Role And Mechanism Of Cullin7 Ubiquitin Ligase In Docetaxel Resistance Of Non-small Cell Lung Cancer

Background:Lung cancer is one of the highest morbidity and mortality cancer in the world,which is a serious threat to human health.It has been show that the incidence of lung cancer in China is 35.23/100000 and the mortality rate is27.93/100000.Lung cancer mainly divided into small cell lung cancer?SCLC?and non-small cell lung cancer?NSCLC?,of which NSCLC accounted for 80%.About 70% Patients have been diagnosed as lung cancer with locally advanced or distant metastasis.Therefore,chemotherapy is an important clinical treatment for lung cancer,which has great significance for prolonging the survival time and improving the life quality of patients.Docetaxel?DTX?,as the third generation of chemotherapy drugs,is an important first-line and second-line drugs for the treatment of NSCLC,which has significant antitumor effects and mainly act on microtubules.It is widely used in clinical treatment of breast cancer,NSCLC,ovarian cancer and so on.It can promote cell tubulin polymerization and inhibit of microtubule depolymerization,which results in abnormal regulation of microtubule dynamics and activates the spindle assembly checkpoint.And then,it would block the normal cell mitosis,resulting in cell cycle arresting in G2/M phase and inducing cell apoptosis.However,docetaxel has the characteristic of chemoresistance and that do great influence on the long-term treatment.Cullin7?Cul7?is the core scaffold protein of the SCF?Skp1-Cullin-F-box?E3 ubiquitin ligase complexes.Recent studies show that Cul7 is closely associated with the occurrence and development of malignant tumors.Prof Yue et al.reported that 3-M gene mutation syndrome associated with the 3M complex which is composed of mutually disjoint mutations?Cul7,OBSL1,CCDC8?and FBXW8.3M complex pathway and downstream molecule CUL9 play the important role in maintaining genomic integrity and microtubules dynamics.The abnormal expression of Cul7 can lead to disorder of microtubule dynamics,tissue mitosis and cytokinesis.Knockdown of Cul7 enhances the sensitivity of lung cancer cells to non toxic doses of paclitaxel.It has been reported that Cul7 is highly expressed in NSCLC,which can promote the proliferation,invasion and metastasis of NSCLC cells.High expression of Cul7 was also found in NSCLC chemoresistance cell lines.However,there is no clear evidence about the relationship between Cul7 and drug resistance.Objective:This subject aim to detect the expression of Cul7 in NSCLC docetaxel-resistanec cell lines and observe the changes of chemosensitivity in Cul7 knockdown or overexpressing NSCLC cells.In order to provide new clue about the mechanism of NSCLC chemoresistance,we would confirm correlation between Cul7 and docetaxel drug resistance and find its possible molecular mechanisms.All these work will also provide new ideas on intervention strategies of clinical oncology docetaxel resistance.Methods:1.Detect the half inhibition concentration(IC₅₀)and resistance index in NSCLC cells and its corresponding docetaxel-resistance cells that was provided by the laboratory of University of Michigan Professor Max Wicha of the United States.2.The expression and localization of Cul7 in NSCLC parental and resistant cells were detected by the means of Real-time PCR,Western Blot Western blot and immunofluorescence.3.Use flow cytometry to observe the apoptosis and cycle change of NSCLC parental and drug-resistance cells under the same concentration of docetaxel.We gradually increase the dosing concentration of docetaxel and etoposide in NSCLC cells,and then detect the expression of Cul7 by means of Western Blot.4.Plasmid mediate Cul7 sh RNA interference in A549,H358,H1299,A549 DTX and H358 DTX cells.p CDNA3-HA-Cul7 was transfected into A549 and H1299 cells by using liposome Lipofectamine 2000 to overexpress Cul7.Detect the efficiency of interference and overexpression by applying Real-time PCR and Western Blot.5.Use MTS to test the drug sensitivity to docetaxel?paclitaxel etoposide in A549 and H1299 cells that has been interfered and overexpressing Cul7.6.The expression of Cul7 and Survivin in A549,A549 DTX,A549shNeg,A549 sh Cul7,A549 p CDNA3 and A549HACul7 were detected by using Western Blot.Use immunohistochemistry to detect the expression of Cul7 and Survivin in non small cell lung cancer tissues.7.Use si-RNA to knock down Survivin in H1299 DTX and H1299HACul7 cells,and detect the interference efficiency by using Real-time PCR and WesternBlot.Detect the sensitivity of transfected H1299 DTX and H1299HACul7 cells to docetaxel by using the method of MTS.Results:The IC₅₀ for A549 DTX or H358 DTX was 18-or 11-fold higher than that in both parental cells.We showed that the expression of Cul7 m RNA and protein was significantly increased in both A549 DTX and H358 DTX cell lines.Immunofluorescence results showed that the Cul7 fluorescence signal in nuclear was stronger than in cytoplasm,and the Cul7 expression in docetaxel-resistant cells was higher than in parental cells.Flow cytometry showed the same docetaxel concentration,resistant rate to apoptosis was significantly less than the parental cells?P<0.05?.Consistent with the trend of Cul7 expression,survivin protein also highly expressed in docetaxel-resistance NSCLC cells.Meanwhile,we also found that knockdown of Cul7 resulted in decreasing of survivin protein in NSCLC cells and overexpression of Cul7 resulted in increasing of survivin protein.Knockdown of Cul7 increases sensitivity to docetaxel,paclitaxel and etoposide in NSCLC cells?P<0.05?.Moreover,Knockdown of Cul7 in A549 DTX and H358 DTX cells restored docetaxel sensitivity?P<0.05?.In H1299 DTX and H1299HACul7 cells transiently silenced Survivin,is found to enhance the sensitivity of tumor cells to docetaxel?P<0.05?.Knockdown of Cul7 in A549 DTX cells resulted in the up-regulation of Cleaved-Caspase3.Conclusion:These results suggest that Cul7 is involved in the development of docetaxel resistance.Cul7 may be a potential therapeutic target for drug-resistant lung cancer and Survivin may be the downstream regulatory gene of Cul7 for resulting in docetaxel resistance.