The Effect Of WNT6down Regulation On The Proliferation And Growth Of MDA-MB-231Cells

Objectives:To investigate the effect of WNT6down regulation on proliferation andgrowth of breast cancer cells (MDA-MB-231),and define the function of WNT6inTriple Negative Breast Cancer(TNBC),for illuminating the molecular mechanisms ofTNBC.Methods:We chose triple negative breast cancer MDA-MB-231cell line as theexperimental subject,the WNT6as the target. In our essay, we designed four groupsincluding siRNAl、siRNA2、siRNA3, which with different targeted sequences, and thescramble siRNA(scRNA). We transfected the siRNA into MDA-MB-231cellsthrough Lipofectamin2000respectively. Then detected the expression of WNT6byRT-PCR and Western bloting, at last chose the group which one has the best silenceeffect as experimental group. Using RT-PCR and Western bloting assay to detect theexpression of WNT6of experimental group on the mRNA and protein level, and thenot transfection cells and scRNA as control. MTT assay was used to estimate the effectof WNT6down regulation on proliferation and growth of MDA-MB-231cells, therebydrew the growth curve in each group. Finally, soft AGAR colony experiment was usedto observe anchor dependenciest of cell lines for each group.Results:We discussed the expression of mRNA for WNT6in each group analysed byRT-PCR.It was observed that WNT6could be obviously down-regulated in the groupswhich are transfected siRNAl、 siRNA2、 siRNA3. Compared to the cell lineMDA-MB-231without transfection, reduce about50%(p<0.01)、40%(p<0.05) and20%(p<0.05) respectively. Western bloting assay showed that the expression of thewnt6encoding protein reduce about45%(p<0.01)、28%(p<0.05)and10%(p>0.05),compared to the control group. Besides we found that siRNAl has the best silence effect. So we chose the one as experimental group in the subsequent experiments, andthe not transfection cells and scRNA as control. After detected WNT6expression inexperimental groups, through RT-PCR and Western bloting assay, WNT6mRNA andprotein expression reduced nearly50%and40%respectively, showing statisticalsignificance(p<0.05), compared to the control groups. MTT assay indicated that thecells after transfected48h and72h, the proliferation rate reduced about48%and49%respectively. Compared to the control groups, the growth of MDA-MB-231cells wereclearly suppressed in experimental group, showing statistical significance(p<0.05).Compared to the control groups, the colony forming efficiecy of the experimentalgroup was slow, which showing statistical significance(p<0.05). The results suggestedthat not only the colony forming speed of experimental group was slower than thecontrol groups, but also the colony numbers and clone formation ability was poor.Conclusions:The down-regulation of WNT6expression could inhibit theproliferation and growth of breast cancer cell lines MDA-MB-231.