| Lung cancer is the leading cause of death from cancer in the world, about 80% of which are non-small cell lung cancers (NSCLC). NSCLC has become the number one cancer killer in the worldwide. Despite improvements in early diagnosis and treatment responses by emerging technologies and newly developed chemo/targeted therapies, the high mortality rate has not been markedly changed. The overall 5-year survival for NSCLC patients is still low (15%) and the recurrence rate is too high, even in early-stage groups. Although attempts have been made to identify important genes and pathways in contribution to tumorigenesis of NSCLC, most of the studies are focused on the molecular network of protein-coding genes. However, knowledge of genomic aberrations associated with non-coding genes such as microRNA and their contributions to NSCLC is relatively limited.MicroRNAs (miRNAs) represent an abundant group of short, non-coding RNAs with the potential to target and silence multiple genes across diverse biological processes, such as cell differentiation, proliferation, growth, mobility, and apoptosis. Tons of exciting evidences have assigned an important regulatory role for miRNAs in several human diseases, including cancer. MiRNAs are directly involved in cancer initiation and progression by repressing the expression of important cancer-related genes and thereby function as tumor suppressors or oncogenes. Accumulating data have suggested miRNAs to be aberrantly expressed in lung cancer especially in NSCLC. Some of them are associated with cancer classification, diagnosis, and clinical prognostic informationTo gain further insight into the biological role of miRNAs in NSCLC, we selected several potential tumor suppressor microRNAs including let-7g, miR-7, miR-34a, miR-101 and miR-146 to investigate the effects of them on the cell proliferation of NSCLC cell line A549. We found that miR-7 and miR-34a inhibited cell proliferation of A549 cells, while no significant changes were observed in let-7g, miR-101 and miR-146 treated cells. Furthermore, recent reports have demonstrated miR-7 to be a potential tumor suppressor in several human cancers, such as brain, breast cancer, and pituitary adenoma. Overexpression of miR-7 could inhibit the expression of its target genes like EGFR, IRS1, PAK1, and thus negatively regulated the cancer cell growth. However, the expression and biological function of miR-7 in NSCLC remain to be further elucidated.In view of this, we screened for the expressions of miR-7 in NSCLC tissues, and investigated the effects of miR-7 on NSCLC cells. Furthermore, potential new targets of miR-7 as well as its biological function were explored in this study.PartⅠThe Expression and Biological Function of microRNA-7 in Non-Small Cell Lung CancerMiRNAs, as one panel of hot molecules, are emerging as key regulators in pathogenesis of cancer, by regulating the expression of their target genes at transcriptional and/or translational level. Mature miRNAs profoundly influence a wide variety of pathways, thereby acting as oncogenes or tumor suppressors. Indeed, a number of miRNAs, such as miR-155, let-7, miR-21, miR-34a and miR-7 have been identified to be dysregulated in some cancers and functionally associated with cancer cell proliferation, differentiation and apoptosis. Among them, miR-7 is suggested to be a putative tumor suppressor in breast cancer and glioblastoma, and overexpression of miR-7 inhibits the tumor cell growth by downregulating its targets such as EGFR, IRS1 and PAK1. Our experimental data also suggested that miR-7 could reduce the cell proliferation of NSCLC cell line A549. However, the expression profile, biological function and molecular mechanism of miR-7 in lung cancer, especially in NSCLC, remain to be further elucidated.In order to explore the expression profile of miR-7 in NSCLC, we screened for the expression of miR-7 in 23 pairs of primary NSCLC tissues by realtime PCR and found that miR-7 was downregulated significantly in NSCLC tissues compared to that in adjacent normal lung tissues (p=0.01561). These results were also validated as well as in NSCLC cell lines (A549, H1299, and H1355) compared with normal human lung fibroblast cell MRC5. Therefore, it suggested that miR-7 was frequent downregulated in both NSCLC tissues and NSCLC cell lines.Given the low expression of miR-7 in NSCLC tissues and cell lines, we further evaluated biological functions of miR-7 in NSCLC cancer cells. A549 and HI299 cells were transfected with mature miR-7 duplex mimics (miR-7) or its negative control duplex mimics oligonucleotide (miR-NC) without specifically targeting any human gene products. Our results showed that enforced expression of miR-7 significantly not only decreased the cell proliferation and cell colony formation but also induced cell cycle arrest in NSCLC cell line A549 and H1299 in vitro, while inhibition of miR-7 partially rescued the negative regulation effects of miR-7. In addition, in order to investigate whether miR-7 inhibits the migration of lung cancer, we constructed the lenti-virurs vector expressing miR-7 and established stable miR-7 expressing A549 and H1299 cell lines. We observed significant decreases in cell migration in stable miR-7 expressing A549 and H1299 cells compared with mock vector control ones. Furthermore, we also found that increased level of miR-7 could markedly reduce their tumorigenicity in vivo.In summary, miR-7 was frequently downregulated in NSCLC and overexpression of miR-7 inhibited the growth of NSCLC cells both in vitro and invo, indicating that miR-7 served as a tumor suppressor in NSCLC cells.PartⅡPSME3 Downregulated by microRNA-7 in Non-Small Cell Lung Cancer CellsTo explore the underlying molecular mechanism responsible for lung cancer growth suppression caused by miR-7, we performed bioinformatics’analysis to search for miR-7 target genes. Three prediction algorithms (PicTar, Target Scan, miRanda) were used to identify putative target genes and forty-nine candidate genes were commonly predicted to be the possible targets of miR-7. Among them, six possible targets functioning as oncogenes or anti-apoptosis genes including PSME3, SP1, MNK1, RAF1, SATB1 and ATP2B2 were selected for further analysis.To investigate the putative miR-7 targets, we cloned the 3’UTR of these six target genes into the reporter plasmid downstream from luciferase and performed reporter assays using 293T cells. The relative luciferase activity of reporter containing the potential binding site was reduced by miR-7 in PSME3, SP1, MNK1, RAF1 and SATB1 except of ATP2B2. qRT-PCR assay showed that only the mRNA levels of PSME3 and RAF1 were down-regulated in both A549 and H1299 cells when these cells were transfected with miR-7 mimics. Importantly, significant decreases in PSME3 protein levels were also detected in both miR-7-transfected A549 cells and H1299 cells. Next, we further analyzed the possible 3’UTR binding sequences of PSME3 using TargetScan and revealed that it was highly conserved in human, chimpanzee, monkey, mouse, cow, horse, hedgehog and elephant. To determine whether PSME3 is regulated by miR-7 through direct 3’UTR interactions, a 3’UTR fragment including binding site (wild-type and mutant) were constructed and cloned into the region immediately downstream of the luciferase reporter gene in pGL3-con vector. For luciferase activity assays, HEK 293T cells were co-transfected with the PSME3-3’UTR-Reporter plasmid and miR-7. The relative luciferase activity of wild-type 3’UTR reporter was remarkably reduced by miR-7. In contrast, mutant reporter fully rescued miR-7 repression of luciferase activity.Taken together, the results of this section indicated that miR-7 suppressed PSME3 expression through direct 3’UTR interactions, which strongly implicates a role of miR-7 in the negative regulation of PSME3 expression in NSCLC.PartⅢThe Expression and Biological Function of PSME3 in Non-Small Cell Lung CancerBased on the downexpression of miR-7 in NSCLC cancer tissues and the direct regulation of miR-7 on PSME3, we hypothesized that there may be an inverse relationship between PSME3 and miR-7 expression in NSCLC. Therefore, we examined the expressions of PSME3 and miR-7 in 23 cases of human NSCLC tumor samples and their matched adjacent normal tissues by qRT-PCR analysis. Interestingly, we found that mRNA level of PSME3 in NSCLC tumor samples was significantly overexpressed compared with their matched adjacent normal tissues (p=0.0003). Contrarily, expression of miR-7 was significantly (p=0.0297) reduced in NSCLC tissues compared with their matched controls. Pearson’s correlation analysis revealed a significant inverse relation between levels of PSME3 and that of miR-7 in human NSCLC tissue samples (r=-0.3548, p=0.0156). In addition, we further analyzed their expressions in three different NSCLC cell lines A549, H1299 and H1355, in all of which miR-7 expressions were also down-regulated while PSME3 expressions were up-regulated compared with normal human fetal lung fibroblast cell MRC5. To further explore whether overexpression of PSME3 protein is a common molecular alteration related to lung cancer especially to NSCLC. We examined PSME3 expressions by immunohistochemistry in tissue microarray of 50 pairs of NSCLC samples and 42 pairs of Small Cell Lung Cancer (SCLC) samples. The relative expression level of PSME3 was scored by two independent pathologists. A total of 100%(50 of 50) of NSCLC cancer tissues showed a net gain of immunoreactivity for PSME3, while all matched normal lung tissues (p<0.001) and 42 cases of SCLC cancer tissues (p<0.001) were much more weakly stained in all matched normal lung tissues (p<0.001) and in 42 cases of SCLC cancer tissues (p<0.001) when compared with NSCLC cancer tissues. Next, we analyzed the association of PSME3 expression with patients’age, race, tumor volume, grade, and stage. The average levels of PSME3 immunoreactivity exhibited a positive correlation with tumor volume (p=0.048), but without significant positive correlation with tumor Gleason score or stage. Importantly, PSME3 expression was significantly different between NSCLC and SCLC (p=0.003), with much higher level in NSCLC than that in SCLC. In addition, to further validate the expression of PSME3 in protein level, we analyzed 23 pairs of resected tumor samples and NSCLC cell lines by western blot assay. Similarly, we found that protein levels of PSME3 in NSCLC tumor samples were significantly higher compared with that in matched adjacent normal tissues. The same results were also proved in NSCLC cell lines A549, H1299 and H1355 compared with normal normal human fetal lung fibroblast cell MRC5. These data indicated that PSME3 protein was overexpressed in NSCLC and might become an independent diagnostic factor for NSCLC.To directly address the involvement of PSME3 in lung carcinogenesis, we treated A549 and H1299 cells with SiRNA specific to PSME3 pool (PSME3 SiRNA). PSME3-SiRNA transfected cells showed inhibited proliferation, induced cell cycle arrest at G1 phase, decreased colony formation and reduced the capacity of cell migration compared to mock transfected cells, while inhibition of miR-7 increased the expression of PSME3 and promoted cell proliferation, progression of cell cycle, and colony formation in H1299 cells. Furthermore, the effects of miR-7 inhibitor were also partially rescued by knock down of PSME3 in colony formation and cell cycle arrest assays in H1299 cells. These data indicated that PSME3, down-regulated by miR-7, might positively regulate and promote cell growth, while silence down of PSME3 gene inhibited tumor growth, which is consistent with the effect of miR-7 overexpression.p21/WAF1 (p21), also known as cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1, is encoded by the CDKN1A gene, Since it has been reported that p21 is degraded in a PSME3-dependent and ubiquitin-independent way. We aimed to investigate whether miR-7 affected the p21 protein expression by downregulation of PSME3 gene. H1299 cells were transfected with miR-7, miR-7 inhibitor, or/and PSME3-SiRNA, and then analyzed by western blot. It showed that miR-7 upregulated the expression of p21 protein, which was consistent with inhibition of PSME3 gene. On the contrary, inhibition of miR-7 resulted in the reduced expression of p21 gene, which was partially rescued by silence down of PSME3 gene by SiRNA. These data indicated that miR-7 could increase the expression of p21 in H1299 cells, at least in part, through downregulation of PSME3.C-MYC and Cyclin D1 are two central and key molecular integrators of cell proliferation and cell cycle, which are frequently overexpressed in tumors including lung cancer. To explore the influence of miR-7 and PSME3 on the expression of C-MYC and Cyclin D1, H1299 cells were transfected with miR-7 and PSME3-SiRNA. In realtime PCR assay, both miR-7 and inhibition of PSME3 reduced the mRNA levels of C-MYC and Cyclin D1. This data indicated that downregulation of PSME3 or/and overexpression of miR-7 suppressed the expression of C-MYC and Cyclin D1 directly or indirectly, and induced cell cycle arrest in NSCLC cells.In conclusion, miR-7 is downexpressed in NSCLC. As a novel target of mir-7, PSME3 is inversely upregulated in primary human NSCLC. These findings for the first time indicate that downregulation of miR-7 in NSCLC may contribute to the malignant phenotype by maintaining a high level of PSME3. Moreover, overexpression of miR-7 or knockdown of PSME3 significantly decreased the cell proliferation, cell cycle progression, colony formation and cell migration in NSCLC cells in vitro and reduced tumorigenicity in vivo. Thus, miR-7 and its target PSME3 represent two potential new functional molecular markers in cancer. Interventions targeting them may ultimately be explored in novel strategies for NSCLC treatment. |
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