The Effect Of Exosomes On The Sensitivity Regulation Of Lung Cancer Cells To Cisplatin

Lung cancer is one of the most common tumors, which is the first reason of the male cancer-related death and the first in the disease incidence and mortality of the world[1]. Thus it is so obvious that it is meaningful about deep-study of lung cancer. In terms of treatment, cisplatin-based chemotherapy is the main treatment of clinical practice. The patient’s sensitivity to cisplatin is the key to cancer therapy, and therefore the problem of drug resistance formation has become a hotspot of cancer therapy.As the most common and first-line used drugs in chemotherapy of non-small cell lung cancer, the primary or secondary cisplatin resistance is a major obstacle to the treatment of lung cancer, as well as five-year survival rate of patients with late (III or IV) lung cancer remains low[2]. There are a variety of points about the mechanism of cisplatin resistance, including the abnormal of cisplatin accumulation and discharge of cells, dysregulation pathway associated with cisplatin resistance, chromosomal abnormalities, DNA damage repair function, cellular detoxification enhancement, inhibition of apoptosis and angiogenesis, surrounding cytoskeleton and extracellular matrix density abnormalities[3-5]. Thus, the formation of cisplatin resistance is formed by a variety of factors involved in various mechanisms. Any kind of mechanism can not be fully used to explain the phenomenon of cisplatin resistance formation in lung cancer, and thus further to improve the study of the mechanism of cisplatin resistance of lung cancer is important.The ways of transmitting information between cells mainly include autocrine, paracrine and endocrine. In the past twenty years, researchers found that one of the mechanisms is microvesicles[7]. The types of microvesicles are various depending on the cell type, source and microenvironment, include exosomes, tolerosomes, prominosomes and so on[8]. Exosomes is released following by the fusion of the multivesicular body (muhivesicular body, MVB) with the cell membrane, which are goblet cell membrane microvesicles, the diameter of which is about30nm to90nm. The complex composition of exosomes includes variety of RNAs, lipids and proteins. Exosomes could be released by a variety of tumor cells, including dendritic cells, intestinal epithelial cells and so on[9]. There are a serious of functions of exosomes, inducing induction of immune tolerance, discharge of the cell garbage and information transmittion. Tumor-derived exosomes may be existed in plasma, urine, saliva, milk, bronchoalveolar lavage fluid and other types of body fluids[9]. Exosomes plays an important role in physiological and pathological states. Exosomes can be used as a new tumor marker, providing a new approach for the treatment of cancer biology[10].In recent years, early research of cisplatin-based chemotherapy were focused on genes of drug resistance and molecular mechanisms of drug resistance, while the cell microenvironment was proposed now. Some researchers show that exosomes play a role in drug resistance[11-12]. Our research selected exosomes as the effect factor of cell microenvironment study to prove that exosomes regulate the sensitivity of cancer cells to cisplatin preliminary.The findings will be described in three parts as follows. Part Ⅰ:Extraction and identification of lung cancer A549cells derived exosomesObjective:Exosomes are released into the extracellular followed by microvesicles intracellular multivesicular bodies (MVBs) fusion with the cell membrane, which are goblet membrane nanoscale. A variety of cells are found to secrete exosomes, such as renal cancer cells, dendritic cells, B cells, intestinal epithelial cells and so on. Exosomes can be existed in various forms of body fluids, such as blood, urine, saliva, milk and so on[9]. The methods of separation and purification of exosomes are mainly ultracentrifugation, sucrose density gradient centrifugation and immunomagnetic bead extraction[9]. Current research on lung cancer cells secrete exosomes is not much. In order to further study exosomes secreted by lung cancer cells, A549cells-derived exosomes were extracted, isolated, purified and identificated.Methods:The method of the combination of ultracentrifugation and ExoQuick-TC was used to extract exosomes of culture supernatant of A549cells; Transmission electron microscopy (TEM) was used to observe the morphology and diameter size distribution of exosomes; Western blot was selected to detect CD63protein expression in the exosomes.Results:Exosomes can be secreted by lung cancer A549cells, the diameter of which is about30to100nm. Exosomes are micromembrane vesicles, which show high expression of CD63protein.Conclusion:Lung cancer A549cells derived-exosomes could be extracted by the method of the combination of ultracentrifugation and ExoQuick-TC. Part Ⅱ:The role of exosomes in the regulation of the sensitivity of lung cancer cells to cisplatinObjective:The late research shows that exosomes can influence the biological function of the cells by mediating informations between cells, including angiogenesis, proliferation, invasion, metastasis, and drug resistance of tumor cells[13-17]. The purpose of this part of the study was to demonstrate that A549cells treated with cisplatin can secrete more exosomes and exosomes secreted by A549cells during cisplatin treatment could decrease the sensitivity of lung cancer cells to cisplatin. This study could provide a new idea for the treatment of lung cancer.Methods:CCK-8assay was used to detected the inhibition of cisplatin on A549cells and the IC50; the electron microscope was used to observe the morphological changes of A549cells treated with cisplatin; the exosomes were quantified by BCA protein quantification; the process of the exosomes swallowed by A549cells were observed with electron microscope; the exosomes of A549cells were extracted before and after cisplatin treatment and after A549cells were pretreated with the two kinds of exosomes, CCK-8was used to detect the cisplatin inhibition of cells, as well as, cell morphology was observed by an electron microscope.Results:After the cells were treated with cisplatin, the cell viability decreased and the cell morphology was changed, the IC50was about3μg/mL.The average amount of exosomes secreted by each cells increases.When co-cultured with cells, exosomes could enter the cell; with exosomes pretreatment, the IC50of A549cells to cisplatin increased and the morphology of corresponding cell also changed.Conclusion:Reduction the secretion of exosomes and the information transmission between cells may improve the efficacy of cisplatin, which provides a new idea for the study of treatment of lung cancer. Part Ⅲ:The mechanism of sensitivity regulation of lung cancer cells to cisplatinObjective:Studies have reported that exosomes mediating RNA could participate in the information transmission, which affects the biological function of the receptor cells[10]. The main part of the study was to investigate the effect of exosomes on the sensitivity regulation of lung cancer cells to cisplatin at the level of RNA. As to RNAs, we mainly chose those miRNAs and mRNAs associated with cisplatin resistance, and those related with other common chemotherapy drugs were also studied.Methods:After A549cells were treated with cisplatin, RNAs were extracted from the cells and and exosomes. RT-PCR was used to detect the expression levels of six miRNAs (miR-21, miR-98, miR-133b, miR-138by, miR-181a, miR-200c). The correlation analysis between the expression levels of miRNAs in cells and exosomes was done. After A549cells were pretreated with exosomes, RNAs were extracted and the intracellular expression levels of six miRNAs and mRNAs (ERCC1, BRCA1, RRM1) associated with cisplatin were detected by RT-PCR, while the expression levels of RNAs associated with other chemotherapy drugs (TUBB3, STMN1, TYMS, TOP2A, EGFR, PDGFR, ERBB2, IGFLR, KIT) were also detected. After A549cells were treated with cisplatin follow by pretreatment with exosomes for3h, intracellular RNAs were extracted, expression levels of six miRNAs and mRNAs were detected by RT-PCR.Results:After A549cells were treated with cisplatin, the expression levels of miRNAs in cells are changed and the expression levels of exosomal miRNAs are also changed. The change of expressions of exosomal miRNAs is greater than the change in cells. After the exosomes were embraced by A549cells, the expression levels of miRNAs in cells change, while the expression levels of mRNAs associated with cisplatin also change. After A549cells were treated with cisplatin follow by pretreatment with exosomes for3h, expression levels of six miRNAs and mRNAs in cells are changed.Conclusion:Exosomal RNAs might be mediated between cells resulting in reducing the sensitivity of A549cells to cisplatin, which provides a new way for the further study of the mechanisms of lung cancer resistance and also provides a new method for the treatment of lung cancer.