| Oral cancer is the general term for malignant tumors that occur in the mouth,and most of them are squamous cell carcinoma,the so-called mucosa heteromorphosis.In clinic,oral cancer includes cancers of the gums,tongue,soft and hard palate,jaw bone,the mouth floor,oropharynx,salivary gland,lip and maxillary sinus and cancer that occurs in facial skin mucous membrane,etc.Oral squamous cell carcinoma is one of the most common malignant tumors in the head-and-neck and is the eighth most malignant tumor in the world.The incidence of oral cancer in China is reported to be 3.29 cases per 100,000.Because of its special position,oral squamous cell carcinoma was not easily detected in the early phase.Once confirmed,five-year survival rate is low,which is mainly attributed to its few early detection and early prevention.Cancer cells were usually already transferred to cervical lymph node when the oral cancer was diagnosed.It is difficult to completely surgically removed in such patients,nevertheless,oral cancer is not sensitive to radiation and chemotherapy.Therefore,the prognosis of these patients are often not very optimistic.In order to improve the cure rate and survival rate of oral cancer patients,the study of head-and-neck squamous cell carcinoma invasion,lymph node metastasis and anti-apoptosis mechanism is needed.An important factor affecting the prognosis of tumor is whether the tumor metastases occur.Tumor metastasis is the process by which the tumor migrates from the primary site to other organs through the blood and lymph channels.Chemokine is a kind of protein with low molecular weight that enables white blood cell to migrate to inflammation region,which can combine with the relevant receptors,and makes the tumor cells migrate to the signal source along the increasing chemokine concentration,thus regulating tumor cell migration and invasion.CXCR4 is one of the chemokines.As the only receptor of CXCL12,CXCR4 is the most characteristic chemokine receptor that has been extensively researched.Although different studies have shown that CXCR4 is related to tumor metastasis,there are few studies on oral squamous cell carcinoma.Therefore,CXCR4 may be a new target for tumor therapy,and targeted intervention therapy for CXCR4 positive tumor tissues may become a new approach to the treatment of tumor.Muller A et al have applied relative antibodies to block CXCR4,while Liang Z have employed RNA silencing CXCR4 technology.Both strategies can significantly inhibit the metastasis of tumor cells.Using chemokine receptor antagonist,inhibitor to downregulate the expression of chemokines,could block the chemokine ligand and receptor signal pathways,and therefore inhibit tumor cell migration and invasion,proliferation,apoptosis,and other biological effects.Therefore,in order to investigate CXCL12-CXCR4 biological axis in the role of oral squamous cell carcinoma metastasis,we used the quantitative RT-PCR and immunohistochemical assay,protein immunoblotting to detect the expression of CXCR4 in oral squamous cell carcinoma tissue with or without lymph node metastasis in different stages,and analysis the relationship of CXCR4 expression and clinical pathological features of oral squamous cell carcinoma.At present,chemokines and corresponding receptors are found in more and more scientific studies,and they have the ability to promote tumor proliferation,invasion and migration.CXCL12 / CXCR4 biological axis,consists of stromal derived factor 1(SDF-1,also called CXCL12)and its receptor CXCR4,has been confirmed to play its vital role in directional migration,invasion and metastasis in breast cancer,malignant melanoma,sarcoma,prostate cancer and other malignant tumors.The invasion and metastasis of oral squamous carcinoma is also the result of the involvement and interaction of multiple genes and multiple factors.Therefore,to explore the molecular mechanism of human oral squamous cell proliferation and metastasis,and investigate effective therapeutic targets will be an important breakthrough in the treatment of patients with oral squamous carcinoma.Chemokines CXCL12 is a stromal cytokine derived factor that plays an crucial role in the growth,metastasis and angiogenesis of tumor cells.CXCR4 is the receptor of CXCL12.CXCR4 is a highly conserved 7 transmembrane G protein coupling receptor,and CXCL12-CXCR4 axis plays an important role in the proliferation,invasion and metastasis in many tumors.Using tongue cancer as the research object,this study is aimed to investigate the expression of CXCL12 / CXCR4 biological axis in normal mucosa,and tongue cancer,to further understand the relationship of CXCL12 / CXCR4 biological axis and the strongest CXCR4 inhibitor during the occurrence,development,invasion and metastasis of tongue cancer,which could provide new research direction for tongue cancer treatment and prognosis.In this study,the molecular mechanism of CXCR4 antagonist TN14003 on the modulation of CXCL12/CXCR4 biological axis in the growth,invasion and metastasis of tongue cancer cells was studied.The research is divided into three parts: The first part: we aimed at 75 cases with complete clinical data sample after head-and-neck surgery and biopsy from Tianjin tumor hospital in 2010-2016.The related factors influencing the tongue cancer postoperative survival rate were analyzed.Through the complete pathological data of oral squamous carcinoma specimens,the normal oral mucosal tissue and benign tumor tissues were used as control.The tumor tissue samples of oral squamous cell carcinoma were categorized according to the clinical pathological stage(Ⅰ,Ⅱ,Ⅲ,Ⅳ).According to the differentiation degree,samples can be divided into high and low two groups.According to the presence of lymph node metastasis,samples were divided into metastasis group and non-metastasis group.Total RNA and total protein of tumor tissues were extracted and qRT-PCR were used to detect the expression of CXCL12 and CXCR4 gene,while Western blotting were applied to investigate the expression levels of CXCL12 and CXCR4 protein.In addition,the expression of CXCL12 and CXCR4 was detected by immunohistochemical assay through the fixation,embedding and slicing of tumor and its adjacent tissues.The second part: The human oral squamous cell carcinoma was divided into the control,TN14003 high,middle and low concentration group at different time points.The specific antagonist TN14003 was used to block CXCR4.Cell proliferation was determined by MTT assay and colony formation.Flow cytometry AnnexinV/PI staining and TUNEL assay was applied to detect cell apoptosis,and Transwell assay was used to investigate cell migration and invasion.Western blotting was used to test the expression of CXCL12-CXCR4 and related signal pathway proteins.In the third part,human oral squamous carcinoma cells were transplanted in armpits of nude mice to establish xenograft model.Mice were divided into blank,control and TN14003 groups.The animals were executed after 4 weeks of administration,and the size of tumor tissue was detected.Western blotting and immunohistochemistry were used to detect the expression of CXCR4 in tumor tissues to verify whether this effect was achieved through changing the CXCL12-CXCR4 level.Results CXCR4 level was detected in the tumor tissues of different stages,and the expression of CXCR4 was significantly increased in phase Ⅱ,Ⅲ and Ⅳ,compared with phase Ⅰ(P<0.05).Compared with phase Ⅱ,Ⅲ,CXCR4 expression in phase Ⅲ and Ⅳ was significantly increased(P<0.05).Compared with phase Ⅲ,CXCR4 expression of phase Ⅳ was significantly increased(P<0.05).CXCR4 gene expression was detected in tumor tissues with metastasis and non-metastasis,and the expression of CXCR4 gene in tumor tissues was significantly increased compared with that of the benign control group(P<0.05).Compared with the metastatic tumor tissues,the expression of CXCR4 was significantly reduced in the non-metastatic tumor tissues(P<0.05).CXCR4 gene expression was also detected in lymph node metastasis and non-metastasis group,and compared with no lymphoid transfer group,the CXCR4 gene expression of lymphoid transfer group was significantly increased(P < 0.05).Flow cytometry results showed that the apoptosis rate increased significantly in high concentration group compared with control group(p < 0.05)after the treatment of TN14003 for 24 h,the cell proliferation of low concentration and high concentration group were increased significantly compared with the control group after the treatment of TN14003 for 48 h and 72 h(p < 0.05).Colony formation results showed cells in high concentration group were significantly suppressed compared with control after the treatment of TN14003 for 24 h,the cell proliferation rates in low concentration and high concentration groups were inhibited significantly compared with control after the treatment of TN14003 for 48 h and 72 h,respectively(p < 0.05).q-PCR and western blotting results showed that the expression of CXCR4 gene and protein in high concentration group was significantly reduced after the treatment of TN14003 for 24 h.The expression of CXCR4 gene and protein in low concentration and high concentration groups were inhibited significantly compared with control after the treatment of TN14003 for 48 h and 72 h(p < 0.05).There was no significant difference in the expression of CXCL12 among these groups.The growth of oral squamous cell carcinoma was significantly inhibited in nude mice treated with TN14003 for 4 weeks.The results of q-PCR and Western blotting showed that the expression of CXCR4 was significantly lower than that in the control group and the blank group(p<0.05)in the tumor tissues of the TN14003 treatment group.There was no significant difference in the expression of CXCL12 among the groups.Conclusions 1.In oral squamous cell carcinoma,CXCR4 is directly related to tumor staging.The expression of CXCR4 increases with tumor stages.CXCR4 was directly related to tumor metastasis,and CXCR4 expression in metastatic tumor tissues increased significantly.CXCR4 expression was significantly increased in lymphoid tissues of patients with tumor metastasis.2.TN14003 treatment can significantly inhibit the proliferation and invasion of oral squamous cell carcinoma cells and promote cell apoptosis.3.TN14003 treatment can significantly inhibit the growth of oral squamous cell carcinoma in vivo.4.The effect of TN14003 on oral squamous cell carcinoma in vivo and in vitro was achieved by inhibiting CXCR4 expression but not the inhibition of CXCL12 expression. |
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