| BackgroundLung cancer is the leading cause of cancer-related deaths worldwide,which is characterized by a low survival and high relapse rate after surgery.NSCLC,the most frequently occurring category of lung cancer,accounts for approximately 80%of all cases.Tumor invasion and metastasis are the main factors responsible for NSCLC treatment failure.Previous studies have shown that the tumorigenesis of lung cancer is involved in both genetic and epigenetic alterations,including the activation of oncogenes and/or the suppression of tumor suppressor genes.Plenty of evidence shows that noncoding miRNAs play key roles in the progression of lung cancer.MiRNAs have been associated with various types of cancer,which involved in many important physiological and pathological processes,including development,differentiation and tumorigenesis.By binding to the 3'-UTR of mRNA,miRNA suppresses protein synthesis through mRNA degradation or translational repression.As a tumor suppressor gene in several cancer types,miR-29a can affect tumor cell growth,migration,invasion and apoptosis.To date,some genes have been identified as miR-29a targetgenes,including CDC42,CLDN1,c-MYC,KDM5B,TNFR1.However,the function and underlying mechanism of miR-29a in regulating lung tumorigenesis are still to be further investigated.As a member of the RAS oncogene family,NRASis one of the most frequently activated oncogenes in human cancer.NRAS is implicated and functionally altered in various cancer and has potential roles in the regulation of cancer cell growth,survival,migration,invasion,and angiogenesis.Oncogenic NRAS promotes tumorigenesis through activation of multiple downstream pathways,including PI3K/AKT,MAPK/ERK,and NF-?B pathway.Thus,NRAS silencing has become an efficient therapeutic strategy in tumors,but it is still far from optimal and novel therapeutic strategies are needed urgently.ObjectiveIt has been reported that miR-29a may play roles in lung cancer via affecting different signaling pathways.In our study,we aimed to further identify the roles of miR-29a and its molecular as well as cellular mechanisms in lung cancer.We investigated its correlation with clinical parameters of patients,and how miR-29a overexpression affects the proliferation,invasion,and invasion of lung cancer cells.At the same time,the drug sensitivity of cisplatin in lung cancer cells overexpressing miR-29a was discussed.Our results revealed a novel mechanism of miR-29a in lung cancer,and it possessed a potential to be used as a novel strategy to develop miR-29a-based therapeutics.Method(1)A total of 38 NSCLC specimens and adjacent normal tissues were collected.The expression of miR-29a in lung cancer tissues and adjacent normal tissues was detected by qRT-PCR.Then,we analyzed the orrelation between miR-29a and clinical parameters.(2)The lentiviral plasmid was used to infect human lung cancer cell line H1299 and A549,and the cell line stably overexpressing miR-29a or miR-NC was established after screening with puromycin.The total RNA of each cell line was extracted and the expression of miR-29a was detected by fluorescence quantitative PCR.The cell proliferation activity was detected by CCK-8 assay and the colony formation assay was performed by CCK-8 assay.The invasion and migration of cells were detected by transwell chamber assay.(3)Screening target genes of miR-29a using bioinformatics software.The expression level of NRAS in normal human tissues and lung cancer tissues was detected by RT-PCR,and the correlation between miR-29a and NRAS was analyzed.The NRAS wild-type(WT)plasmid or the NRAS mutant(MT)plasmid was constructed and transfected into 293T cells with miR-29a or miR-NC mimics.Luciferase activity was measured 48 hours later.MiR-29a or miR-NC were transfected into H1299 and A549 lung cancer cell lines respectively.NRAS protein level was detected by western blot.(4)Cell viability of H1299/miR-29a and H1299/miR-NC cell lines was tested by CCK8 kit at different concentrations of cisplatin.The proliferation activity of lung cancer cells of H1299/miRr-29a group,H1299/miR-29a group overexpressing NRAS and H1299/miR-29a were detected under cisplatin concentration of 5?M.Flow cytometry was used to detect the apoptosis rate.Caspase-3 activity kit was used to detect caspase-3 activity.(5)A subcutaneous tumorigenic model was established in nude mice.The tumorigenicity of H1299/miR-29a and H1299/miR-NC cells was measured.The volume of tumor mass was measured regularly and the growth curve was drawn.The mRNA and protein expression levels of NRAS in subcutaneous xenografts were detected by real-time PCR and Western blot.Results(1)The miR-29a expression in tumor tissues was significantly lower compared with those controls;miR-29a expression in stage ?-? lung cancer tissues was significantly lower than those in stage ? and ?.Correlation analysis of clinical parameters showed that the expression level of miR-29a was related to tumor staging,regardless of sex/age.(2)Overexpression of miR-29a in H1299 and A549 cells in vitro could inhibit the proliferation and colony formation,while significantly reduce invasion and migration..(3)Luciferase assay showed that microRNA-29a markedly suppressed luciferase activity in NRAS 3'-UTR wild type.We found that the NRAS expression was downregulated at the protein level imiR-29a-treated cells.The average expression level of NRAS was significantly higher in tumor tissuesthan that in normal tissues.Spearman rank correlation analysis showed that levels ofNRAS and miR-29a in lung cancer was negatively correlated.These results demonstrate that NRAS is a directtarget of miR-29a.(4)Overexpression of MiR-29a in H1299 cells significantly increased the chemosensitivity of cisplatin treatment,and forced expression of NRAS reversed miR-29a-induced chemoresistance of cisplatin to lung cancer.The combination of miR-29a and cisplatin significantly induced apoptosis,whereas forced expression of NRAS partially reversed the effect of some miR-29a plus cisplatin treatment.Caspase-3 activity was significantly up-regulated following miR-29a and cisplatin combination therapy compared to miR-29a alone or cisplatin alone,whereas forced expression of NRAS attenuated the cysteine during therapeutic treatment Aspartase-3.(5)Nude mice tumorigenic model showed that overexpression of miR-29a inhibited the formation of subcutaneous tumors in nude mice,tumor growth slow.In subcutaneous xenografts of miR-29a overexpression group,levels of NRAS mRNA and protein were significantly lower than those in the control group.ConclusionMiR-29a plays a role of tumor suppressor in lung cancer tissues and low expression in tumor tissues.MiR-29a inhibits the proliferation,invasion,migration and tumor growth of lung cancer cells through the target gene NRAS and enhances the sensitivity of lung cancer cells to cisplatin. |
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