[The effect of LAMP2A shRNA on the resistance of breast cancer cells to paclitaxel].
OBJECTIVE
To prepare a recombinant lentiviral carring human lysosome-associated membrane protein type 2A (LAMP2A) gene shRNA, establish MDA-MB-231 cell line with a low expression of LAMP2A and observe the change in cell resistance to paclitaxel.


METHODS
Four shRNAs were designed according to the sequencing analysis of LAMP2A mRNA. The pGLV-EGFP-shRNA lentiviral vector was established by gene recombination technology and was confirmed by DNA sequencing. The lentiviral vector and the packaging vector were used to cotransfect the HEK293T cells to obtain the lentivirus against LAMP2A mRNA, and the titer of the virus was determined. The constructed shRNA lentivirus was applied to infect human breast cancer cell line MDA-MB-231, and then the cells were screened with puromycin for two weeks. The inhibitive efficacy of the shRNA on the LAMP2A protein was determined by Western blotting. After the breast cancer cell line with a low expression of LAMP2A was treated with three different concentrations of paclitaxel (1, 10, 100 nmol/L), MTT assay was performed to observe the difference in the proliferation ability between the control group and the low LAMP2A expression groups.


RESULTS
DNA sequencing revealed that the recombinant lentiviral plasmid was correctly constructed with the virus titer reaching 2×10(8); TU/mL. The LAMP2A protein expression in the obtained breast cancer cell line dropped drastically. After the treatment with paclitaxel at 10 and 100 nmol/L respectively, the drug resistance of cells with a low expression of LAMP2A was notably weaker than that of the control group (P<0.05).


CONCLUSIONS
The recombinant shRNA lentiviral vector against human LAMP2A gene was successfully constructed, and the breast cancer cell line MDA-MB-231 transfected with it stably expressed a low level of LAMP2A. It was proved that the down-regulation of LAMP2A could reduce the resistance of breast cancer cells to paclitaxel.
Department of Clinical Biochemistry, Southwestern Hospital, Third Military Medical University, Chongqing 400038, China.
Qi
Q
Han
Sha
S
Chen
Mingzhen
M
Yang
Zhujun
Z
Zhang
An
A
Chen
Chuanmin
C
Hu
Shuhui
S
Li
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000502:physiology; D052119:Lysosomal-Associated Membrane Protein 2 / Q000235:genetics* / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics*; D019562:Viral Load
D000970:Antineoplastic Agents; D052119:Lysosomal-Associated Membrane Protein 2; D034741:RNA, Small Interfering; D017239:Paclitaxel