| The research of tumor microenvironment has shifted people’s attention from the tumor parenchymal cells to the niche where tumor cells localized,pointing out new directions and strategies for cancer treatment and research.The tumor microenvironment contains not only extracellular matrix(ECM)of non-cellular components,growth factors,chemokines and inflammatory factors,but also many different types of cellular components,such as mesenchymal stem cells(MCS),differentiated fibroblasts,macrophages,endothelial cells,adipocytes,etc.Among them,mesenchymal stem cells are a type of adult stem cells with multi-directional differentiation ability and the ability to specifically migrate to damaged tissues,chronic inflammation sites.Mesenchymal stem cells have broad application prospects in tissue engineering and cell therapy.However,there were also increasing evidences that MSC,under the stimulation and induction of tumor cells,could activate and differentiate into cancer-associated fibroblasts(CAF)and promote the development of cancer.In-depth understanding of the interaction between MSC and tumor cells can not only present new targets for cancer treatment,but also use tissue engineering and gene therapy techniques to transform the "dark side" of MSC into the beneficial side for human health.Lung cancer is one of the most malignant tumors in China and the world.Despite the advancement of treatment technology,lung cancer still accounts for a very high proportion of cancer-related deaths.The development of lung cancer is accompanied with abundant vascularization and fibrosis,suggesting the enrichment of MSCs in lung cancer tissue,as MSCs usually localized in the perivascular region.The purpose of this study is to explore the interactions between lung cancer MSC and tumor cells and the downstream mechanism,which will enhance our understanding of the targeted treatment of lung cancer.In this study,we firstly isolated MSC from human lung cancer tissues and verified their characteristics via fibroblastic morphology,cell surface markers,the ability of multi-directional differentiation.The results showed that the isolated adherent mesenchymal cells had a uniform morphology and grew in spiral shape,similar to fibroblasts.They do not express the markers of endothelial cell,hematopoietic cells,macrophages,but express markers of mesenchymal cell.The results of immunofluorescence assays showed that these isolated mesenchymal cells expressed mesenchymal markers but not epithelial markers.Under specific induction,these cells have the ability to differentiate into adipocytes and osteoblasts.Therefore,it can be verified that the isolated cells are MSCs.Furthermore,we explored the effect of lung cancer MSC on lung cancer cells through nude mice subcutaneous co-transplantation tumor formation experiment.The experimental results showed that lung cancer MSC significantly promoted lung cancer in vivo tumorigenicity.Then we explored the miRNA changes of lung cancer cells after co-cultured with lung cancer associated MSCs through miRNA chips.We found a variety of inhibitory miRNA were downregulated,of which miR-194-5p caused our research interest,because of it is significantly down-regulated after co-cultured.Furthermore,we transfected lung cancer cells with lentivirus and mimics to make lung cancer cells overexpress or inhibit the expression of miR-194-5p to conduct functional studies.The results showed that miR-194-5p can significantly inhibit the proliferation and invasion ability of lung cancer cells.Through bioinformatics technology,we predicted that the downstream target gene of miR-194-5p was LIN28 B,and it was verified by Western blot technology.Further we used cytokine chips to try to find out the mechanism of lung cancer MSC regulate the miRNA changes of lung cancer cells,the results show that lung cancer MSC can secrete b FGF to down-regulate the expression of miR-194-5p in lung cancer cells to promote the proliferation of lung cancer cells.However,the specific molecular mechanism of how b FGF regulates miR-194 expression changes is still in progress.Through the above research,our subject identified the presence of lung cancer associated MSC in lung cancer tissues,and it promoted the proliferation of lung cancer cells.Further research showed that the lung cancer MSCs can regulate lung cancer cells by secreting b FGF and regulate the expression of miR-194-5p-LIN28 B axis,leading to the development of lung cancer.This research explores the interaction between lung cancer MSC and lung cancer cells and its preliminary mechanism from the perspective of tumor microenvironment,and is expected to deepen the understanding of the lung cancer microenvironment in regulating the development of lung cancer.Intrahepatic cholangiocarcinoma(ICC)is a primary tumor originating from the intrahepatic bile duct epithelial cells and it is the second most common primary liver tumor.Therefore,exploring the molecular mechanism of ICC development is of important clinical significance.This thesis is consisting of the following two parts:Graphene is a new type of carbon nanomaterial with one-atom-thick hybrid.Recently its unique physical and chemical property has attracted the attention of scientists from all over the world.In the field of biology and medicine,graphene oxide(GO),a derivative of graphene,is characterized by its good biocompatibility,large specific surface area and easy surface modification,have made a greater application in drug delivery,biological imaging,biosensors,antibacterial materials,cancer diagnosis and treatment.GO has the advantages of simple preparation,low cost,and easily largescale production.However,GO cannot effectively adsorb double-stranded DNA or RNA,which greatly limits its application in gene transfection field.In this study,we grafted positively charged Polyethyleneimine(PEI)onto GO,so the surface of GO could electrostatically adsorb the negative RNA,therefore,we want to explore whether the functionalized GO-PEI can be a novel efficient low-toxicity,low-cost gene delivery system including RNA.The abnormal expression of miRNA is closely related to the occurrence and development of various tumors,including ICC.And the analysis of miRNA expression is important for the early diagnosis,treatment and prognosis of ICC.The use of functional GO-PEI to carry chemotherapy drug or si RNA to inhibit tumor growth and invasion has been reported by our collaborators.However,the use of PEIGO as a carrier of multiple repressive miRNAs to inhibit the proliferation of ICC cells has not been reported.Through high throughput miRNA microarrays analysis and the TCGA of human ICC bulk sequencing data,we revealed some down-regulated miRNAs in ICC tissues compared with peri-tumor tissues,including miR-122-5p,miR-194-5p,let-7c-5p and miR-125b-5p.Further in vitro and in vivo functional experiments showed that GO-PEI could efficiently carry these repressive miRNAs into tumor cells and further inhibit the proliferation of ICC cells and tumor spheres and enhanced the sensitivity to sorafenib treatment.Finally,we demonstrated that GO-PEI could efficiently inhibit the ICC proliferation in vivo.In addition,the combination of drug treatment is a main strategy for clinical tumor treatment.We tried to combine β-lapachone with Palbociclib to investigate whether the combination therapy could inhibit the proliferation of ICC cells and tumor spheres.This part of research focused on the development of novel nanocarrier of repressive miRNAs and combined drug treatment strategy to inhibit ICC cells. |
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