Mechanisms On Nasopharyngeal Carcinoma Invasion And Metastasis Inhibited By CXCR7 Interference And Its Proliferation Inhibited By Plumbagin Via ROS

To better elaborate our research,this thesis is divided into two parts.In the first part,we studied on the invasion and metastasis inhibition of human nasopharyngeal carcinoma by CXCR7 interference.In the second part,we studied mechanisms on inhibition of nasopharyngeal carcinoma cell proliferation by plumbagin,the Chinese herb monomer,via ROS.Part 1 : Effects of inhibiting CXCR7 gene expression on the invasion and metastasis of human nasopharyngeal carcinoma BackgroundNasopharyngeal carcinoma is one of the most common head and neck malignant tumors in our country.Characteristic of poor differentiation,high malignant degree,and high metastasis tendency,nasopharyngeal carcinoma seriously endangers the life of suffering patients.Although many treatment methods exist,no one is perfect.Currently,it is still not optimistic for nasopharyngeal cancer treatment with advanced metastasis.Therefore,elucidating the invasion and metastasis mechanisms of nasopharyngeal carcinoma is one of the important factors for the effective treatment of nasopharyngeal carcinoma.As a new discovered receptor of SDF-1 in recent years,Chemokine receptor7(CXCR7)is not only involved in the regulation of a variety of biological processes,such as embryonic development,chemotaxis and stem cell homing,but also modulates tumor behavior.Many studies have discovered that the expression of CXCR7 in tumor tissues is higher than that in normal tissues,and downregulation of CXCR7 not only inhibits the growth of hepatocellular carcinoma and breast cancer cells,but also prevents the migration or invasion of ovarian cancer cells and colon cancer cells,However,it is less understood how CXCR7 affects the proliferation,apoptosis,migration and other biological behaviors in nasopharyngeal carcinoma cells.In this research,we intended to collect clinical samples and confirmed the distribution of CXCR7 in nasopharyngeal carcinoma tissues by immunohistochemical method.Besides,we revealed the correlation between CXCR7 and clinical stage and prognosis of nasopharyngeal carcinomare.Then we investigated the function and mechanism ofCXCR7 in the regulation of invasion and metastasis of nasopharyngeal carcinoma.ObjectiveTo verify the efficacy and explore the mechanism of CXCR7 on the invasion and metastasis of nasopharyngeal carcinoma(NPC).To lay the foundation for further researches on CXCR7-targeting molecular therapy and transformation application.Methods1.Immunohistochemical method was used to detect the expression of CXCR7 in nasopharyngeal carcinoma.The relationship between CXCR7 expression and the clinical stage,distant metastasis was analyzed by chi square test.2.By using RNA interference technology,CXCR7-sh RNA lentiviral vector was constructed and used to infect CNE2 human nasopharyngeal carcinoma cells.Besides the experimental group with CXCR7-sh RNA lentiviral vector,two control groups were set up: the negative control group with empty lentiviral vector and the blank control group without any treatment.Protein expression of CXCR7 in cells from all three groups was examined by western blot.3.Proliferation and invasiveness of CNE2 cells were measured by MTT assay and Transwell chamber test,respectively.At the same time,the expression levels of AKT,P-AKT and MMP7 protein in each group were detected by western blot.Results1.Immunohistochemistry result showed that CXCR7 was highly expressed in nasopharyngeal carcinoma,and the clinical stage and distant metastasis rate of CXCR7positive(+)patients were much higher than those of CXCR7 negative(-)patients(P<0.05).2.The expression level of CXCR7 protein of CNE2 cells in the experimental group was significantly lower than those in the negative control group and blank control group(P<0.01).3.MTT result showed that,the growth rate of CNE2 cells in the experimental group was significantly lower than those in the negative control group and blank control group(P<0.05).Transwell test result showed that,the number of cells who crossed over Matrigel in experimental group was significantly less than those in the negative controlgroup and the blank control group(P<0.01).In addition,western blot result showed that the expression level of P-AKT and MMP7 in experimental group was significantly lower than those in control groups(P<0.01).ConclusionThe expression of CXCR7 is closely related with the clinical stage and distant metastasis of nasopharyngeal carcinoma;CXCR7 inference can inhibit the proliferation and invasiveness of nasopharyngeal carcinoma cells in vitro.The underlying mechanism involves AKT activation and MMP7 expression regulated by CXCR7.Part 2 : Mechanism of plumbagin in inhibiting the proliferation of Nasopharyngeal carcinoma cell via ROS BackgroundAs a head and neck cancer,nasopharyngeal carcinoma is sensitive to chemotherapy.Chemotherapy is crucial to patient who is intolerant of radiotherapy and surgery.However,current commonly used chemotherapeutic drugs are not ideal.Plumbagin is the active ingredient of plumbago,a common Chinese folk herb with multiple pharmacological effects.Studies have shown that plumbagin has strong anti-tumor effect in lung cancer,liver cancer,breast cancer.However the effects and mechanism of plumbagin on human nasopharyngeal carcinoma up to now is not clear.ROS(Reactive oxygen species),a key redox signaling molecule in the cell,not only plays a role in tumor induction,but also exhibits anti-tumor effect.Under normal circumstances,ROS is maintained within a stable range in human body and plays active roles in anti-inflammatory,antibacterial and other aspects.When this balance is disrupted,ascending ROS level in the cell promotes cell transformation and results in malignant occurrence.In recent years,with in-depth understanding of the biological function of ROS,its role in killing tumor cell has gradually been recognized.At present,many traditional chemotherapeutic drugs have been found to induce anti-tumor activity by inducing ROS accumulation.However,the role of ROS in the treatment of NPC by plumbgain has not been reported.ObjectiveTo observe the effects of plumbagin on proliferation of human nasopharyngeal carcinoma,and to investigate the mechanism of ROS in nasopharyngeal carcinoma cell cycle blocking by plumbagin,laying a good experimental foundation for clinical application of plumbagin against nasopharyngeal carcinoma.MethodsHuman nasopharyngeal carcinoma cell lines 6-10 B was used in the study.Cell proliferation of nasopharyngeal carcinoma cells with different dose of plumbagin or NAC,a scavenger of ROS,was examined by MTT assay.ROS generation was detected by DCFH-DA probe.Cell cycle of 6-10 B with plumbagin/ROS was measured by flow cytometry.Cell cycle related Aurora A,Confilin,P-Confilin(ser3),P-GSK3?(ser9)and nuclear transcription factor STAT3(total protein and phosphorylated form)were detected by western blot.ResultsOur results showed that plumbagin inhibited remarkably the proliferation of nasopharyngeal carcinoma cells in a dose-dependent manner.ROS level increased significantly in 6-10 B cells treated with plumbagin.NAC reversed the increase of ROS in 6-10 B cells and countbalanced the proliferation inhibition of nasopharyngeal carcinoma cells by plumbagin.Plumbagin led to G2/M cycle arrest in 6-10 B cells,with expressions of P-GSK3?(ser9)upregulated and cell cycle related Aurora A?Confilin?P-Confilin(ser3)and STAT3 downregulated,and phosphorylated STAT3 inhibited.Meanwhile,the scavenging of ROS blocked G2/M arrest and inhibit the downregulation of the above cycle associated protein induced by plumbagin.ConclusionPlumbagin inhibits the proliferation of nasopharyngeal carcinoma cells by arresting cells in G2/M phase via mechanisms including GSK3?/STAT3 signal and the downstream of cycle associated protein inhibition caused by ROS upregulation.