The Role And Mechanism Of CX3CL1 In Spinal Metastasis Of Non-small Cell Lung Cancer

Background and objectiveAt present,lung cancer is a kind of malignant tumor with the highest morbidity and mortality in the world.Among all the diagnosed cancer patients,11.6% are lung cancer patients.Meanwhile,due to environmental pollution and smoking,the incidence of lung cancer is still rising.In 2018,the world's new cases of lung cancer reached a staggering 2.1 million people,about 1.8 million people died of lung cancer accounting for 18.4 percent of all cancer deaths.Due to the deepening of lung cancer research,clinical diagnosis and treatment of lung cancer have been improved,but the complications of lung cancer are still difficult to control,the death rate has been high.Currently,the clinical classification of lung cancer is mainly based on different cell sources.Non-small cell lung cancer(NSCLC)accounts for over 80%,and the rest is small cell lung cancer(SCLC).Adenocarcinoma is the most common type of non-small cell lung cancer,followed by squamous cell carcinoma and large cell lung cancer.NSCLC accounts for the vast majority of lung cancer patients,and bone metastasis of lung cancer is also common in NSCLC patients,so that all tumor samples selected in this experiment are NSCLC.Complications caused by metastasis of lung cancer are one of the main causes of death of patients.Not only metastatic lesions will occur in the middle and advanced stage of lung cancer,but some patients with early lung cancer will also have a high proportion of recurrence and metastasis even after radical treatment.Bone is one of the most common metastatic sites of lung cancer,and about 30% of lung cancer patients will eventually develop bone metastasis.Once bone metastasis occurs,the patient's quality of life will be seriously affected.In patients with bone metastasis,spine metastasis is the most common,the proportion can reach more than 50%.Lung cancer spinal metastasis in the pathological manifestations of bone lysis destruction,often accompanied by vertebral collapse,fracture,bone pain,severe paralysis can occur,greatly reduce the patient's survival time which bring great pain to the patients.At present,the mechanism of the occurrence,invasion and metastasis of lung cancer is not clear.According to the existing experience,it can be considered that the occurrence of lung cancer and bone metastasis is a multi-factor,multi-step process related to a variety of genes.First of all,oncogene activation or tumor suppressor gene mutation leads to the increase of abnormal cells.However,once the immune function of the body is low,the corresponding immune monitoring function cannot be performed,and the mutated cells in the body cannot be identified in time,so as to eliminate the mutant cells in the body and curb the occurrence of tumors.After the formation of the tumor,through epithelial mesenchymal transformation,the primary tumor lesion falls off into the blood and forms circulating tumor cells,which flow to bone tissue with the blood.With the disorder of the interaction between osteoblasts and osteoclasts,the bone microenvironment is severely damaged,laying a foundation for the proliferation of tumor cells in the metastatic lesion and eventually forming the bone metastatic lesion.Chemokine ligands,a class of small molecule chemokines,play an important role in inflammatory response and have been verified by more and more experiments in the process of tumorigenesis.Chemokines can be classified according to the different residues between the initial cysteine pairs.CX3 chemokine ligand 1(CX3CL1)is the only member of the chemokine CX3 C family,which can be divided into membrane-bound and soluble forms,giving CX3CL1 complex characteristics.In addition to playing an important role in inflammation,diabetes and other diseases,CX3CL1 has been proved to be highly expressed in glioma,breast cancer,renal clear cell carcinoma and other tumors,and can promote the proliferation,invasion and metastasis of corresponding tumors.However,the role and mechanism of CX3CL1 in lung cancer and bone metastasis are still unclear.We are aimed at studying CX3CL1 at the molecular,in vitro,and in vivo levels to reveal the molecular mechanism of spinal metastasis in NSCLC,so as to provide theoretical basis for clinical targeted therapy and provide new ideas and methods for clinical treatment of spinal metastasis in NSCLC.Research contents and methods1.Clinical sample and case selection:Firstly,eligible patients with lung cancer and spinal metastasis who underwent surgery in our hospital were included in the study.50 patients with primary lung cancer and 35 patients with NSCLLC spinal metastasis were selected.The expression of CX3CL1 in spinal metastasis and primary lung cancer was detected by immunohistochemistry and Western Blot.2.Molecular and cytological studies:In the previous experiment,we used A549 cell line to inject BCLB/c nude mice through the left ventricle,and selected spinal metastasis for isolation and culture.After six times of screening,we obtained A549L6 cells with high spinal metastasis in lung cancer.A549L6 cell lines with overexpression and knockdown of CX3CL1 were constructed by lentivirus infection.Transwell,cell adhesion,and cck8 assay were used to detect the effect of CX3CL1 intervention on the tumor properties of lung cancer cells.3.In vivo studies: The ability of metastasis,proliferation and tumorigenesis of lung cancer cell line induced by CX3CL1 knockout was tested in vivo using mouse tumor metastasis model(left ventricular tumor injection model).4.Studies on the regulation of bone metabolism: The osteoclast differentiation experiment was used to study the bone destruction mechanism mediated by CX3CL1.5.CX3CL1 regulation of downstream gene expression and signaling pathways in lung cancer cells: Finally,the expression of downstream genes of CX3CL1 was detected by transcriptome sequencing.Results1.We found significant differences in CX3CL1 expression between spinal metastasis and primary lung cancer.A549L6-sh cell lines with CX3CL1 knockdown were successfully constructed by infecting A549L6 cell lines with CX3CL1 shRNA lentivirus.2.By using mouse tumor metastasis model(left ventricular tumor injection model),we found that compared with control cells,the tumorigenic ability and metastasis ability of A549L6-sh cells were significantly reduced.3.By intervening the expression of CX3CL1 in target cells,we found that CX3CL1 could promote the adhesion and invasion ability of A549L6 cells,but had no significant effect on the proliferation ability.4.We added CX3CL1 protein into the osteoclast differentiation study system,and found that CX3CL1 can significantly promote the differentiation of osteoclasts.5.By transcriptome sequencing,we found that CX3CL1 may promote the expression of downstream genes,such as SERPIND1 and ALDH1A1,after binding with its specific receptor CX3CR1.ConclusionBased on the above experiments,we think that CX3CL1 may play an important role in spinal metastasis of lung cancer.CX3CL1 can promote the invasion and metastasis of lung cancer cells induce osteoclast differentiation to break the balance of bone microenvironment and mediate the bone destruction caused by bone metastasis of lung cancer.This study provides a new theoretical basis for the molecular mechanism of spinal metastasis and bone destruction in lung cancer.