| Research background:According to the latest data released by WHO in 2020,breast cancer(BC)has exceeded lung cancer and has become the highest incidence rate of malignancy in the world.It is predicted that by the end of 2021,more than 570000 women will be newly confirmed as breast cancer,and more than 90000 women will die of breast cancer.In recent years,although the treatment of breast cancer is constantly updated and iterated,whether in the diversity of chemotherapy programs,or in the update of surgical methods and multidisciplinary comprehensive treatment such as radiotherapy,targeted therapy,immunotherapy,etc.,it has brought more benefits for breast cancer patients,greatly prolonging the overall survival rate and disease recurrence free time of patients,but there are still 30% ～ 40% of patients died due to recurrence and metastasis.Looking for an effective therapeutic target for breast cancer and improving the prevention and treatment effect of breast cancer has always been a major medical research topic.The occurrence,development and metastasis of malignant tumor are realized by the continuous proliferation of malignant cells.Cell proliferation includes four phase of cell cycles in which a large number of genes participate.In previous studies,our team screened a candidate gene,DYNLT1,by comparing the transcription differential genes in two pairs of breast cancer drug-resistant tissue samples.DYNLT1 gene encodes the kinetin light chain Tctex 1,which is one of the non-catalytic auxiliary components of the kinetin complex in the cytoplasm.It is mainly involved in the connection of kinetin to cargo and adaptor proteins that regulate the function of kinetin.It provides motor for vesicles and organelles and makes them move retrogradely along microtubules in cells.Studies have shown that DYNLT1 protein can participate in a variety of cell functions,including membrane organelle transport,mitotic spindle orientation,nuclear migration and cell migration.However,the role of DYNLT1 in breast cancer has not been reported.In order to further explore the role of dynlt1 in breast cancer,this study intends to intervene the expression of DYNLT1 in cells,animals and clinical tissues through gene editing,and observe the effect of DYNLT1 on the growth of breast cancer cells and paclitaxel sensitivity,so as to provide a new target for the treatment of breast cancer.Methods:1.Using the bioinformatics data of the public platform to clarify the correlation between the expression of DYNLT1 and clinical prognostic indicators(such as OS,RFS).2.Construction of breast cancer cell lines ZR75-30 and BT 549 stably knockdown DYNLT1 by lentiviral sh RNA system;The MDA-MB-231 breast cancer cell line overexpressing DYNLT1 was constructed by transfecting the overexpression vector.3.CCK8 and clone formation were used to detect the proliferation of breast cancer cells after downregulation and overexpression of DYNLT1;The changes of cell cycle at different time points(0h,4h,8h,12 h,18h and 24h)were detected by flow cytometry;The changes of cell cycle regulatory proteins were detected by q RT PCR and WB assay.4.In the breast cancer cells with knockdown and overexpression of DYNLT1,they were treated with various cycle inhibitors(thymidine,hydroxyurea,mimosine,nocodazole)respectively.The changes of cell cycle were detected by flow cytometry.The expression of cell cycle regulatory proteins was detected by q RT-PCR and WB assay.5.Paclitaxel,a cycle specific chemotherapeutic drug,was given to breast cancer cells with knockdown and overexpression of DYNLT1.CCK8 assay was used to detect the changes of cell toxicity to paclitaxel after intervention of DYNLT1 expression;Apoptosis was detected by flow cytometry,and the expression of apoptosis related proteins was detected by WB assay.6.The relationship between the expression of protein binding to DYNLT1 and cell cycle was analyzed by immunocoprecipitation and mass spectrometry.7.The nude mice with knockdown or overexpression of DYNLT1 were divided into two groups: the treated group and the non-treated group.Paclitaxel(paclitaxel concentration was 1.2mg/ml,10ul/g)was injected intraperitoneally every two days according to body weight.The body weight and tumor growth of nude mice were measured and recorded.After tumor collection,the expressions of DYNLT1,Ki67,cycle and apoptosis pathway related proteins were detected by immunohistochemistry.8.Collect the basic clinical data,pathological data and follow-up data of breast cancer patients,and analyze the correlation between the expression of DYNLT1 and the clinical characteristics of breast cancer combined with immunohistochemical results.Result:1.Using TCGA data analysis,we found that the expression of DYNLT1 in normal tissues was significantly lower than that in tumor tissues in most tumors,including breast cancer;At the same time,the survival curve analysis showed that high expression of DYNLT1 in breast cancer significantly shortened the overall survival time(P < 0.001)and disease recurrence free time(P < 0.001).2.Through CCLE(https://portals.broadinstitute.org/ccle)The results showed that the expression of DYNLT1 was higher in ZR75-30 and BT 549 breast cancer cell lines,but lower in MDA-MB-231 breast cancer cell lines.In ZR75-30 and BT 549 cells,DYNLT1 sh RNA lentivirus was infected respectively.q RT-PCR and WB results showed that the expression of DYNLT1 in ZR75-30 DY-SH and BT5 49 DY-SH cells was 80.3% lower than that in control cells(ZR75-30 NC-SH and BT 549 NC-SH),respectively±5.7%(p<0.01)、83.2%±2.7%(p<0.01);Meanwhile,the overexpression vector of DYNLT1 was transfected into MDA-MB-231 cells.q RT-PCR and WB results showed that the expression of DYNLT1 in MDA-MB-231 DY-Flag cells was 2.0% higher than that in MDA-MB-231 NC-Flag cells ±2 times(P < 0.01).These results suggest that the stable cell lines with DYNLT1 knockdown and overexpression were successfully constructed.CCK8 proliferation experiment and plate cloning experiment showed that:compared with the control cells(ZR75-30 NC-SH and BT 549 NC-SH),the proliferation ability of ZR75-30 DY-SH and BT 549 DY-SH cells was significantly inhibited after DYNLT1 knockdown;After overexpression of DYNLT1,the proliferation of MDA-MB-231 DY-Flag cells was significantly stronger than that of control cells(MDA-MB-231 NC-Flag).3.Flow cytometry analysis showed that compared with the control group(ZR75-30NC-SH),the proportion of G2/M was 7.60%,significantly lower than 20.30% of the control group(P < 0.01);Compared with the control group(ZR 75-30 NC-SH),the ratio of G2/M was 14.89% after dnlt1 knockdown,which was significantly lower than 35.46%in the control group(P < 0.01).4.The protein bound to DYNLT1 was analyzed by IP,the results of Mass spectrometry analysis showed that DYNLT1 was the most abundant in G2/M phase cell cycle regulation pathway.5.CCK8 test showed that the sensitivity of the stable cell line with DYNLT1 knockdown was higher than that of the control group,and the IC50 of ZR 75-30 cells were10 ug/ml in the control group and 4.5 ug/ ml in the knockdown group;Flow cytometry also showed that the apoptotic of the cells with DYNLT1 knockdown was higher than that of the control group at the same drug concentration(66% and 82% respectively in the control group and knockdown group at 7.2 ug/ml,p<0.05);The IC50 of BT 549 cells were12.5 ug/ml in the control group and 6.0 ug/ml in the knockdown group;Flow cytometry also showed that the number of apoptotic cells increased after DYNLT1 knockdown compared with the control group at the same concentration(17.7% and 45.8% in BT 549 cells at 5.4ug/ml,respectively,p<0.05).5.In vivo experiments showed that: in the model of in situ tumorigenesis of ZR75-30 cell fat pad knockdown,the growth rate of tumor in the knockdown group was significantly lower than that in the control group;After paclitaxel intraperitoneal injection,the growth rate of tumor in both the control group and the DYNLT1 knockdown group was significantly slower than that in the control group,but the tumor shrinkage in the DYNLT1 knockdown group was significantly larger than that in the control group(P <0.01).The results of immunohistochemistry(IHC)showed that the expression of DYNLT1 protein in the tumor tissue of nude mice was significantly lower than that in the control group;At the same time,the expression of Ki67 in DYNLT1 knockdown group was significantly lower than that in the control group.In the untreated group,there was no significant difference in Caspase3,BCL2 and other related apoptotic proteins between the knockdown group and the control group.After paclitaxel intervention,the expression of Caspase3 in DYNLT1 knockdown group was significantly increased,while the expression of anti apoptotic protein BCL2 in the knockdown group was significantly decreased.6.Through immunohistochemical staining of 33 cases of breast cancer clinical specimens,including 10 cases in SD group,11 cases in PR group and 12 cases in PD group,the results showed that the proportion of breast cancer with high expression of DYNLT1 was 30% in SD group,9% in PR group and 83.3% in PD group.Conclusion1.DYNLT1 is highly expressed in breast cancer,and its expression is negatively correlated with the overall survival time and relapse free time of breast cancer patients.2.DYNLT1 promotes the proliferation of breast cancer cells mainly by affecting cell cycle G2/M phase.3.Overexpression of DYNLT1 significantly attenuates the chemosensitivity of breast cancer cells to paclitaxel. |
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