The Role Of Let-7c In Non-small Cell Lung Cancer Proliferation And Cisplatin Resistance

Lung cancer is the leading cause of cancer-related death worldwide. The incidence and mortality are appeard to be increasing these years. Lung cancers are divided into two histopathological types:non-small cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), which represent approximately80%and20%of tumors, respectively. In general, surgery is believed to offer the best prospects for cure for early stage NSCLC.However, most patients with NSCLC are diagnosed in the advanced stage (ⅢB or Ⅳ) and lose opportunity to receive sugery. The preferred treatment for the unresectable NSCLC patient is platinum-based two-drug chemotherapy.Despite improvements in early diagnosis and newly developed chemo/targeted therapies that improve treatment responses, the overall5-year survival for NSCLC patients remains low (15%) and the recurrence rate is high, even in early-stage groups. Lack of early detection and chemotherapy resistance are both contributing factors to disfavorable prognosis observed in lung cancer. Thus, advances in both of these areas are likely to lead to improved outcomes.MicroRNAs are a class of endogenous single-strand and highly conserved non-coding small RNAs. They regulate negatively the expression of target genes by translational repression or mRNA degradation through binding to the3’UTR of target genes. Several lines of evidence suggest that miRNAs are frequently deregulated in human malignancies and function as tumor suppressors or oncogenes. miRNAs modulates cell survival and growth through targeting tumor suppressor genes (TSGs) and oncogenes. Furthermore, miRNA also could modulate the cell response to chemotherapy. Taken together, many miRNAs have been identified as potent regulators of cell viability and drug sensitivity, and represent a new class of potential thereauptic target and diagnostic biomark.The Previous studies have revealed the importance and potential role of miRNAs in NSCLC. MiRNAs, including miR-21, let-7, miR-34and miR-145, have been demonstrated to play important regulatory roles in NSCLC. The lethal-7(let-7) is the first known human miRNA. Let-7family members are found decreased in various human cancer with few exceptions and function as tumor suppressors, including lung, prostate and colon cancer. Emerging evidences demonstrate an important role of let-7c in regulating diverse cellular processes including proliferation, apoptosis, migration and invasion. The dysregulation of miRNAs are involved in iniaition, progression, response to chemetherapy and prognosis.The effect and possible mechanisms of let-7c in NSCLC remains large unknown. Here we explored the role of let-7c in NSCLC proliferation and resistance to DDP to provide theoretical basis for the development of therapeutic method based on let-7c. Charper1Let-7c inhibits NSCLC cell proliferation by targeting HOXA1Objective:To explore underlying mechanisms by which let-7c suppresses NSCLC cell proliferation.Methods:The expression level of let-7c was quantified by qRT-PCR. A549and H1299cells were transfected with let-7c mimics to restore the expression of let-7c. The effects of let-7c were then assessed by cell proliferation, colony formation and cell cycle assay. Mouse experiment was used to confirm the effect of let-7c on tumorigenicity in vivo. Luciferase reporter assays were performed to identify the target gene of let-7c. qRT-PCR and Western blot were used to explore the regulatory effect of let-7c on target gene and possible mechanisms.Results:HOXA1is identified as a novel target of let-7c. MTS, colony formation and flow cytometry assays demonstrated that forced expression of let-7c inhibited NSCLC cell proliferation by inducing G1arrest in vitro, which is consistent with the inhibitory effects induced by knockdown of HOXA1. Mouse experiments demonstrated that let-7c expression suppressed tumorigenesis. Furthermore, we found that let-7c could regulate the expression of HOXA1downstream effectors CCND1, CDC25A and CDK2. Conclusions:Let-7c inhibits NSCLC cell proliferation and tumorigenesis by partially direct targeting HOXA1pathway. Charpter2A let-7c/JMJD1A double-negative feedback loop inhibit H1299cell growthObjective:To explore the mechanisms of let-7c inhibiting NSCLC cell growth based on the charpter1Methods:Luciferase reporter assays, qRT-PCR and Western blot were performed to explore the effect of let-7c on JMJD1A. The expression level of let-7c was quantified by qRT-PCR. Immunocytochemical staining was used to detect the expression of JMJD1A in NSCLC tissue and para-carcinoma tissue. The effects of JMJD1A knockdowon on the H1299grwoth was assessed by cell proliferation, colony formation, cell cycle assay and mouse experiment. Luciferase reporter assays and/or Western blot were performed to identify the effect of let-7c and JMJD1A on EZH2.Results:JMJD1A was identified as a novel target of let-7c; JMJD1A was upregulated in NSCLC tissues compared with in the para-carcinoma tissues by immunohistological staining. The expression of JMJD1A is unrelated to the clinical pathological characteristics (eg, age, sex, metastasis and pT). The expression level of JMJD1A was negative associated with the overall survival of NSCLC patients. MTS assay and colony formation assay demonstrated that JMJD1A knockdown inhibited H1299cell proliferation. Cell cycle assay demonstrated that JMJD1A knockdown induced G1arrest. Mouse experiments revealed that JMJD1A knockdown suppressed the tumorigenesis of H1299in vivo. JMJD1A also modulated the expression of let-7c; and then let-7c and JMJD1A generated a double-negative feedback loop. The let-7c/JMJD1A loop regulated the expression of EZH2.Conclusion:The let-7c/JMJD1A double-negative feedback loop inhibits H1299cells proliferation and one of possible mechanisms is through the downregulation of EZH2.Charpter3Let-7c sensitizes cisplatin-resistant A549cells by targeting ABCC2and Bcl-XLObjective:To explore the role of let-7c in cispaltin-resistant A549(A549/DDP) cells.Methods:A549/DDP cells was transfected with let-7c mimics, let-7c inhibitor or negative control (NC). IC50was determined by MTS assay. The apoptosis was explored by flow cytometry and hoechst33342staining. Messenger RNA and protein level were detected by qRT-PCR and Western blot. Luciferase assay was performed to determine whether ABCC2and Bcl-XL are direct targets of let-7c.Results:The IC50of DDP in A549and A549/DDP cells were18.03±1.418μM and69.03±5.086μM, respectively (P<0.001). the IC50value of DDP in A549/DDP cells transfected with let-7c mimics, NC and let-7c inhibitor was29.83±1.810μM,66.84±2.870μM and99.03±5.446μM, respectively. Flow cytometry and hoechst33342staining showed that DDP-induced apoptotic cells in let-7c transfected A549/DDP cells was markedly increased compared to NC-treated cells, and downregulation of endogenous let-7c prevented DDP-induced apoptosis in A549/DDP cells. Bioinformatics analysis, luciferase reporter assays, qRT-PCR and Western blot demonstrated that ABCC2and Bcl-XL are targets of let-7c. Knockdown of ABCC2and Bcl-XL could mimic the effect of let-7c restoration on A549/DDP cells sensitivity to DDP.Conclusion:Ectopic expression of let-7c could increase A549/DDP cells sensitivity to DDP, and one of the mechanisms was through directly targeting ABCC2and Bcl-XL...