Molecular Mechanism Of STIP In The Proliferation Of Non-small Cell Lung Cancer

Lung cancer is one of the most common type of malignant tumors. In recent years, the incidence and mortality rates of lung cancer were increasing. The major types of lung cancer are classified histologically as small cell lung cancer and non-small cell lung cancer(NSCLC). NSCLC accounts for 80%-85% of all lung cancer cases. In our country, lung cancer has become the leading cause of cancer-related mortality, and with a 5-year survival rate of only 10%. It mostly based on operation treatment, but 80% of NSCLC patients are diagnosed at an advanced stage and lost the chance of surgical procedure. Chemotherapy is a major treatment for advanced lung cancer, but with the increase of chemotherapy, the drug resistance rate will be higher. At present, there is no ideal treatment for advanced lung cancer, therefore, it is imperative to identify new molecular targets that play a role in the pathogenesis of NSCLC to design novel preventi ve/therapeutic strategies to control this malignancy.STIP(septin and tuftelin-interacting proteins) protein is a kind of nucleoproteins containing multiple structural domains, and can self-aggregate into rod-shaped polymers termed stiposomes in the nucleoplasm. In C.elegans, the knockdown of STIP via RNA interference causes developmental arrest of early embryos at the 16-cell stage, this lethal phenotype can be readily rescued with either human or Drosophila STIP, highlighting its conserved essential biol ogical function across metazoans from worms to flies to humans. Proteomic studies identified STIP as a spliceosome-associated factor that mediates the release of the lariat intron in pre-m RNA, affecting intracellular gene expression. However, the biological roles and molecular functions of STIP in cancer remain unknown.In this study, we reported that STIP is overexpressed in clinical NSCLC samples compared to adjacent normal lung samples and that STIP maybe plays a critical role in NSCLC progression. In order to clarify its biological effects in NSCLC cells, we constructed lentiviral vector-mediated siRNA to interference STIP expression and using MTT assays and colony formation assays to detect the effect of STIP on cell proliferation. The data suggest that the downregulation of STIP inhibited the proliferation of NSCLC cells. Because of cell proliferation depends on cell cycle progression, so then we further explore the impact of STIP on cell cycle. STIP depletion in A549 and H460 cells led to cell cycle arrest at the G2/M phase. To further elucidate the molecular changes associated with the observed G2/M arrest in The effect of STIP on the expression of G2/M-related proteins suggested that cell cycle arrest is associated with the downregulation of Cyclin B1、CDK1 and Cdc25 C. Furthermore, we found a decrease in the dephosphorylation of CDK1 at at Thr-14/Try-15. We found downregulation of STIP resulted in a significant decrease in protein expression. Collectively, these results provide evidence that STIP knockdown in NSCLC causes cell cycle arrest at the G2/M phase by affecting the expression and activation of the CDK1-Cyclin B1 complex. In general, cell cycle arrest is accompanied by the induction of apoptosis. We demonstrated that STIP knockdown in A549 and H460 cells induced apoptosis by observing nuclear morphological changes and by performing flow cytometry analysis using Annexin V and PI double staining. The mechanism of STIP knockdown inducing apoptosis, on the one hand was to increased the expression of Bax and decreased that of Bcl-2; on the other hand was to activate Caspase-9 and Caspase-3, resulting in procedural cell death. Proteomic studies identified STIP as a compo nent of the nuclear spliceosome, functions in pre-m RNA processing by excising introni c nucleic acids. To identify the STIP-mediated gene expression changes, RNA sequencing was performed on NSCLC cells. Compared with the control treatment, STIP knockdown resulted in the differential expression of 566 identified genes, including 490 upregula ted genes and 76 downregulated genes. These genes are involved in multiple processes associated with cancer, including the MAPK, Wnt, PI3K/AKT, and NF-kB signaling pathways. The data demonstrated that STIP knockdown inhibits the tumorigenicity of NSCLC in vivo. Collectively, our results suggest that STIP might be a novel potential diagnostic marker and therapeutic target for NSCLC.