AC1MMYR2 Impaired Paclitaxel-induced Cell Invasion And Metastasis

Objective: In recent years, paclitaxel(taxol) is a widely used front-line chemo-drug for many types of cancer. Latest research indicates that continued treatment with taxol, may lead to tumor cell dissenmination and extravasation. Mi RNAs play an important role in tumor formation and metastasis. Using mi R-21 antisense oligonucleotide in combination with low dose taxol can effectively improve the therapeutic effect, and inhibit tumor metastasis. However, the specific mechanism remains unclear. To investigate the potential mechanisms of taxol-induced tumor metastasis, and whether combining mi R-21 small molecule inhibitor--AC1MMYR2 with taxol could be explored as a means to improve the effect of chemotherapy and limit tumor metastasis.Methods: 1. According to the TCGA data, we analyse the expression level of mi R-21 and CDK5 in normal and tumor tissues. We use In situ hybridization and Immunohistochemistry technology to detect the expression of mi R-21 and CDK5 in 58 pairs of breast cancer tissues with metastasis or not, to study the relationship between the expression pattern of mi R-21 and CDK5 and tumor metastasis. 2. MDA-MB-231 and U87 VIII cells were divided into three groups: control group, low dose taxol group(0.5 μM) and high dose taxol group(2 μM). The expression level of E-cadherin, β-catenin, vimentin and CDK5 were determined by Western blot, we use Real-time PCR to detect the expression level of mi R-21. 3. MDA-MB-231 and U87 VIII cells were divided into four groups: control group, taxol group(2 μM), AC1MMYR2 group and taxol plus AC1MMYR2 group. Immunoflurescence was used to detect the number of invadopadia. Cell invasion number and migration ability of two cells were evaluated by Transwell and Wound Healing Assay, respectively. 4. MDA-MB-231 and U87 VIII were incubated with AC1MMYR2 for 24 h, RNA sequence assay, GO assay and KEGG assay were employed to identify the potential biological target of mi R-21 in this process. MDA-MB-231 and U87VIIIcells were divided into four groups: control group, taxol group, AC1MMYR2 group and taxol plus AC1MMYR2 group, Western blot was used to detect the expression levels of CDK5, CDK5RAP1 and p-FAKSer732. MDA-MB-231 and U87 VIII were transfected with either sh CDK5 lentivirus or a CDK5 overexpression plasmid, and to construct cells that CDK5 was stably knocked down or over-expressed. The cells were divided into three groups: control group, taxol group and taxol plus AC1MMYR2 group, using Western blot to evaluate the expression of E-cadherin, β-catenin and vimentin. U87 VIII was divided into four groups: control group, taxol group, AC1MMYR2 group and taxol plus AC1MMYR2 group, Immunofluorescence staining was used to confirm the expression level of E-cadherin, N-cadherin, β-catenin and Tubulin-α and subcellular location. 5. MDA-MB-231 and U87 VIII were divided three groups: control group, AC1MMYR2 group and AC1MMYR2 plus taxol group, using Western blot to evaluate the expression level of CDK5RAP1, Egr-1 and P39. MDA-MB-231 and U87 VIII were divided into four groups: control group, Ig G group, AC1MMYR2 group and AC1MMYR2 plus taxol group, the association between p39 and CDK5 was observed by co-immunoprecipitation. MDA-MB-231 and U87 VIII were divided into four groups: control group, AC1MMYR2 group, taxol group and taxol plus AC1MMYR2 group, subcellular location of p39 was confirmed using Immunofluorescence staining. Western blot was used to evaluate the expression of p39 in nucleus and cytosol. 6. Constructing MDA-MB-231 orthopic tumor model, to investigate the effect of AC1MMYR2 on high dose taxol-induced tumor metastasis in vivo. 5*106 MDA-MB-231 cells transduced with luciferase lenti-virus were injected into mammary fat pads of each nude mouse. The mice were randomly assigned to four groups(n=8 per group): PBS group, AC1MMYR2 group, taxol group and combination-therapy group. Using the animal Living Image Technology to detect the bioluminescence of tumor and lung. Lung metastasis was identified by H&E staining. Immunohistochemistry technology was used to detect the expression levels of CDK5 and β-catenin.Results:1. According to the TCGA data, we found that both mi R-21 and CDK5 are highly expressed in tumors compared to normal tissues, the difference was statistically significant(P<0.05). In situ hybridizatioon results revealed that stronger mi R-21 staining were observed in invasive carcinoma samples compared to the non cases, and CDK5 were expressed in all 39 invasive carcinoma samples tested. But only 7 of 19 non-lymph node metastasis samples expressed CDK5. 2. Compared to control group, the expression level of E-cadherin was elevated in low dose taxol group, but β-catenin and vimentin were decreased. However, in high dose taxol group, the expression level of E-cadherin was significantly decreased, while β-catenin and vimentin were substantially increased(P<0.05). In addation, the expression of mi R-21 and CDK5 were significantly increased in high dose taxol group, the difference was statistically significant(P<0.05). 3. In MDA-MB-231 and U87 VIII cells, immunofluorescence staining results revealed that taxol significantly enhanced invadopodia number, invadopodia number increased almost 2~3 fold compared to the control group in MDA-MB-231 cells. AC1MMYR2 treatment dramatically reduced the number of cells capable of forming invadopodia in MDA-MB-231 and U87 VIII cells, and in AC1MMYR2 plus taxol group, only a few invadopodia were observed. Transwell assay results showed that cell invasion number was noticeable increased in high dose taxol group(P<0.05), while cell invasion number was decreased about 45~55% after AC1MMYR2 treament, compared to high dose taxol group. Wound Healing Assay revealed that taxol increased migration ability of the two cells, directional migration was obviously impaired in AC1MMYR2 group or combined with taxol group(P<0.05). 4. According to RNA sequence assay, GO assay and KEGG assay, we noticed that CDK5RAP1(CDK5 regulatory subunit associated protein 1) was the only one target gene that was foud in both in MDA-MB-231 and U87 VIII cells. Western blot showed that, taxol treatment significantly increased the expression levels of CDK5, and downstream target, p-FAKSer732(P<0.05). Both AC1MMYR2 group and AC1MMYR2 plus taxol group, the expression of CDK5 and p-FAKSer732 was decreased(P<0.05). Moreover, CDK5RAP1 expression was significantly increased(P<0.05). In both cells transfected with sh CDK5 lentivirus, the sh RNA-mediatedCDK5 silencing was able to increase the expression level of E-cadherin, while in AC1MMYR2 or combined with taxol groups, expression of mesenchymal markers β-catenin and vimentin were significantly decreased. Conversely, in MDA-MB-231 and U87 VIII cells that transfected with CDK5 overexpression plasmid, Western blot results showed CDK5 overexpression blocked the function of AC1MMYR2, the expression of E-cadherin was decreased in AC1MMYR2 and combination groups(P<0.05), while the expression level of β-catenin and vimentin were increased(P<0.05). We observed stronger staining of E-cadherin in the AC1MMYR2 plus taxol treated U87 VIII cells, compared to control group, while nucleus distribution of β-catenin reduced, and the fluorescence of N-cadherin was reduced. 5. Western blot results revealed that in AC1MMYR2 group and combination group, the expression level of CDK5 was decreased while the expression of CDK5RAP1 and Egr-1 were significantly increased, but the expression level of p39 in AC1MMYR2 group and combination group were reduced(P<0.05). Co-immunoprecipitation showed that CDK5 bound to p39 in control group, but this association ceasedin in AC1MMYR2 group and combination group. Moreover, nucleus staining of p39 was significantly reduced after AC1MMYR2 treament in the two cells. Western blot results showed that the expression of p39 was decreased in nucleus, while the expression of p39 was increased in cytoplasm. 6. Successfully established breast cancer orthotopic model. Living Image results showed us that, taxol suppressed tumor growth to some extent compared to PBS group, but increased lung metastasis formation. Tumor growth was suppressed after AC1MMYR2 plus taxol treatment, but lung metastasis was significantly reduced. H&E staining got the same results. Immunohistochemistry results revealed that stronger staining of CDK5 and β-catenin were observed in taxol group, however, the expression level of CDK5 and β-catenin were significantly reduced in AC1MMYR2 and combination groups.Conclusion So far, only few examples of small moleculestargeting oncogenic mi RNAs have been reported. Our work demonstrated that AC1MMYR2 appared to be a promising strategy in combating taxol-induced metastasis, which highlighted the potential fordevelopment of therapeutic modalities for better clinic taxol application.