short chain(<200 nt)and long chain(>200 nt)according to the length.In this kind of data,the excavation of different non-coding RNAs and its mechartism research is increased in-depth.Lots of studies have demonstrated that a short chain of ncRNA(such as miRNAs)and a long chain of ncRNA(such as IncRNAs)play an important role in multiple cancers,which has given rise to be targets for therapeutic research.miRNAs is a class of small,non-coding RNAs,and could regulate the biology processes of different diseases and play a potential role as diagnostic and prognostic biomarkers of cancers.IncRNAs belong to a novel heterogeneous class of ncRNAs,and are involved in various biological processes,including imprinting,histone-code regulation and proliferation of cancer cells,through regulation of gene expression.What kind of role the ncRNAs play in NSCLC and whether they serve as valuable biomarkers of NSCLC,these questions are great important for the patients and cancer therapy,which remains unclear.Objectives:In my study,we focused on investigating the role and the underlying molecular machanism of three ncRNAs(miR-638,miR-204 and hncRNA Meg3)on NSCLC.In additional,we discussed that whether abnormal expression of another ncRNAs(let-7)can predict prognosis in patients with multiple cancers.Methods:Part 1 and Part 2:The expression of miR-638(miR-204)and SOX2(SEXI)were dectected in NSCLC tissues by using RT-PCR.Cell invasion,proliferation and apoptosis were investigated by using transwell,MTT(CCK8)and flow cytometry assays.Luciferase reporter assay was used to confirm the regulatory function of miR-638(miR-204)on the target gene SOX2(SIX1),The expression levels of EMT markers were examined by using western blot.Part 3:Stable clones with up-regulation of Meg3 were generated with plasmid via lentivirus technology.Knock-down of Meg3 were generated via small RNA interference technology.IC50,cell cycle and apoptosis were investigated by using MTT and flow cytometry assays.The expression levels of p53,p-catenin and survivin were measured by using westerm blot.Meg3 expression was detected in blood samples collected from patients with advanced lung cancer that had received cisplatin-based chemotherapy by using RT-PCR.Part 4:A meta-analysis was performed by searching PubMed,EMBASE and ISI Web of Science database.Pooled hazard ratios(HRs)and corresponding 95%confidence intervals(Cl)were calculated.Subgroup analyses were conducted for cancer type and ethnicity.Results:Part 1:Low miR-638 and high SOX2 were associated with tumor size,metastasis and stage of NSCLC.Down-regulated miR-638 promoted cell invasion and proliferation,while the effect of miR-638 high expression was reverse.Furthermore,miR-638 could regulate SOX2 by binding to its 3’-UTR.Silencing of SOX2 partially abolished the enhancement of cell invasion and proliferation induced by down-regulated miR-638.Subsequently,aberrant miR-638 expression could modulate the expression levels of EMT markers.Part 2:miR-204 expression was decreased and SIX1 expression was increased,and both of them were associated with tumor size,metastasis and stage of NSCLC.miR-204 could regulate cell invasion and proliferation.Furthermore,we identified that SIX1 was a candidate target of miR-204.Silencing of SIXI partially weakened the promotion of cell invasion and proliferation induced by down-regulated miR-204.Dysregulation of miR-204 expression could regulate the expression levels of EMT markers.Part 3:The expression levels of Meg3 were significantly lower in cisplatin-resistant A549/DDP cell,compared with those in parental A549 cell.Furthermore,up-regulation of Meg3 was able to re-sensitize A549/DDP cell to cisplatin.Meanwhile,down-regulation of Meg3 could decrease the sensitivity of A549 cell to cisplatin.The results of the present study also demonstrated that the Meg3-mediated chemosensitivity enhancement was related to the induction of cell cycle arrest and increased cell apoptosis,through regulation of p53,p-catenin and survivin,which were target genes of the Wnt/β-catenin pathway.Part 4:A total of 1757 cases of multiple cancers were involved for this meta-analysis.The HR of low let-7 expression in multiple cancers was 1.80(95%Cl:1.18-2.76),for lung cancer HR=1.99(95%Cl:1.17-3.40).A subgroup analysis was performe.d about ethnicity,combined HR was 1.61(95%Cl:0.84-3.11)for Asians,and 1.94(95%Cl:1.11-3.39)for non-Asians.Conclusion:In summary,our results indicate that:(1)Down-regulation of miR-638 induces EMT and promotes NSCLC cell invasion and proliferation by regulating SOX2.(2)miR-204 functions as a tumor suppressor by regulating SIXI in NSCLC.(3)IncRNA Meg3 may have a crucial role in the development of cisplatin resistance in NSCLC.(4)Low expression of let-7 predicts poor prognosis in patients with multiple cancers,especial in lung cancer. |
