The Effect Of Combination Of Stathmn1 RNAi And Anticancer Drugs On Cell Proliferation

Stathminl protein is a cytoplasmic protein which is universial in vertebrate cells. It encodes a protein about 19 kd composed of 149 amino acids. The activity of Stathminl protein is regulated by phosphorylation and dephosphorylation in the different stages of mitosis. Stathminl plays an enormous role in the regulation of the dynamic system of cell microtubules by binding with microtubules directly to accelerate the process of depolymerization of microtubules or binding with free tubulin to prevent the process of microtubule polymerization. Thus it can also regulate cell proliferation and differentiation efficiently.Taxol is one of the hottest anticancer drugs after doxorubicin and cisplatin. It has been reported in the current that taxol plays an important role in the treatment of a wide variety of tumor such as ovarian cancer, the lung cancer, the bladder cancer, and the breast cancer, etc. The target of drug taxol is cytoskeletal microtubules system. It affects the dynamic balance of the microtubule polymerization and depolymerization after binding with the N-31 amino acids and amino acids 231 to 231 of P-tubulin which promotes not only the polymerization of the tubulin and the stabilization of microtubule structure, but also inhibites the depolymerization of the tubulin. The combination hinders the formation of a mitotic spindle that leads to the abnormal arrangement of microtubules and the formation of stable non-functional microtubule bundles which results in the accumulation of cells in the G2/M phases, and ultimately leads to the inhibition of cell growth and the acceleration of cell apoptosis. DPPT (deoxypodophyllotoxin) has caused the attention of the researchers as a new anticancer drugs in recent years by comparision to taxol. It can strongly inhibit tublin aggregation and play an efficient role against cancer by integrating colchicines domain which is located on the tubulin proteins,cell cycle arrest in the G2/M phases.To find experimentally the effect of the two anti-cancer drugs on the dynamic changes of the assembly of microtubule in the progress of normal cell proliferation, the mouse myoblast cell C2C12 was used as the experimental cell lines, and respectively exposed to different concentrations of taxol and DPPT for 48 hours. After the treatment of two anti-cancer drugs, the expression of Stathminl gene was detected, the morphological appearance was observed. The cell survival rate was determined by MTT assay, total protein and mRNA was extracted. The result showed celluar survival rate is roughly presents parabolic as drug concentration increased, the death rate of cell was lower when it was exposed to the drug concentration of 0.1 μM and 100μM. Cell death rate increased with the increase of concentration in a certain range of concentration. It was different that cell survival rate was lowest when it was exposed to the drug concentration of 1μM of DPPT, but cell survival rate was lowest when it was exposed to the drug concentration of 0.5μM of taxol. In general, the effect of DPPT on the cell death was lower than taxol. The expression of STMN1 was assayed by Western Blot, STMN1 protein expression respectively decreased 70.6% and 77.9% relative to the control group. The expression of mRNA was assayed by RT-PCR, Stathminl expression respectively decreased 60.6% and 79.9% relative to the control group. Thus, the effect of taxol on the treatment of cancer is higher than the effects of DPPT.To study the effect of Stathminl expression on the cell proliferation, the expression vectors of Stathminl human shRNA interference were constructed. The efficiency of shRNA was detected in A549. Then these recombinant plasmids were transfected into A549 by Iipofection, the effect of interference plasmid on the level of down-regulation of Stathminl gene expression was tested. In addition, migration of human lung adenocarcinoma cell A549 transfected by the interference plasmids was detected by wound healing assay. The result showed STMN1 protein expression of cells transfected by STMN1/hshRNA1 and STMN1/hshRNA3 respectively decreased 82.2% and 78.4% relative to the control group, STMN1 protein expression of cells transfected by STMN1/hshRNA2 only decreased 34.5%. The levels of mRNA were assayed by RT-PCR, Stathminl expression respectively decreased 59.1% and 54.7% relative to the control group. However, mRNA expression of cells transfected by STMN1/hshRNA2 decreased 32.6%. Thus Stathminl shRNA1 and shRNA3 interference vectors were contructed successfully. Interference vectors of Stathminl shRNA could be used in further experiments. The wound healing assay result showed that the migration rate of cells transfected by STMN1/hshRNA1 and STMN1/hshRNA3 was slower than cells transfected by STMN1/hshRNA-control. It indicated that low level of Stathminl expression could supress cell A549 migration.This experiment has also detected the influence of combination of Stathminl RNAi and drugs on the cell proliferation. The results revealed that the survival rate of A549 under 0.5μM taxol was 28.5% while under the same concentration of DPPT, it was 45.8%. However, while STMN1/hshRNA interference combining with 0.5 μ-M taxol and DPPT separately, the survival rate of A549 decreased to 21.2% and 32.4%. If combined with STMN1/hshRNA3 interference,0.5μM taxol and DPPT would make the survival rate of A549 change to 27.2% and 31.7%. Therefore, RNAi could reduce drug concentration in tumor treatment.