The Effect On The WNT Signaling Pathway Of The Human Breast Cancer Stem Cell Of RNA Interference Inhibited STAB1

Background:Breast cancer is one of the most common malignant tumor in women，recurrenceand metastasis are the main cause of treatment failure. It is studied that there is asmall number of cancer stem cells in tumors, which is the root cause of tumorigenesis,recurrence and metastasis. CSC is in regulating network of a series of signaltransduction pathways, which contains the wnt/β-cateninwnt signaling pathway.SATB1（special AT rich sequence binding protein，SATB1）a nuclear matrix bindingprotein in tissue-specific expression, and it causees the tumorigenesis, recurrence andmetastasis, which can regulate the wnt/β-cateninwnt signaling pathway.Objective：To observe the change of the wnt-1、β-catenin and discuss how theSATB1affect the wnt/β-cateninwnt signaling pathway of CSC, we inhibit the SATB1by RNAi.Methods:1Synthetize4kinds of lentiviral vectors which contain the RNA interferencesequence on SATB1according to the SATB1gene coding sequence in GenBankand the design principles.2To gain the negative control and explore the infect condition, we infected theMCF-7with the NC-GFP lentiviral vectors.3Infect4groups of MCF-7with4kinds of lentiviral vectors which contain theRNA interference sequence on SATB1,Examine the SATB1mRNA and SATB1protein by fluorogenic quantitative polymerase chain reaction(FRQ-PCR), andWestern blot, select the most efficient MCF-7cell in RNA interference withSATB1(MCF-7/siSATB1).4Sort the CSC from MCF-7、MCF-7/siControl and MCF-7/siSATB1according tothe special immune maker CD44+CD24-of breast cancer CSC by flowcytometry. 5Examine the SATB1mRNA、wnt-1mRNA、β-catenin mRNA from the threegroups of CSC above.The wnt-1and β-catenin is the key molecule in theWnt/β-cateninwnt signaling pathway.Results:1、Construct and sequence the recombined lentivirus SATB1-RNAi-LV successfully:SATB1/GV115-RNAi#1、SATB1/GV115-RNAi#2、SATB1/GV115-RNAi#3、SATB1/GV115-RNAi#4，and the lentivirus titer is1.0×109TU/ml.2、GFP fluorescent protein was highest over70%after SATB1-RNAi-LV infectMCF-7cell3-4days.3、The CTof the SATB1mRNA of the group of SATB1/GV115-RNAi#4is28.91±1.56,the protein optical destiny of it is5.382±1.21，which is significantlylower than the other, so we selected the group of SATB1/GV115-RNAi#4as theMCF-7/siSATB1.4、The immune maker CD44+CD24-express in the MCF-7、MCF-7/siControl�'�MCF-7/siSATB1:①MCF-7：4.39%；②MCF-7/siControl：4.31%；③MCF-7/siSATB1：2.59%。And extract the RNA after sort the CSC from MCF-7、MCF-7/siControl and MCF-7/siSATB1.5、Examine the SATB1mRNA、wnt-1mRNA、β-catenin mRNA from the threegroups of CSC by FRQ-PCR:the SATB1mRNA of three groups is26.738±0.622、26.958±0.940、29.18±0.556（P＜0.05）in that order, it showsthat SATB1of the group of MCF-7/siControl has decline86.3%. Wnt-1mRNAof three groups is28.628±0.604、28.883±0.680、30.095±0.762（P＜0.05）in thatorder, it shows that Wnt-1of the group of MCF-7/siControl has decline63.3%.β-catenin mRNA of three groups is30.073±0.1.126；28.55±0.32；29.21±0.64（P＜0.05）in the order, it shows that SATB1of the group of MCF-7/siControl hasdecline69.3%.Conclusion:RNAi inhibit the SATB1of the CSC, and it can affect the the wnt mRNA andβ-catenin mRNA of the wnt signaling pathway.