| ObjectivesPrevious research by the research group showed that Liuwei Dihuang Pill can inhibit the growth of tumors in mice with triple negative breast cancer,prolong the survival of tumor-bearing mice and inhibit tumor metastasis,but the molecular mechanism of tumor metastasis inhibition is still unclear.This study investigated the effects of Liuwei Dihuang Pill on the migration of MDA-MB-231 cells and the expression of MAP3K1,SOX2,FOXD3 and KLF4 in triple negative breast cancer cells MDA-MB-231 after MAP3K1 and SOX2 silencing.To further clarify the molecular mechanism of Liuwei Dihuang Pill in inhibiting migration of triple negative breast cancer.Methods1.Preparation of drug-containing serum:The research group selected SPF adult SD rats,and used Liuwei Dihuang Pill to administer three doses of low(1.07g/kg/d),medium(5.35g/kg/d)and high(10.70g/kg/d).After the day,blood was taken from the abdominal aorta after anesthesia,and the drug-containing serum of Liuwei Dihuang Pill was separated.2.Design and screening of RNAi plasmids:(1)SOX2-RNAi plasmid design:Take the target gene as the target gene,and design 3 pairs of RNAi target sequences based on the RNA interference(RNAi)sequence design principles.Directed clones form small interfering RNAs that bind to endogenous mRNA.Recombinant vector transfer plasmids containing silenced fragments of the target gene were constructed,and PCR and sequencing of the recombined plasmids were performed to determine whether they contained silenced fragments.Results: The silenced fragments of the target gene were successfully transcribed and translated into the vector transfer plasmid.Three pairs were inserted The sequence is exactly the same as the designed target gene fragment.Re-introduction into MDA-MB-231 cells to achieve no or low expression of the target gene in the cell.(2)Screening of SOX2-RNAi plasmid:Design and construct an RNAi interference sequence of 1 negative plasmid and 3 SOX2 sites,transfect them into breast cancer cell MDA-MB-231 in an instant,and observe the transfection after 6 hours,48 hours After extracting the protein and measuring the protein expression level,the most obvious decrease in expression was the plasmid with the best silencing effect.3.Cell migration analysis:Real Time Cellular Analysis(RTCA),RTCA xCELLigence DP developed by Essen Biotechnology and the CIM-Plate16 migration detection plate used together.The bottom of the upper chamber is a PET microporous membrane integrated with a micro gold electrodesensor.The sensor that reaches the bottom of the upper chamber through the microporous membrane causes the impedance of the sensor to change.The cell migration curve is recorded by software and computer real-time calculation of the impedance change of the sensor,and the cell index is studied.(CI),analysis of changes in the effects of cell migration capacity through an increase or decrease in cell index.4.Effect of drug-containing serum on RTCA and protein expression after silencing SOX2:Screen the best plasmid for silencing SOX2,add it to the medium containing breast cancer cells MDA-MB-231 in different serum groups,determine cell migration by RTCA method,and detect breast cancer cell MDA-MB-231 cells by Western blotting Expression of MAP3K1,SOX2,FOXD3,and KLF4 protein levels;Immunofluorescence was used to detect the expression of MAP3K1,FOXD3,and KLF4 cells in breast cancer cell MDA-MB-231 cells and their fluorescence levels.Results1.The effect of serum containing Liuwei Dihuang Pill(containing medicated serum,the same below)on the migration of MDA-MB-231 cells:(1)Preliminary screening of MDA-MB-231 cell migration in triple negative breast cancer cells:The MDA-MB-231 cell migration index was measured by real-time label-free method for about 48 hours.The results showed that compared with 20,000,40,000 and 80,000 cells,60,000 migration indices were the highest,and the difference was statistically significant(P<0.05),indicating 60,000.The cells are best suited for subsequent experiments.(2)Effect of drug-containing serum on migration of MDA-MB-231 cells:Compared with the control group,Liuwei Dihuang Pills containing serum high,medium and low dose groups and paclitaxel positive drug group can reduce the migration ability of breast cancer cells MDA-MB-231,indicating that the paclitaxel positive drug group,containing Liuwei Dihuang Wan The serum low-dose group,the middle-dose group and the high-dose group all inhibited the migration of triple-negative breast cancer MDA-MB-231.In the Liuwei Dihuang Pill-containing serum group,the high-dose group of Liuwei Dihuang Pill had the most inhibitory effect.Well,the difference was statistically significant(P < 0.05).2.The effect of drug-containing serum on the migration of MDA-MB-231 cells after silencing SOX2:(1)Screening of SOX2-RNAi plasmid:Western blotting method was used to screen out the SOX2-RNAi(ID: 69217-1),SOX2-RNAi(ID: 69218-1),SOX2-RNAi(ID: 69219-2),and negative control(ID: CON-313)can silence the expression of SOX2 protein in triple-negative breast cancer cells MDA-MB-231,of which SOX2-RNAi-17(ID: 69217-1)has the best silencing effect,and the difference is statistically significant(P <0.05).(2)Effect of drug-containing serum on MDA-MB-231 cell migration after silencing SOX2:Compared with the negative plasmid group,the migration index of the positive plasmid+paclitaxel group,positive plasmid+high,medium and low dose drug-containing serum groups decreased,the difference was statistically significant(P <0.05).Compared with the positive plasmid group,the migration index of the positive plasmid+paclitaxel group,positive plasmid+high,medium and low dose drug-containing serum groups decreased,the difference was statistically significant(P <0.05).3.Effect of medicated serum on expression of related proteins in MDA-MB-231 cells after silencing SOX2(1)Effect of medicated serum on the expression of MAP3K1,FOXD3,and KLF4 in MDA-MB-231 cells after silencing SOX2:Compared with the positive plasmid group,the expression of MAP3K1 in the control group was reduced,and the positive plasmid + paclitaxel group and the positive plasmid +The expression levels of MAP3K1 protein in the high-,medium-,and low-dose drug-containing serum groups were all increased,and the difference was statistically significant(P <0.05).Compared with the negative plasmid group,the expression of MAP3K1 protein in the positive plasmid + paclitaxel group,the positive plasmid + high,medium,and low dose drug-containing serum groups were all increased,and the difference was statistically significant(P <0.05).(2)Effect of medicated serum on the expression of SOX2 protein in MDA-MB-231 cells after silencing SOX2:Compared with the positive plasmid group,the expression of SOX2 in the control group increased.The expression of SOX2 protein in the medium and low dose drug-containing serum groups was reduced,and the difference was statistically significant(P <0.05).Compared with the negative plasmid group,the expression of SOX2 protein in the positive plasmid + paclitaxel group,the positive plasmid + high,medium,and low dose drug-containing serum groups were all reduced,and the difference was statistically significant(P <0.05).4.After silencing SOX2,the effect of drug-containing serum on protein immunofluorescence expression in MDA-MB-231 cells:Effect of medicated serum on the immunofluorescence of MAP3K1,FOXD3,and KLF4 in MDA-MB-231 cells after SOX2 silencing: MAP3K1,FOXD3,and KLF4 after silencing all had different fluorescence intensities in the eight groups.MAP3K1 and FOXD3 are localized and expressed in the cytoplasm and nucleus,mainly cytoplasm,and KLF4 is mainly nucleus.Compared with the positive plasmid,the fluorescence intensity of MAP3K1 decreased in the control group,and the fluorescence intensity of MAP3K1 increased in the positive plasmid +paclitaxel group,the positive plasmid + high,medium,and low dose drug-containing serumgroups,and the difference was statistically significant(P < 0.05).Compared with the negative plasmid group,the MAP3K1 fluorescence intensity of the positive plasmid + paclitaxel group,the positive plasmid + high,medium,and low dose drug-containing serum groups were all increased,and the difference was statistically significant(P <0.05).Conclusion1.The four interference sites constructed have a reduced effect on the expression of SOX2 protein,and SOX2-RNAi-17 has the best silencing effect.2.Liuwei Dihuang Pill can inhibit the metastasis of triple negative breast cancer cells MDA-MB-231 cells,and the high dose of six Dihuanghuang pills has the best inhibitory effect.3.After silencing SOX2,Liuwei Dihuang Pill can up-regulate the protein expression of MAP3K1 in triple negative breast cancer cell MDA-MB-231 cells,down-regulate the expression of SOX2 protein in triple negative breast cancer cell MDA-MB-231 cells,and up-regulate the triple negative mammary gland.Protein expression of FOXD3 in cancer cell MDA-MB-231 cells up-regulated protein expression of KLF4 in triple negative breast cancer cell MDA-MB-231 cells.Therefore,Liuwei Dihuang Pill can inhibit the metastasis of triple-negative breast cancer,and it is likely to inhibit the metastasis of triple-negative breast cancer by regulating the protein expression of MAP3K1,SOX2,FOXD3,and KLF4.MAP3K1,SOX2,FOXD3,and KLF4 may be important targets for the prevention and treatment of triple-negative breast cancer. |
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