| Foreword:Nasopharyngeal cancer(NPC)is a malignant tumor derived from nasopharyngeal epithelium.It is ranked 24 th in common cancers according to its incidence.70% of new cases of nasopharyngeal cancer occur in East and Southeast Asia,especially Southern China,such as Guangdong,Guangxi,Hunan,Fujian,Jiangxi,and Hong Kong,is a high incidence area for nasopharyngeal cancer.The incidence of males is generally 2-3 times higher than that of females.The age group with high incidence is 40-50 years old.Because the malignant tumor of the patient's nasopharynx is relatively hidden,the early clinical symptoms are not typical.About 70% of the patients were already stage III or IV when they were diagnosed.In addition,most patients with nasopharyngeal carcinoma belong to poorly differentiated or incompletely differentiated squamous cell carcinoma,with a high degree of malignancy,and prone to distant tumor metastasis outside the nasopharynx.The NCCN guidelines recommend concurrent chemoradiotherapy and adjuvant chemoradiotherapy or concurrent chemotherapy followed by induction chemotherapy as a treatment.According to statistics,almost 98% of NPCs are closely related to EBV,in which EBV latent membrane protein 1(LMP1)has been proved to play an important role in the process of cell differentiation to control and promote the occurrence and development of nasopharyngeal carcinoma.It can directly transmit cell pathways through various signals in nasopharyngeal cancer cells,such as TRAFs & NF-?B,PI3K-A kt/PK B,MAPK,transporter PRA1,etc.,regulate and control the normal proliferation and growth of nasopharyngeal cancer cells,Migration,differentiation,immunity and apoptosis,thereby controlling the occurrence and development of cancerous cells.The latest research results of nasopharyngeal carcinoma of LMP1 show that miR-155 can promote the proliferation,migration and invasion of nasopharyngeal carcinoma cells in vitro,but the effect on radiotherapy of nasopharyngeal carcinoma cells is still unclear;and the high mobility group protein B1(HMGB1)was recently found to be overexpressed in human nasopharyngeal carcinoma tissues,and its expression level is related to the patient's overall survival and disease-free survival,and the mechanism of promoting HMGB1 and the role of promoting HMGB1 in NPC are Little is known;in addition,circulating tumor cells(CTC)and EBV DNA and RNA as a promising biomarker for nasopharyngeal cancer are also constantly being valued,but the relationship between these several markers and the metastasis of nasopharyngeal cancer No research.Object:In order to further explore the molecular mechanism of EBV LMP1 and its related factors miR-155,HMGB1 in the occurrence and development of nasopharyngeal carcinoma,analyze the role of EBV DNA and RNA,CTC in the diagnosis of nasopharyngeal carcinoma metastasis,look for gene therapy and judge the nose Molecular target of pharyngeal cancer prognosis.We first designed and constructed a stable nasopharyngeal carcinoma cell line transfected with LMP1 eukaryotic expression vector,and evaluated the effects of LMP1 on the proliferation and migration of nasopharyngeal carcinoma cells.By detecting the expression of miR-155 in nasopharyngeal carcinoma cells transfected with LMP1 and the sensitivity of miR-55 gene suppression to nasopharyngeal carcinoma cell radiotherapy,the miR-155 was evaluated for the proliferation,migration,invasion and in vitro radiotherapy of nasopharyngeal carcinoma cells Impact,to explore the correlation between miR-155 and LMP1.By detecting HMGB1 in human nasopharyngeal carcinoma specimens and EBV-infected nasopharyngeal carcinoma cells,the promotion of LMP1 on HMGB1 and the regulation of HMGB1 on the proliferation of nasopharyngeal carcinoma cells in vitro were evaluated.The levels of EBV DNA and RNA(LMP1,LMP2,BART,and EBER1)in the plasma of patients with nasopharyngeal carcinoma were detected,and the relationship between EBV DNA,RNA and CTC and tumor metastasis in CTC cells in the peripheral blood of patients was evaluated.Method:(1)Construct a recombinant LMP1 expression vector and transfect CNE-2 cells,and then observe the transfection rate and changes in LMP1 transcription and protein expression levels by real-time quantitative PCR and Western blotting;transfection of pEGFP-LMP1 with CNE-2 Cloning of nasopharyngeal carcinoma cells,CCK8 detection of cell activity,cell migration ability by cell scratch test;cell invasion ability by cell invasion test;quantitative analysis of protein expression of HMGB1 and P65 by Western blotting method;afterwards,qPCR was used to determine LMP1 Transcription level of miR-155 in expressed CNE-2 cells;transfect CNE-2 and HONE1 cells with miR-155 mimic,and conduct cell invasion experiments to determine the effect of miR-155 on nasopharyngeal carcinoma invasion;use RNeasy Mini kit Isolate and extract total cell mRNA from CNE-2 cells,use mirVana? miRNA isolation kit to extract miRNA in CNE-2 cells;CCK8 detects miR-155 mimics proliferation after transfection of CNE-2 cells,and uses miR-Cell proliferation after 155 inhibitor;determine the survival rate of normal CNE-2 cells or CNE-2(LMP1)cells after miR-155 inhibition under radiotherapy.(2)Following the principle of being informed,84 patients with nasopharyngeal carcinoma were biopsied,52 patients were positive for EBV and 32 were negative for EBV,and the complete clinical data before radiotherapy or chemotherapy were recorded in these patients.These samples were tested for HMGB1 expression and its relationship with LMP1 DNA levels.Afterwards,the transfer of EBV-infected CNE-2 cells from the nucleus to the cytoplasm and the release of HMGB1 were evaluated,and the effect of HMGB1 on the proliferation of CNE-2 cells and the effect of RAGE-specific siRNA transfection on the proliferation of CNE-2 cells were determined.(3)Collect data of 250 patients with nasopharyngeal carcinoma,including 136 cases with metastasis and 114 cases without metastasis.Peripheral venous blood was taken before treatment,CTC was counted,and real-time quantitative PCR method was used to detect Plasma LMP1,LMP2,BART and EBER1 levels,to assess the relationship between DNA/RNA and tumor metastasis.Result:(1)The expression of LMP1 protein transfected with the recombinant vector was significantly higher than that of the control group 14 days after the pEGFP-LMP1 plasmid was transfected into CNE-2 cells.The pEGFP-LMP1 eukaryotic vector was successfully constructed;CCK8 detection showed that LMP1 overexpressed CNE-2 The cell proliferation activity was higher than that of the control group,with statistical difference;in the cell scratch experiment,the LMP1 overexpressing CNE-2 cells were greater than the control group at 12 h and 24 h time points,with statistical difference,P<0.01;cell invasion In the experiment,the number of CNE-2 cells overexpressing LMP1 across the membrane was significantly higher than that of the control group at 12 h and 24 h,with statistical difference,P<0.01;LMP1 overexpressing CNE-2 cell group HMGB1 and P65 protein expression Both were higher than the control group,and the single factor analysis of variance showed statistical differences,respectively: P=0.0058;P=0.02;comparing the transcription level of miR-155 between CNE-2 and HONE1,it was found that there was no significant statistical difference,P>0.05,but the transcription level of miR-155 in LMP1 overexpressing CNE-2 cells was 4.5 times that of CNE-2,P<0.001.Using miR-155 mimic to transfect CNE-2 and HONE1 cells,miR-155 transcription levels increased by 11.22 and 11.63 times,respectively.In the cell invasion experiment,miR-155 mimic was transfected with CNE-2 and HONE1 cells.After 24 h,the number of stained cells crossing the stromal membrane was significantly higher than the number of transmembrane cells of the transfected control group,with statistically significant differences,P<0.001.By CCK-8 detection,miR-155 simulated transfection can significantly promote the proliferation of CNE-2 cells,P<0.05.Compared with miR-Con cells,transfection of miR-155 inhibitors significantly reduced the level of miR-155 in CNE-2 cells P<0.05;P<0.01,cell proliferation also decreased significantly,P<0.05.After radiation treatment with 0 or 4.0 Gy,after transfection with miR-155 inhibitor,CNE-2(LMP1)cells formed fewer colonies than those transfected with miR-Con,P<0.01.It was more significant in CNE-2(LMP1))cells transfected with miR-155 inhibitors than in miR-Con cells,P<0.01.Regardless of exposure to 4.0 Gy radiation,miR-155 knockdown did not significantly reduce the number of colonies in CNE-2(Con)cells,P>0.05.(2)The HMGB1 protein level in the EBV positive group was also significantly higher than that in the EBV negative group(P<0.001).HMGB1 mRNA levels were significantly correlated with LMP1 DNA levels in 52 EBV positive human samples,R2=0.5640,P<0.0001.HMGB1 in the supernatant of CNE-2 cells infected with EBV also increased significantly within 12 to 48 hours after infection(P<0.05,P<0.01,or P<0.001),which was greater than 0.5 mg/mL HMGB1 treatment Promote the proliferation of CNE-2 cells(P<0.05 or P<0.01 of 0.5,1,3 mg/mL HMGB1),and there is a dose-dependent(P<0.05).This effect of promoting cell proliferation is also time-dependent(36 or 48 hours after treatment,P<0.05 or P<0.01).After 60 nM transfection of siRNA-RAGE,HMGB1 significantly inhibited the proliferation of CNE-2 cells(P<0.05 or P<0.01 after 36 or 48 h).(3)The average CTC value of the transfer group was significantly higher than that of the non-transfer group,P<0.0001.The plasma EBV DNA and RNA levels of patients with metastatic nasopharyngeal carcinoma were significantly higher than those of non-metastatic nasopharyngeal carcinoma.The relative levels of CTC,LMP1 DNA,BART or EBER1 were significantly correlated with tumor stage and nodule stage(R2> 0.25,regardless of tumor stage or lymph node stage,four biomarkers).Conclusion:(1)The recombinant plasmid of pEGFP-LMP1 can effectively transfect CNE-2 cells and be stably expressed in CNE-2 cells.EBV LMP1 plays an important role in the occurrence and development of nasopharyngeal carcinoma.It may promote the proliferation,invasion and migration of nasopharyngeal carcinoma by activating the HMGB1/NF-kb signaling pathway.miR-155 is associated with EBV-positive nasopharyngeal carcinoma,and its upregulation is associated with LMP1,while miR-155 can promote the proliferation,migration and invasion of nasopharyngeal carcinoma cells in vitro,and inhibition of miR-155 can make nasopharyngeal carcinoma cells more effective for radiotherapy sensitive.(2)HMGB1 is significantly increased in nasopharyngeal carcinoma tissues in vivo or in vitro.HMGB1 is associated with the malignant state of nasopharyngeal carcinoma.The high expression of LMP1 caused by EBV infection promotes the proliferation of nasopharyngeal carcinoma cells in vitro through HMGB1/RAGE signaling.(3)The plasma DNA and RNA levels of patients with EBV-infected nasopharyngeal carcinoma were significantly increased.LMP1 DNA and EBER1 RNA are associated with nasopharyngeal carcinoma metastasis. |
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