| BackgroundLung cancer is the most common cause of cancer-related death worldwide. Lung cancers are divided into two histopathological types: non-small cell lung cancer(NSCLC) and small-cell lung cancer(SCLC). NSCLC accounts for 80~85% of all lung cancers and is generally diagnosed at an advanced stage and total resection is difficult, also it is easy to recurrence after surgery. Radiotherapy and chemotherapy are often used as auxiliary means after the operation. But they not high specially kill tumors cells, also may produce side effects. Exploring a new treatment of NSCLC,especially the mechanism of its occurrence, development and evolution on gene level,finding an effective target of gene therapy is the hotspot in recent years.In recent years, the systematical researchers are concentrating on long non-coding RNAs(lncRNAs), which are defined as transcripts greater than 200 nucleotides in length without obvious protein coding functions. Recent evidence suggests that the inconsequential “dark matter†of the genetic transcription may play a major role in regulating multiple biological processes, including cell growth, cell cycle progression,differentiation, and apoptosis. Furthermore, a large and growing body of literature has indicated the involvement of lncRNAs in a variety of diseases, especially cancer.Recent studies have demonstrated that dysregulation of several lncRNAs is related to human cancers, such as breast cancer, gastric cancer, lung cancer, prostate cancer, etc.Moreover, those lncRNAs that have been associated with cancers are often aberrantly expressed and play functional roles in carcinogenesis, tumor growth, invasion,metastasis, recurrence and resistance, indicating that lncRNAs may act as oncogenes or tumor suppressor genes in the development of cancers. So we predict that lncRNAs could act as a new marker of diagnosis and judgment of prognosis and therapeutic effect of tumor, meanwhile as a new therapeutic target in cancer gene therapy.Wnt/β-catenin signaling pathway plays a crucial role in regulating multiple aspects of tumor development, including lung cancer. In a previous study, we observed thatlncRNA AK126698 was significantly downregulated in A549/DDP cells compared with parental A549 cells. In addition, we previously predicted that FZD8, the most important Wnt receptor, might be a target of AK126698. However, the clinical importance of lncRNA AK126698 and molecular mechanisms controlling its effects are unknown.ObjectiveThe aim of our study is to investigate the correlation between AK126698 and the clinicopathologic features of NSCLC patients, and then to prove that FZD8 is the target gene of AK126698, and participates in the regulation of NSCLC cell proliferation, migration and drug resistance, thereby better elucidating the mechanisms of NSCLC and providing therapeutic targets for the treatment of NSCLC.Methods1. We examined the expression of lncRNA AK126698 in 56 non-small-cell lung cancer(NSCLC) tissues samples and three NSCLC cell lines using quantitative real-time PCR(qRT-PCR).2. Gain and loss of function approaches were used to evaluate the biological function of AK126698 in NSCLC cells. The effects of lncRNA AK126698 on cell proliferation, migration and apoptosis were investigated.3. The mRNA and protein expression of FZD8 were determined by qRT-PCR and western blot after changing the AK126698 levels in NSCLC cells.4. Axin1, β-catenin, c-myc, cyclin D1 and E-cadherin protein expression is assayed by Western blot.5. The levels of lncRNA AK126698 and FZD8 m RNA were determined in two lung cancer cell lines(A549 and NCI-H520) and their cisplatin-resistant variant A549/DDP and NCI-H520/DDP by using qRT-PCR. Proteins of FZD8 are represented by Western blot.6. The effects of AK126698 on the proliferation and chemosensitivity of cancer cells was investigated, using both gain- and loss-function studies.7. The effects of FZD8 on NSCLC to cisplatin were examined by inhibiting the expression of FZD8.8. The correlation between AK126698 level and chemoresistance was further investigated in clinical NSCLC specimens Results1. LncRNA AK126698 was significantly downregulated in NSCLC tissues compared with paired adjacent non-tumor tissue samples. Meanwhile, lower expression of AK126698 was detected in A549, NCI-H520 and H1299.2. Lower AK126698 expression was associated with larger tumor size and advanced tumor stage.3. Ectopic AK126698 expression inhibited cell proliferation and migration and induced apoptosis. Conversely, decreased AK126698 expression promoted cell proliferation and migration and inhibited cell apoptosis.4. We demonstrated that the Frizzled-8(FZD8), a receptor of Wnt/β-catenin pathway, was a target of AK126698.5. AK126698 could inhibit the activation of Wnt/β-catenin pathway, which was demonstrated by measuring of the expression levels of Axin1, β-catenin, c-myc, cyclin D1 and E-cadherin.6. LncRNA AK126698 levels were dramatically downregulated in the cisplatin-resistent human non-small-cell lung cancer(NSCLC) cell lines A549/DDP and NCI-H520/DDP compared with their respective parental cell lines, accompanied by increased FZD8.7. Overexpression of AK126698 increased the sensitivity of A549/DDP and NCI-H520/DDP cells to cisplatin, while silencing AK126698 reduced the sensitivity of cells to cisplatin in A549 and NCI-H520.8. LncRNA AK126698 increasing chemosensivity was associated with inhibition of cell proliferation and apoptosis enhancement through negative regulation of FZD8 expression.9. AK126698 expression levels were significantly lower in NSCLC tissues with high levels of FZD8 and ERCC1, a marker for cisplatin-resistance.ConclusionThe study found that lncRNA AK126698 inhibits the proliferation and migration of NSCLC cells by targeting FZD8 to suppress the Wnt/β-catenin signaling pathway and lncRNA AK126698 reverses the cisplatin resistence of NSCLC cells, at least in part,through the regulation of FZD8 expression. It may provide a new target for therapeutic intervention of NSCLC. |
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