| Part Ⅰ Talin2 knockdown regulates the invasion of human breast cancer cells MDA-MB-231 via alteration of the tumor microenvironmentObjective:Immunohistochemical detection was performed on postoperative tissue samples of breast cancer patients to understand the distribution of Talin2 protein in breast cancer tissues and adjacent tissues.By studying the effects of Talin2 gene knockdown on the migration and invasion of MDA-MB-231 cells,on the AKT signal transduction and glucose and lactate dehydrogenase content in the tumor microenvironment,to explore the feasibility of Talin2 gene knockdown in the treatment of breast cancer.Methods:obtaining postoperative tissue samples from untreated breast cancer patients,Immunohistochemistry(IHC)was performed using tissue microarray technology after pathological confirmation.Talin2 in human breast cancer MDA-MB-231 cells was knocked down with shRNA,and then the knockdown effect of Talin2 was confirmed by RT-qPCR and Western blot analysis.Cell invasion ability of MDA-MB-231 cells with Talin2 knockdown was observed by transwell invasion method,and cell migration ability was observed by scratch test.The contents of glucose and lactate dehydrogenase in the cells were measured by kits,and the expression levels of AKT,pAKT,mTOR,pmTOR,HIF-1α,total S6K,and pS6K were detected by Western blot analysis.Results:Breast cancer tissues is a 5.6 times higher expression of Talin2 than adjacent normal tissue samples.The levels of endogenous Talin2 protein were significantly lower in the two groups of MDA-MB-231 cell lines that Talin2 were successfully knocked down,and the expression level of Talin2 mRNA was significantly reduced.The invasive ability of MDAMB-231 cells decreased after Talin2 knocked down in the two groups,and the migration ability decreased significantly after 48 hours.The levels of glucose and lactate dehydrogenase in the cells were decreased.Western blot analysis showed that the levels of AKT,pAKT,mTOR,pmTOR,HIF-1α,total S6K,and pS6K in the cells were decreased.Conclusions:Compared with the normal tissues,the expression of Talin2 is higher in breast cancer tissues.Talin2 knockdown may reduce the basal glucose and LDH levels by down-regulating AKT/mTOR signaling,which affects the tumor microenvironment.As a result,the invasion and migration ability of MDA-MB-231 cells was significantly reduced.Therefore,Talin2 knockdown may be a new approach for the breast cancer treatment.Part Ⅱ 18F-FDG metabolic changes to evaluate talin2 knockdown on the inhibitory effect of human breast cancer cell MDA-MB-231Objective:To observe the inhibitory effect and 18F-FDG uptake changes of human breast cancer cells before and after talin2 knockdown,to explore the feasibility of observing the therapeutic effect of talin2 knockdown through 18F-FDG metabolism changes.Methods:Human breast cancer cells line MDA-MB-231 with talin2 knockdown were cultured and subcultured,then added with 18F-FDG in sugar free medium,incubating the cell at 37℃for 30min,60min,90min and 120min.After washing and centrifugation,the cell CPM count(B)and the supernatant CPM count(F)were measured with a gamma meter.The cell-binding rate of 18F-FDG was calculated.MDA-MB-231 cell Talin2 knockdown in logarithmic growth phase was inoculated into the subcutaneous site of 6-week-old severe combined immunodeficiency Scid mice.The tumor grew for 4 weeks and the tumor size was measured.18F-FDG was injected into the tail vein of mices bearing human breast cancer cell line MDAMB-23 1 or human breast cancer cell line Talin2 shRNA2 knockdown.The mice were killed by cervical dislocation in three periods,the percentage of intake per gram of different tissues and organs(%ID/g)was measured and statistically analyzed.3 tumor-bearing mice of each of the above two types were randomly selected and their tumor tissues were taken out.The protein expression level of Talin2 in these tissues was measured by the Western blot.At the same time,the human breast cancer cells MDA-MB-231 and Talin2 shRNA2 knockdown were subcultured for 28 days,and the Talin2 protein in these cell was measured by the Western Blot.Results:The cell binding rate of 18F-FDG for the Talin2 shRNA knockdown human breast cancer MDA-MB-231 cells increased gradually with time.At 90min and 120min,compared with the control group,the cell binding rate of 18F-FDG in talin2 shRNA knockdown group decreased(P<0.05).In vivo distribution experiment of tumor bearing mice,it was found that the uptake of 18F-FDG in tumor tissue of the mice in the Talin2 shRNA2 knockdown group was lower than that in the control group at different periods of 60min,90min and 120min(P<0.05).Tumor model growth inhibition experiments show:the Talin2 shRNA2 knockdown mice has slow tumor growth and small size.Within 2 weeks,compared with the control group,the inhibition of tumor growth was more obvious with the prolongation of time.But after 2 weeks,the tumor growth rate of Talin2 shRNA2 knockdown mice increased slowly.The tumor tissues of the above 2 types of tumor-bearing mice were measured by the Western Blot,the Talin2 protein in tumor tissues of Talin2 shRNA knockdown group was found lower than that of the control group.At the same time,after subculturing 28 days,Western blot also showed that the expression level of talin2 protein in the Talin2 shRNA knockdown group was lower than that in the control group.Conclusions:Talin2 knockdown can significantly inhibit the growth of breast cancer.The inhibition was weakened in the later stage.The cell binding rate of 18F-FDG in breast cancer cells with talin2 knockdown was decreased,and the uptake of 18F-FDG in tumor tissue was decreased.It is suggested that 18F-FDG PET/CT can be used to monitor the efficacy and prognosis of Talin2 knockdown treatment. |
PDF Full Text Request