| Objective: The drug resistance mechanism of tumor is complicated biological process which is mediated by a lot of genes,included multilevel and involved in mixed factors.generally involving chemotherapy drug efflux,tumor cell abnormal apoptosis,cell cycle changing,and so on.Nucleolin is an important multifunctional protein in cell nucleus,which participates in the progression of DNA synthesis,RNA transcription,ribosomes formation,the translation of m RNA and the formation of micro RNA.These function affects tumor cell proliferation,apoptosis and tolerance of chemotherapy drugs.In this study,we wanted to evaluate whether NCL participate in cell drug resistance mechanisms in CA46 through different expression level of nucleolin involvement.Methods:1、 CA46-C23-knockdown(CA46-C23-KD)cell and CA46-C23-overexpression(CA46-C23-OE)cell was established by Lentivirus vector mediated transfection,and a multi-drug resistance cell line(CA46/ADR)cell model was established through long-term induction of low concentration of adriamycin.2、 adriamycin IC50 of CA46-C23-KD、 CA46-C23-OE and CA46/ADR cell models were detected by cell proliferation method(MTS).3、BCL-2 m RNA stability was performed by actinomycin D test in CA46-C23-KD and CA46-C23-OE cell,NCL and BCL-2 m RNA interaction was performed by RNA-protein immunecoprecipitation(RIP)in CA46 cell.4、 Cell cycle was detected in CA46-C23-KD by flowcytometry.Cyclin D3 and cyclin E1 was detected by westernblot in CA46-C23-KD and CA46-C23-OE cells.The interaction between NCL and cyclin D3、 cyclin E1 was performed by protein immunecoprecipitation(CO-IP).5、The interaction between NCL and P-gp or MDR1 m RNA was performed by CO-IP and RIP in CA46/ADR cell.6、 Micro RNA-221 was detected by real-time fluorescence PCR in CA46-C23-KD,CA46-C23-OE and CA46/ADR cell models.NCL and pri-mi RNA-221 interaction was performed by RNA-protein immunecoprecipitation(RIP)in CA46 cell.Results:1、 Compared with negative control group,NCL m RNA and C23 protein expression decreased in CA46-C23-KD cell,NCL m RNA and C23 protein raised significantly in CA46-C23-OE cell,P-gp and C23 expression are raised in CA46/ADR cell.2、The IC50 of CA46-C23-KD cell to adriamycin was 0.147±0.02 μg/ml,significantly lower than the control group(CA46-C23-KNC)0.301±0.04μg/ml(p<0.05).While The IC50 of CA46-C23-OE cell to adriamycin was 0.679±0.06μg/ml,significantly higher than the control group(CA46-C23-ONC)0.383±0.08μg/ml(p<0.05).The IC50 of CA46/ADR to adriamycin was 1.874±0.13 μg/ml,while IC50 of CA46 to adriamycin was 0.215±0.05 μg/ml,The IC50 of CA46/ADR to adriamycin(ADR)raised about eight times than CA46 cell.3、 The BCL-2 m RNA stability of CA46-C23-KD is lower than the negative control group(CA46-C23-KNC),their half-life of the BCL-2 m RNA were 4.8±0.15 hours VS 3.2±0.18 hours,and the BCL-2 m RNA stability of CA46-C23-OE significantly enhanced than the the negative control group(CA46-C23-ONC),their half-life of the BCL-2 m RNA were 6.9±0.13 hours VS 5.2±0.21 hours.C23 enhanced the stability of BCL-2 m RNA by combined directly.RIP result confirmed that C23 protein could interact with BCL-2 m RNA directly.4、 The ratio of G0/G1 phase increased,S phase reduced in CA46-C23-KD cell.Cyclin D3 increased and cyclin E1 reduced in CA46-C23-KD cell,cyclin D3 had no obvious changed and cyclin E1 increased in CA46-C23-OE.And then,the CO-IP results showed C23 protein could direct interact with cyclin D3 and cyclin E1.5、NCL and MDR1/P-gp increased in CA46/ADR cells,but CO-IP and RIP result did not confirm that C23 protein could interact with P-gp or MDR1 m RNA directly.6、 Micro RNA-221 raised in CA46-C23-OE and CA46/ADR cell,but reduced in CA46-C23-KD cell.RIP result confirmed that C23 protein could interact with pri-mi RNA-221 directly.conclusion:1、 C23 combined with BCL-2 m RNA directly,which could increase the BCL-2 m RNA transcription though increaseing the stability of the BCL-2 m RNA.This combination could resist the pharmacological actions of adriamycin.in CA46 cells through antiapoptotic mechanisms.2、C23 combined with cyclin D3 and cyclin E1 directly,and regulated the expression of cell cycle protein to increase S phase cycle.Which could result to resist adriamycin in CA46 cells though accelerating the process of cell cycle.3、C23 was correlated with P-gp,but C23 could not be combined with P-gp or MDR1 m RNA directly.4、C23 could increase the expression of micro RNA-221 by combining pri-mi RNA-221 directly,which could effect drug resistant to tumor cell though micro RNA-221 target genes. |
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