Study On The Mechanism Of NF-?B Promoting The Occurrence And Development Of Lung Adenocarcinoma By Regulating ABCE1

Introduction Lung cancer is one of the most common lethal cancers in humans,with the highest incidence and mortality among many cancers.Due to the complicated etiology and metastasis of lung cancer,which poses a great threat to human health.From a pathological point of view,lung cancer can be divided into non-small-cell Lung Carcinoma and small-cell Carcinoma,and non-small-cell Lung Carcinoma can be further divided into squamous-cell Carcinoma,Adenocarcinoma and large cell carcinoma.Adenocarcinoma of the lung is the most common,accounting for about40% of all lung cancers.Although many advances have been made in the treatment of lung adenocarcinoma,lung adenocarcinoma remains one of the most aggressive and fatal tumor types,with an overall survival rate of less than 5 years.Therefore,it is of great significance to develop new targeted gene therapy and apply it to the clinic to improve the clinical treatment of lung adenocarcinoma.Smoking is a major environmental risk factor for lung cancer,and many genes have been reported to be involved in the development of lung adenocarcinoma.It has been reported that ABCE1(ATP-binding cassette transporter E1)is related to non-small-cell lung carcinoma,and its expression is related to the clinical stage of non-small-cell lung carcinoma.As a member of ABC transporter family,ABCE1 is mainly expressed in the cytoplasm.It is understood that the Ribonuclease L(RNase L)inhibitor protein can be encoded by the ABCE1 gene,because RNase L can combine with RNA and RNA degradation within the cell,thus Ribonuclease inhibitor L can through regulating 2-5amp/Ribonuclease L(2-5 a/RNase L)pathway suppressing RNA degradation and the purpose of inhibiting cell apoptosis.It has been confirmed that the high expression of ABCE1 can promote the growth and invasion of tumor cells,while inhibition of the expression of ABCE1 protein can reduce the vitality and proliferation of tumor cells,suggesting that ABCE1 is crucial to the progression of lung adenocarcinoma metastasis.Therefore,the study of ABCE1 may be important to reveal the molecular mechanism of human malignant tumor development.Epidemiological investigations and clinical studies have shown that there is a strong relationship between inflammation and the occurrence of tumors.Inflammation plays an important role in all stages of tumor evolution.Smoking and environmental pollutants have been found to damage DNA in bronchial epithelial cells and induce inflammation,which in turn contributes to the development of lung cancer.NF-?B(Nuclear factor-kappa light chain enhancer of B cells)is a Nuclear transcription factor involved in the regulation of gene expression,as well as a key regulatory factor in the production of pro-inflammatory cytokines and chemokines,which is involved in the regulation of more than 200 genes.NF-?B is an essential regulator in the development of immune system,skeletal system and epithelial cell.Studies have shown that NF-?B is activated in malignant tumors,and activated NF-?B contributes to tumor survival by driving proliferation and up-regulating the transcription of anti-apoptotic proteins.Activation of NF-?B signaling pathway has also been observed to induce inflammation and promote tumor formation in early lung adenocarcinoma.In summary,we proposed that NF-?B may be involved in invasion and metastasis of lung adenocarcinoma by upregulating the expression of ABCE1.We planned to observe the expression of transplantation of lung adenocarcinoma tissues,lung cancer cell lines and stable cell lines in nude mice.After ABCE1 was silenced the expression of RNase L was detected,and the proliferaction,invasion,apoptosis and animal induced of lung cancer cells were studied.In depth study ABCE1 pronoter activity and explore the binding sites of NF-?B,thus to explore the regulation effect of NF-?B to ABCE1.Materials and methods Materials:1?Specimens of 48 cases of human lung adenocarcinoma and corresponds paracancerous lung tissue specimens2?Human lung adenocarcinoma cell line A549 and other lung carcinoma cell lines 95-D(high metastatic lung cancer cells of human)3? Balb/c nude female mice aged 4-5 weeks(14-17 g)Various experimental reagents4?Quantitative real-time PCR relative agents5?Immunohistochemistry relative agents6?Western Blot relative agents7?CCK8 relative agents8?Transwell relative agents9?Flow cytometry relative agents10?Tools for Animal tumorigenicity11?Plasmid construction relative agents12?Luciferase reporter assays relative agents13?Ch IP(Chromatin immunoprecipitation)relative agents Methods:1.48 cases of lung adenocarcinoma and corresponding paracancerous lung tissue samples were collected,and cell culture of A549 cells and 95-D cells was conducted.2.The protein expression level of ABCE1 in 48 cases of lung adenocarcinoma and 48 cases of paracancerous specimens was detected by Western Blot.3.The protein expression level of ABCE1 in A549 cells and 95-D cells was detected by Western Blot.4.The m RNA expression level of ABCE1 in 48 cases of lung adenocarcinoma and 48 cases of paracancerous specimens was detected by Real-time PCR.5.The m RNA expression level of ABCE1 in A549 cells and 95-D cells was detected by Real-time PCR.6.A stable cell line A549-sh ABCE1 that stably silenced the expression of ABCE1 was constructed in A549 cells by using the sh RNA expression system.7.The protein expression level of ABCE1 in A549-sh ABCE1 cells was detected by Western Blot.8.CCK8 detected the proliferation ability of A549-sh ABCE1 and A549-p LKO.1cells.9.Invasive ability of A549-sh ABCE1 and A549-p LKO.1 cells was detected by Transwell.10.Apoptosis of A549-sh ABCE1 and A549-p LKO.1 cells was detected by flow cytometry.11.A549-p LKO.1 cells and A549-sh ABCE1 cells were injected into the axilla of nude mice,respectively,to construct xenograft model in nude mice.On day 21,cervical dislocation was performed on nude mice,and tumorigenicity was detected.12.The expression of ABCE1 and RNase L in the tumor was detected by Western Blot.13.The expression of ABCE1 and RNase L in the tumor was detected by Real-time PCR.14.The expressions of NF-?B p65 and p50 in lung adenocarcinoma,paracancerous tissues and lung cancer cells were detected by Western Blot.15.Protein expression of NF-?B p65 and p50 in lung adenocarcinoma and paracancerous tissues was detected by immunohistochemistry.16.Vector construction and luciferase reporter gene were used to detect ABCE1 promoter activity and NF-?B binding site in 95-D and A549 cells.17.The binding of NF-?B p50 and p65 to predicted binding site of ABCE1 upstream promoter in 95-D and A549 cells was detected by Ch IP.Results 1?The expression level of ABCE1 in lung adenocarcinoma tissue was significantly higher than that in paracancerous lung tissue,and ABCE1 was highly expressed in lung adenocarcinoma cells(A549),respectively.Real-time PCR showed the same result as Western Blot.2?CCK8 results showed that the proliferation ability of A549-sh ABCE1 cells decreased significantly after ABCE1 gene silencing compared with the control cell A549-p LKO.1.3?Cell invasion results showed that the invasion ability of A549-sh ABCE1 cells was significantly reduced after ABCE1 gene silencing compared with A549-p LKO.1cells in the control group.4?Flow cytometry results showed that A549-sh ABCE1 cell apoptosis rate was increased after ABCE1 gene silencing compared with control cell A549-p LKO.1.5?In the nude mouse xenograft models,the tumor volume of A549-sh ABCE1 cell group after tumorigenesis was dramatically smaller than that of A549-p LKO.1cell group.6?Western Blot showed that in A549-sh ABCE1 cell group,ABCE1 expression was down-regulated and RNase L expression was elevated;p65 and p50 were highly expressed in lung adenocarcinoma tissues and cell lines.7?Immunohistochemistry results illuminated an aggregation effect of p65,p50 in the nucleus.8?Western Blot and Real-time PCR showed that compared with 95-D,p65,p50 showed high expression following with an increasement of ABCE1 expression in lung adenocarcinoma cell line A549.And the expression of RNase L was decreased.9?Luciferase assays revealed p GL3-488 and p GL3-110 had almost the same promoter activity,which indicated that truncated region in ABCE1 promoter(-488~-110)had no binding site to NF-?B.While the significantly decreased ABCE1 promoter activity in p GL3-47 than that in p GL3-110 demonstrated that binding site NF-?B to ABCE1 promoter lied between-110~-47.The results can be observed consistently in 95-D and A549 cell lines.Very slight promoter activity difference was detected among p GL3-47,p GL3-mut and p GL3-Basic in 95-D and A549.10?Ch IP assay showed that P65 and P50 were able to bind to this putative NF-?B responsive element in 95-D and A549 cells.And this association of P65 and50 with the putative NF-?B responsive element was significantly increased in A549 cell line.Conclusions1.ABCE1 is highly expressed in lung adenocarcinoma and lung adenocarcinoma cell A549.2.The proliferation and invasion ability was decreased and the apoptosis rate was increased in the A549 cell lines silencing ABCE1 expression.3.The tumorigenic rate and volume of animals was decreased in the A549 cell lines silencing ABCE1 expression.4.NF-?B p65?p50 are highly expressed in lung adenocarcinoma and lung adenocarcinoma cell A549.5.High expression of NF-?B up-regulates the expression of ABCE1 in lung adenocarcinoma.