| 1. Background and Objective:Resistance to chemotherapy is a major obstacle for the effective treatment of cancers. Lin28has been shown to contribute to tumor progression, tumor relapse and bad outcomes. However, the relationship between lin28and chemoresistance remained unknown. In this study, we investigated the association of lin28with paclitaxel resistance and identified the underlying mechanisms of lin28in human breast cell lines and breast cancer tissues.2. Methods:(1) To determine whether lin28expression is also associated with chemoresistance in breast cancer, we measured the expression of lin28in five breast cancer cell lines (SK-BR-3, MCF-7, Bcap-37, MDA-231, T47D) by western blot and determined their sensitivities to chemotherapeutic drug paclitaxel by MTT assay. (2) To confirm the level of lin28determine the sensitivities to chemotherapeutic drug paclitaxel, we then knockdown the expression of lin28by transfection of lin28siRNA to T47D cell, and evaluated the sensitivities of the cells to paclitaxel by MTT assay.(3) To further confirm the role of lin28expression in regulating paclitaxel resistance in breast cancer, we established three lin28stably expressing SK-BR-3clones (S1, S24, S27) and one lin28stably expressing Bcap-37clone (B1), then evaluated the sensitivities of these lin28-stably expressing cell lines to paclitaxel by MTT assay.(4) We next determined the effect of Iin28transfection on paclitaxel-induced apoptosis by flow cytometric assay.(5) We measured apoptotic protein PARP gene expression in S24clone treated by paclitaxel by Western blotting.(6) To determine whether Lin28is also associated with relapse or metastasis of breast cancer, we examined the expression of Lin28in tumor tissues from various patients by immunohistochemistry.(7) To further investigate the mechanism of lin28-induced paclitaxel resistance, we measured the expression of MRP-1, p21, Rb, cyclinB1and Akt in three stable lin28expression SK-BR-3clones by western blot.(8) To further investigate the mechanism of lin28-induced paclitaxel resistance, we measured let-7miRNA by realtime RT-PCR in three stable lin28expression SK-BR-3clones.(9) To further confirm the relationship between let-7miRNA and the seneitivity to paclitaxel of lin28-expressing cells, we transfect the pre-let-7a miRNA to S1cell, then evaluated the sensitivities of the cells to paclitaxel by MTT assay.3. Results:(1) Results showed that lin28in T47D cancer cell line was highly expressed, whereas its expression was relatively lower in MCF7, Bcap-37and SK-BR-3cancer cell lines. The further study showed that the expression level of lin28was closely associated with the resistance of the cells to paclitaxel treatment. The IC50values of four cell lines to paclitaxel treatment were48.20μM for T47D cells,4.40μM for MCF-7cells,5.50μM for Bcap-37cells and9.80μM for SK-BR-3cells, respectively.(2) We found the lin28siRNA transfection cells are more sensitive to paclitaxel, the IC50values in different concentrations are lower than the control groups.(3) We found that these stable lin28expression clones were more resistant to paclitaxel compared to the controls cells. The IC50values were9.90μM for the empty transfected SK-BR-3cells and22.30～35.60μM for three stable lin28expression SK-BR-3clones. Moreover, the results showed that, among three stable lin28expression SK-BR-3clones, lin28expression in S1clone was lower than clones S24and S27. The highly-expressing lin28clones (S24and S27) were more paclitaxel-resistant than S1clone. We found that the stable lin28expression clone B1were more resistant to paclitaxel compared to the control cells. The IC50values were6.10μM for the empty transfected Bcap-37cells and28.30μM for the stable lin28expression Bcap-37clones.(4) Transfection with lin28significantly inhibited paclitaxel-induced apoptosis in three stable lin28expression SK-BR-3clones, resulting in a reduction from50.78%to20.47～30.32%and69.88%to27.61～38.92%at the dose of10μM and40μM paclitaxel respectively(p<0.01)(PI staining). We found apoptotic cells were dramatically decreased in lin28stable expression clone S24from42.27%to17.62%and58.81%to24.71%at the dose of10μM and40μM paclitaxel, respectively (P<0.01)(PI-AnnexinV double staining). Transfection with lin28significantly inhibited paclitaxel-induced apoptosis in the stable lin28expression Bcap-37clone, resulting in a reduction from28.3%to5.12%and32.85%to8.56%at the dose of5μM and20μM paclitaxel respectively (PI staining).(5) We measured cleaved PAPR expression, which means to develop apoptosis, was upregulated after paclitaxel treatment in the parental and mock control cells, while in lin28stable expression S24cells, cleaved PARP expression showed no changement.(6) The result showed that, after adjuvant chemotherapy, Lin28expression was dramatically increased. Moreover, Lin28was dramatically upregulated in relapse or metastatic breast cancer tissues, when compared to primary tissues or operative tissues.(7) Western blot analysis showed that p21and Rb were significantly increased in stable lin28expression cancer cells compared to mock control, whereas Cyclin B1and Akt expressions did not alter. There are no alteration in the expression of MRP-1.(8) Real-time PCR analysis also showed that let-7a and let-7b miRNAs were dramatically decreased in stable lin28expression cancer cells compared to mock control.(9) Transfection of S1cells with a let-7a pre-miRNA significantly decreased IC50value of paclitaxel by comparison with the parental group and the pre-miRNA control group (p<0.01).4. Conclusions:(1) Lin28expression was positively related with paclitaxel resistance in breast cancer cells.(2) Lin28transfection induced paclitaxel-resistance in breast cancer cells.(3) Lin28transfection inhibited paclitaxel-induced apoptosis in breast cancer cells(4) Lin28expression is related with relapse or metastasis of breast cancer.(5) Lin28induces the expressions of p21and Rb in breast cancer cells.(6) Lin28inhibits the level of let-7miRNAs in breast cancer cells.(7) The upregulation of let-7miRNA increased the sensitivity of lin28-expressing clone S1to paclitaxel.Our results indicated that lin28expression might be one mechanism of paclitaxel-resistance in breast cancer, and lin28could be a potential target for overcoming paclitaxel resistance of breast cancer.
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