The Effects And Mechanisms Of Alk2Down-regulation On Human Breast Cancer Cell Proliferation, Invasion And Migration

Objective To investigate the effects and possible mechanisms ofALK2down-regulation on human breast cancer cells MDA-MB-231proliferation, invasion and migration, then providing experimental basis forsearching new breast cancer therapeutic targets and therapeutic drugs.Methods The expression of ALK2in MDA-MB-231cells wasdetected by RT-PCR; Adenovirus DN-ALK2was used to infectMDA-MB-231cells. Experimental group is MDA-MB-231/DN-ALK2cells，control group are both MDA-MB-231/RFP cells and MDA-MB-231cells. To detect the ability of proliferation, invasion and migration in therecombinant MDA-MB-231/DN-ALK2cells, MTT, colony-forming,wounding and Transwell invasion assays were carried out. RT-PCR andWestern Blot were used to detect the expression pattern of CTGF on mRNAand protein in three groups.A total of1×106cells were inoculated subcutaneously into each groupof nude mice (n=5) to establish xenograft breast cancer model. The grosstumor volumes were measured dynamically every3days. The mice were sacrificed by the cervical dislocation method and tumors were obtained after34days. Take the subcutaneous tumor to make the tissue slices. Theexpression of Connective Tissue Growth Factor (CTGF) in the three groupswere detected by immunohistochemistry.Results ALK2was highly expressed in MDA-MB-231cells;MDA-MB-231cells were transfected with AdDN-ALK2and AdRFP respectively. After36hours, the fluorescence expression was amount to70%.DN-ALK2was highly expressed in MDA-MB-231/DN-ALK2cells.Compared with control groups including MDA-MB-231/RFP cells andMDA-MB-231cells, the OD value of experimental group was decreasedsignificantly in MTT assay; The colony formation rate was significantlydecreased from the control group’s (91.0±3.6)%and (91.3±5.1)%to theexperimental group’s (34.0±5.3)%(P <0.05); The healing rate of24h wasfell from the control group’s (68.3±1.7)%and (62.3±2.1)%to theexperimental group’s (26.4±0.6)%(P <0.05); The healing rate of48h wasfell from the control group’s (97.3±0.7)%and (94.4±0.9)%to theexperimental group’s (30.5±0.8)%(P<0.05); The cells of experimentalgroup which moved across the matrix barrier was decreased from controlgroup (143.3±2.1) and (142.3±2.5) to（33.3±4.2）(P<0．05). The expressionof CTGF on mRNA and protein was down regulated. The average of opticaldensity was decreased from control group (0.8267±0.3841) and(0.8407±0.5266) to (0.4684±0.1057)(P<0．05). The results in vivo suggested that tumors were observed in the twocontrol groups after13days, but tumors in MDA-MB-231/DN-ALK2wereobserved after16days. After34days, all of the nude mice were sacrificedand measured the volume for tumors. the volume of tumors in controlgroups （2.465±0.250）cm~3and （2.454±0.250）cm~3were bigger than thatin MDA-MB-231/DN-ALK2groups（0.804±0.090）cm3(P<0.05). Theimmunohistochemical assay of tumor tissue section showed that expressionof CTGF in MDA-MB-231/DN-ALK2groups was lower than that in twocontrol groups significantly(P<0.05).Conclusions ALK2was highly expressed in human breast cancerMDA-MB-231cells, DN-ALK2could inhibit the proliferation, invasion,and migration of MDA-MB-231cells in vitro and in vivo, and these effectswere possibaly related with the down-regulation of CTGF.