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Objective:To investigate the expression of USP41 in triple-negative breast cancer (TNBC) and its correlation with malignant phenotype and adriamycin sensitivity.Methods:The expression of USP41 in TNBC resistant cell lines and clinical tissue samples was detected by Western blot and qPCR. Subsequently, the high expression of USP41 molecule was determined, and the role and possible mechanism of USP41 in the malignant phenotype and adriamycin resistance of TNBC were evaluated by cell biological methods such as CCK8, colony formation assay, transwell, Western blot, and CoIP-MS.Results:USP41 expression was significantly higher in triple-negative breast cancer samples than in adjacent non-cancerous tissues. USP41 expression was nearly 40-fold higher in the doxorubicin-resistant cell line MDA-MB-231/DXR, with an IC50 value of 6.86 μM. Interference with USP41 significantly increased the sensitivity of MDA-MB-231/DXR cells to doxorubicin. Interference with USP41 significantly inhibited cell proliferation, colony formation and migration of cells, with a decrease in the number of clones of 30%-80% and a decrease in the number of migrating cells of more than 70%, and the difference was statistically significant. In addition, USP41 knockdown improved the sensitivity of MDA-MB-231 cells to doxorubicin, with an IC50 decrease from 5.49 μM to 2.36 μM and 2.56 μM. CO-IP results showed that USP41 could directly interact with RACK1, and the expression of RACK1 was significantly higher in cancer tissues than in adjacent non-cancerous tissues. Interference with RACK1 inhibited MDA-MB-231 cell proliferation, with IC50 decreasing from 9.87 μM to 4.67 μM and 4.36 μM. Colony formation capacity decreased by more than 30% and the difference was statistically significant. USP41 knockdown decreased cell migration by more than 70% compared to control.Conclusion:High expression of USP41 is associated with malignant surface and adriamycin resistance in TNBC, and RACK1 may be a key molecule in the role of USP41.
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BACKGROUND: TDP43 may be a negative regulator of MAPK signaling pathway under hypoxic-ischemic conditions. However, its effect on JNK and p38 MAPK signaling pathways in osteoarthritis remains unclear.OBJECTIVE: To investigate the expression of chondrocyte lesion-related gene RACK1 in wild type TDP43 involved in osteoarthritis, and to analyze its stress effect. METHODS: Human umbilical cord mesenchymal stem cells were transfected by TDP43 lentivirus, and the ability to differentiate into chondrocytes in vitro was analyzed. Umbilical cord mesenchymal stem cells transfected by TDP43 lentivirus, empty vector and without transfection were co-cultured with chondrocytes for 12 days. The chondrocyte proliferation was detected at 0, 3, 6, 9 and 12 days of co-culture. The chondrocyte apoptosis rate was detected by flow cytometry at 3 days of co-culture. The expression levels of TDP43, RACK1, p38, JNK, AP-1 and cl-xl in chondrocytes were detected by qRT-PCR at 3 days of co-culture. RESULTS AND CONCLUSION: (1) After TDP43 lentivirus transfection, human umbilical cord mesenchymal stem cells could differentiate into chondrocytes. (2) The morphology of chondrocytes co-cultured with TDP43 lentivirus transfected-umbilical cord mesenchymal stem cells showed significant change, and the cells became large with abundant branches. Chondrocytes co-cultured with empty vector transfected- or non-transfected umbilical cord mesenchymal stem cells were spindle-shaped in appearance and showed adherent growth with no morphological changes. (3) After co-cultured with TDP43 lentivirus transfected-umbilical cord mesenchymal stem cells, the apoptosis of chondrocytes was promoted, and the cell proliferation was inhibited (P < 0.05). (4) The expression levels of TDP43, RACK1, p38, JNK, AP-1 and Bcl-xl in the chondrocytes co-cultured with TDP43 lentivirus transfected-umbilical cord mesenchymal stem cells were significantly higher than those in the chondrocytes co-cultured with non-transfected- and empty vector-transfected-umbilical cord mesenchymal stem cells. (5) To conclude, high expression of TDP43 in chondrocytes can activate the expression of RACK1, and further regulate chondrocyte proliferation and apoptosis.
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Purpose To investigate the expression and clinical significance of receptor for activated C kinase 1 (RACK1) and β-catenin in cervical cancer tissues. Methods The expression of RACK1 mRNA and β-catenin mRNA in 126 cases of cervical cancer and adjacent normal tissues was detected by RT-PCR, and the expression of RACK1 and β-catenin protein was detected by SP immunohistochemistry. The correlation between the expression of RACK1 and β-catenin protein and clinicopathological data and prognosis was analyzed. Results The relative expression of RACK1 mRNA in cervical cancer tissues was lower than that in adjacent normal tissues, while the relative expression of β-catenin mRNA in cervical cancer tissue was higher than that in adjacent normal tissues. There was a significant difference between the two groups (P < 0.05) . The positive expression rate of RACK1 in cancer tissue group was lower than that in adjacent tissue group (19.0％ vs 63.5％) , while the positive expression rate of β-catenin was higher than that in adjacent tissue group (69.0％ vs 31.0％) (P < 0.05) . The higher the TNM stage, the lower the differentiation and the lower the RACK1 positive expression rate with lymph node metastasis, the higher the positive expression rate of β-catenin (P < 0.05) .There was a negative correlation between RACK1 and β-catenin protein expression (r = 0.404, P < 0.05) . The survival rates of the patients with strong positive expression of RACK1 protein and weak positive expression of β-catenin protein were higher (42.8％ vs 33.3％, 42.8％ vs 36.4％) , but the difference was not statistically significant (P> 0.05) . There was a significantly difference in the median survival time between the two groups (57.2 vs 34.0, 44.2 vs 29.8, P < 0.05) . Weak positive expression of RACK1 protein and strong positive expression of β-catenin were risk factors for poor prognosis in patients with cervical cancer (HR were 9.654 and 8.882 respectively, P < 0.05) .Conclusion The abnormal expression of RACK1 and β-catenin may be related to the occurrence, development and prognosis of cervical cancer, and affect the lymph node metastasis of cervical cancer.
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Objective To investigate the effect of shRNA-mediated down-regulation of the receptor for activated C kinase 1 (RACK1) gene on the chemotherapeutic sensitivities in human lung adenocarcinoma cell line A549.Methods The shRNA recombinant plasmid targeting to human RACK1 gene was designed and transferred into A549 cells by lipofectin technique.The protein level of RACK1 was measured by Western blot to confirm the function of shRNA plasmid.Drug sensitivities of A549 cells to cisplatin,gemcitabine,pemetrexed and paclitaxel were analyzed by MTT assay.The protein expression of LRP and MRP were detected by Western blot.Results After 24 hours transfection,the relative expression quantity of RACK1 protein in RACK1-shRNA group was 0.267± 0.470,which was significantly lower than that in vector-shRNA group (0.821±0.109) and control group (0.842±0.060) (F =54.438,P ＜ 0.05).The results of MTT showed that the growth of A549 cells in the RACK1-shRNA group was markedly inhibited.The sensitivities of A549 cells to cisplatin and paclitaxel were significantly enhanced compared with that in the vector-shRNA group and control group (P ＜ 0.05).The relative expression quantity of LRP and MRP protein in RACK1-shRNA group were 0.163±0.056 and 0.246±0.050,which were lower than that in vector-shRNA group and control group (F LRP =19.430,F MRP =61.548,both P ＜ 0.05).Conclusion Targeted gene silencing of RACK1 improves the sensitivity of A549 cells to the ascisplatin and paclitaxel medicines,which might be achieved through down-regulation of the expression of LRP and MRP.
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Objective To establish several human umbilical vein endothelial cell ( HUVEC ) strains with over-ex-pression or low expression of receptor for activated C kinase 1 ( RACK1 ) , which will provide an effective tool for future studying the function of RACK1 in arrhythmia.Methods The full-length cDNA sequence of RACK1 gene was amplified and inserted into pIRES2-EGFP.At the same time, designed and synthesised complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence , then subcloned into the plas-mid pGenesil-1 .The HUVEC cells were transfected with these plasmids and screened by using G 418 .And the expression of RACK1 mRNA and protein in the cells were assayed by qRT-PCR and Western blot , respectively . Results RACK1 eukaryotic expression vector and siRNA expression vectors of RACK 1 were constructed success-fully.After a 48 h transfection of HUVEC cells with the recombinant vectors and G 418 selection, the positive cell clones were obtained .qRT-PCR and Western blot showed that over-expression vector and interference vectors could effectively enhanced and knocked-down RACK1 expression in HUVEC strains .Conclusions HUVEC cell strains with over-expression and low expression of RACK 1 have been successfully established .
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Objective To investigate the role of receptor for activated C kinase 1 (RACK1) in vascular smooth muscle cells (VSMCs) proliferation modulated by co-cultured endothelial cells (ECs) and shear stress. Methods Using EC/VSMC co-cultured parallel plate flow chamber system, two levels of shear stress, i.e. low shear stress (LowSS, 0.5 Pa) and normal shear stress (NSS, 1.5 Pa), were applied for 12 h. BrdU ELISA was used to detect the proliferation of VSMCs, and Western blot was used to detect the protein expressions of RACK1 and phosphor-Akt. Under the static condition, RNA interference was used to suppress the expression of RACK1 in VSMCs, and then the proliferation of VSMCs and expressions of RACK1 and phosphor-Akt were detected. By using co-culture model (ECs/VSMCs) and separated culture model (ECs//VSMCs), the effect of ECs on expressions of RACK1 and phosphor-Akt in VSMCs was further analyzed. Results Comparative proteomic analysis revealed that LowSS increased the expression of RACK1 in rat aorta. In vitro experiments showed that LowSS induced the proliferation, expressions of RACK1 and phospho Akt in VSMCs co-cultured with ECs. Target RNA interference of RACK1 significantly decreased the proliferation of VSMCs, and the phosphorylation of Akt. In comparison with ECs//VSMCs (separated culture) group, the expression of RACK1 and phosphor-Akt were both up-regulated in the VSMCs co-cultured with ECs (ECs/VSMCs group). Conclusions The expression of RACK1 in VSMCs was modulated by shear stress and neighboring ECs, which might induce cellular proliferation via PI3K/Akt pathway. The investigation on VSMC proliferation and the involved biomechanical mechanism will contribute to understanding and help preventing the pathogenesis and progress of atherosclerosis.