MTDH Mediates The Sensitivity To Docetaxel Through The PI3K/Akt/mTOR Pathway In NSCLC

Background and ObjectiveLung cancer has the highest case-fatality rate among all cancers in China and over the world. NSCLC is more than 80% of all lung cancers. Due to lack of typical early symptom, most patients are in the metaphase or advanced phase of lung cancer upon confirmed diagnosis, thus losing the chance of an operation. Accordingly, curative treatment is the main means for lung cancer. The most commonly used therapy for lung cancer is chemotherapy. Although new chemotherapy drugs and various combined schemes are constantly emerging recently, many cancers are insensitive to chemotherapy drugs or gradually acquire tolerance to such drugs during chemotherapy, and this is the main cause of failure of treatment and death. Searching and intervention of factors that affect chemoresistance is a hot point of current research in targeted treatment of cancers. Metadherin is a newly found oncogene, and Over-expression in various cancers has been reported. Increase of MTDH expression can activate various signal paths, enhancing tumor cell proliferation and anti-apoptosis capabilities, accelerating growth of new vessels of tumor, and enhancing aggression and metastasis of tumor cells. This research further clarifies correlation between MTDH and chemoresistance of NSCLC through in-vitro experiment, observes the relation between MTDH expression and sensitivity of NSCLC cell to docetaxel, and explores possible mechanism, so as to provide theoretical foundation and molecular target for reversal of tumor chemoresistance.Methods and Materials1. Construct a specific targeted MTDH siRNA in vitro first, and MTDH mRNA fundamental expression level of HBE cell and six NSCLC cell lines (A549, H1299, SPCA1, H1703, PC-9, and H1650) had been detected. Cell line of relatively high MTDH fundamental expression level has been selected and small molecule RNA interference technique has been used for instantaneous transfection of MTDH siRNA. Realtime PCR and western blto has been used to detect MTDH mRNA and protein level.2. We have used MTT method and cloning formation experiment to detect whether lowering of A549 cell MTDH expression level affects sensitivity to docetaxel. To explore whether MTDH affects apoptosis, flow cytometer has been used to analyze apoptosis rate of siMTDH A549 after treatment by docetaxel. To explore whether MTDH affects the cell cycle, flow cytometer has been used to analyze.3. We have used western blot to detect the protein level of bcl-2, bax, LC-3 and p-AKT when lowering the expression of MTDH.4. LY294002 has been used to block PI3K/AKT pathway. Western blot was used to detect the protein level of p-AKT to estimate the blocking efficiency. MTT method was used to detect whether highering of H1299 cell MTDH expression level affects sensitivity to docetaxel Before and after blocking of PI3K/AKT.Results1. Construct a specific targeted MTDH siRNA in vitro first. Transfect into A549 cell and successfully build MTDH downgrade of A549 cells.2. The sensitivity of A549 cell to doceltax was regulated by the expression of MTDH. Downregulation of MTDH enhance the cell apoptosis induced by docetaxel, but not in cell cycle.3. Over-expression of MTDH can activate P13K/AKT/mTOR pathway, hence lower sensitivity of NSCLC cells to docetaxel.ConclusionThrough a series of experiments of this research, we have found that over-expression of MTDH causes increase of resistance of NSCLC cells to docetaxel and such action occurs via PI3K/AKT/mTOR signal path. After reduction of expression of MTDH, sensitivity of NSCLC to docetaxel increases. MTDH inhibitor may become a sensitivity enhancing agent for chemical therapy for NSCLC, providing a new solution for such chemical therapy.