AKR1B10 Promotes Nasopharyngeal Carcinoma Development And Its Molecular Mechanism

Objective:Nasopharyngeal carcinoma(NPC)is one of the most common malignant tumors in Southern China and Southeast Asia,the incidence rate of nasopharyngeal carcinoma was 30?80/10 million per year,particularly in Guangdong and Hongkong.In clinic,high metastasis,high recurrence rate and low differentiation were capital features of nasopharyngeal carcinoma.Outpatients had nearly 88%of the NPC patients with cervical lymph node metastasis.Cervical lymph node metastasis is regarded as one of the significant features of nasopharyngeal carcinoma.Nasopharyngeal carcinoma development is a complicated process with multiple factors and steps,including EB virus infection and other non-viral factors(such as chemical carcinogen,nitrosocompounds).As reports,Aldo-keto reductase family 1 member B10(Aldo-keto reductase 1B10,AKRIB 10)play important roles in nasopharyngeal carcinoma development.But its exact molecular mechanism is still unclear.N,N'-Dinitrosopiperazine(DNP)is one of non-viral factors(such as chemical carcinogen,nitrosocompounds)for nasopharungeal carcinoma.The previous works showed that N,N'-Dinitrosopiperazine promoted nasopharyngeal carcinoma cell invasion and migration through activation of multiple signaling molecules,AKR1B10 is an important part of that.DNP can mediate the expression of AKR1B10 to participate in nasopharyngeal carcinoma development,but its mechanism is not clear.In this paper,at first using immunohistochemistry and ELISA,we analyzed the expression of AKR1B10 in nasopharyngeal carcinoma tissue and serum,clarified the relationship between AKR1B10 level in tissue and serum and the development and metastasis of nasopharyngeal carcinoma.And then using molecular cloning technique,gene transfection,gene interference technology,real-time PCR technology and Western Blotting technology et al.,we investigated that DNP can induce AKR1B10 high expression in nasopharyngeal carcinoma cells.At last,the experiments on cell function were conducted.Brieftly,using clone formation,transwell migration,and invasion assay,we detected AKR1B10 expression in DNP-mediated nasopharyngeal carcinoma metastasis,and then clarified the molecular mechanisms of DNP-involved in metastasis of nasopharyngeal carcinoma through AKR1B10.These provided a new serum screening marker for the target detection and diagnosis of nasopharyngeal carcinoma and metastasis,and provide valuable reference and basis for the screening of targeted therapeutic candidate molecules.Methods:(1)Immunohistochemistry was used to detect AKR1B10 protein expression in nasopharyngeal carcinoma patients with no lymph node metastasis and lymph node metastasis,and analyze on the relationship between AKR1B10 expression and the occurrence and development of nasopharyngeal carcinoma.(2)ELISA method was used to detect the expression of AKR1B10 in the serum of patients with metastatic nasopharyngeal carcinoma or unmetastasis,and the relationship between serum AKR1B10 level and metastasis of nasopharyngeal carcinoma was determined.(3)MTT method was used to detect the growth of 6-10B and 5-8F cells with DNP treatment at different concentrations,to analyze the non toxic concentrations(NCC)of DNP to 6-10B and 5-8F cell.(4)The difference of AKR1B10 protein expression was detected with or without DNP treatment(the control group),and the changes of cell behavior was observed in these groups.(5)Using molecular cloning techniques,we constructed the AKR1B10 expression vector pcDNA 3.1-AKR1B10.pcDNA 3.1-AKR1B10 was transfected into 6-10B cells,and the stable clones were screened.AKR1B10 protein expressions were detected in the control group and the transfection ones,and the cell behavior changes observed.(6)Western Blotting was used to detect AKR1B10 expression in 6-10B after DNP treatment.We clarified that DNP-mediated AKR1B10 expression promotes the invasion and migration of nasopharyngeal carcinoma cell.(7)AKR1B10 in 6-10B and 5-8F cells was silenced by siRNA interference,AKR1B10 expression was detected after DNP treatment,its invasion and migration were detected to clarified that AKR1B10 induced by DNP promotes the invasion and metastasis of nasopharyngeal carcinoma cells.(8)The migration and invasion ability of 6-10B and 5-8F cells with DNP treatment were detected,and the expression of AKR1B10 was also detected.Transwell migration and invasion assay and cloning formation were used to determine whether DNP can promote the migration and invasion of nasopharyngeal carcinoma cells.(9)SPSS 19.0 software and non parametric test were used to statistical analysis on experimental results and data.Results:(1)Immunohistochemical technique was used to detect 20 cases of normal nasopharyngeal epithelial tissues(NNET),20 cases of without lymph node metastasis of NPC(NPC,AJCC/UICC TNM 2009 stage),30 cases of lymph node metastasis of nasopharyngeal carcinoma(LMNPC).Results showed that the positive rate of AKR1B10 in NNET,NPC and LMNPC tissues were respectively 10.0%(2/20),85.0%(17/20),93.3%(28/30).AKR1B10 expression in nasopharyngeal carcinoma(NPC)was significantly higher than that in normal nasopharyngeal tissues(P<0.05)and LMNPC tissue expression higher than without metastasis NPC(P<0.05).These suggest that AKR1B10 increased expression was associated with nasopharyngeal carcinoma(NPC),upregulation expression is related to clinical development of NPC metastasis.(2)The detection of AKR1B10 are composed of two parts,and the relationship between serum AKR1B10 level and the metastasis of NPC is defined.The first part is to detect the serum levels of AKR1B10 expression in 30 cases of healthy control group,8 cases of chronic nasopharyngitis group,71 cases of primary NPC group(no staging).Results show that the median levels of AKR1B10 in healthy control,chronic nasopharyngitis group and primary NPC were respectively 62.73 ng/L?408.01 ng/L?603.52 ng/L.AKR1B10 expressions in nasopharyngeal carcinoma and chronic nasopharyngeal inflammation were increased,and the nasopharyngeal carcinoma group was significantly higher than that in healthy control group and chronic inflammation group(P<0.05).These suggest that AKR1B10 serological level was positively correlated with nasopharyngeal carcinoma.In the second part,70 cases of clear clinical stage(AJCC/UICC TNM 2009 staging)AKR1B10 in nasopharyngeal carcinoma(NPC)of serum specimens from patients were detected,of which no metastasis of NPC patients in 3 cases,lymph node metastasis of NPC patients were 59 cases,distant metastasis NPC patients in 8 cases.The serum median concentrations of AKR1B10 in the three groups were respectively 287.16 ngL?482.08 ng/L?5878.21 ng/L,and there was a significant difference between the three groups(P<0.05).AKR1B10 expression in the distant metastasis NPC was significantly higher than that without metastasis(P<0.05)cases.These suggest that the serum levels of AKR1B10 was associated with nasopharyngeal carcinoma development and metastasis,and it may be used as a serological screening marker for nasopharyngeal carcinoma and evalution of metastasis.(3)MTT assay results showed that in 0-100.0?mol/L,the growth of 6-10B cells had no significant effect(P>0.05),at 150?mol/L or more,DNP on 6-10B cell growth and proliferation was significantly inhibited(P<0.05).0?100?mol/L is a non toxic concentration of DNP to 6-10B cells.The toxicity concentration of DNP to the 5-8F cell was 0?150?mol/L.(4)AKR1B10 expression and cell colonies were detected in 6-10B with DNP treatment.The results showed that AKR1B10 expressions and cell colony numbers significantly increased after DNP treatment compared with the untreated group.(5)Used the application of molecular cloning technology,we successfully constructed AKR1B10 over expression vector pcDNA3.1-AKR1B10,which has effectively expression.pcDNA 3.1-AKR1B10 was transfected into 6-10B cells,and AKR1B10 was detected using Western-blotting.The results showed that the AKR1B10 protein expression was dramatically increased when compared to the control group.Western Blotting analysis of DNP-induced AKR1B10 expression showed that after 6-10B cells being treated with different concentrations of DNP,AKR1B10 expression was significantly increased(P<0.05),showing a time and dose-dependent manner.(6)Migration invasion ability was analyzed by Transwell migration and invasion assay in 6-10B and 5-8F cells with 100 ?M DNP treatment and RNAi-AKRIB10.The results showed that DNP induced 6-10B and 5-8F cell migration ability decreased after blocking AKR1B10(P<0.05),AKR1B10 played a significant role in DNP-mediated migration and invasion.(7)Cell immunofluorescence analysis showed that DNP mediated 6-10B and 5-8F cell AKR1B10 expression,protein mainly localized in the cell cytoplasm expression.Conclusion:(1)DNP enhances the invasion and metastasis of nasopharyngeal carcinoma cells through mediating the expression of AKR1B10.It is suggested that DNP as a chemical carcinogen may be one of the important factors for nasopharyngeal carcinoma metastasis.AKR1B10 may be an important signal molecule in nasopharyngeal carcinoma metastasis.(2)The AKR1B10 levels in tissue and serum showed that in the cervical lymph node metastasis and distant metastasis of nasopharyngeal carcinoma,AKR1B10 expression was dramatically increased when comared primary nasopharyngeal carcinoma tissues and normal nasopharyngeal tissues.This suggests that AKR1B10 expression is a candidated marker for nasopharyngeal carcinoma clinical development,metastasis,as early diagnosis.(3)Gene interference technology,molecular cloning technology,Real-time PCR technology and Western Blotting comfirmed that DNP can induce AKR1B10 high expression.(4)Cloning formation and Transwell migration and invasion assay data showed that AKR1B10 plays an important role in DNP-induced nasopharyngeal carcinoma metastasis,DNP-mediated ARR1B10 expression may be an important factor in nasopharyngeal carcinoma metastasis.