Study on the Effect of PIWIL2 Expression on EMT of Breast Cancer Stem Cells and Its Mechanism

Haiying Liu, Yan Liu, Fang Shan

Abstract


Objective: This study mainly elaborated the specific molecular mechanisms of breast cancer metastasis and EMT, which has important clinical value in judging the prognosis and treatment of breast cancer patients. Methods: A total of 493 patients with breast cancer were studied. The expression of PIWIL2 gene was analyzed by immunohistochemistry and section analysis, and the correlation between gene expression and clinicopathological parameters and prognosis of the patients was investigated. TGF-β1 was used to induce breast cancer cells, and EMT model was constructed at the same time to compare the changes of EMT model cells in the control group and obervation group. The expression of PIWIL2 gene in EMT model was detected by Western bloting. After transfection and RNA interference, PIWIL2 expression was silenced. At the same time, the morphological changes of the cells after the reduction of PIWIL2 gene expression were observed through microscope. The gene changes related to EMT were detected by RT-PCR and Western bloting. The expression of PIWIL2 gene was decreased by invasion test. And the expression of breast cell invasiveness and other related expressions such as MMP-13, MMP-9, VEGF shall also be decreased. The effect of MAPKERK and P13K/AKT pathway on the regulation of PIWIL2 expression was studied by obseving the cellular mophology through the microscope. And RT-PCR and Western bloting were used to detect the expression, including Snail, Vimentin and SIug. The effects of PIWIL2 and the downstream transcription factor SIug in ER pathway in breast cancer cells were analyzed by light microscopy, RT-PCR, Western bloting and Transwell invasion experiment. Results: PIWIL2 expression was negatively correlated with its survival rate in estrogen receptor a negative patients, while was not directly related to estrogen receptor positive patients. After the induction of TGF-β1 in MCF-7 breast cancer cells, the cells were spindle shaped and would lose the intercellular adhesion. With the development of EMT, the inhibition of PIWIL2 gene expression would block the effect of TGF-β1 on EMT in MCF-7 cell lines. The changes of EMT-related genes expression can be shown by RT-PCR and Western Bloting. SiRNA sclienced the Piwi12 expression, which will decrease the cellular invasiveness. At the same time, such matrix metalloproteinase as MMP9 and MMP13 and the mRNA transcription vascular endothelial growth factor was significantly down-regulated. After silencing the expression of SIug, all EMT of TGF-β1 gene will be inhibited, and the estrogen a signal pathway can inhibit the expression of PIWIL2 and the downstream transcription factor SIug. Slug is a common downstream transcription factor of estrogen a and PIWIL2, which also serves as an important bridge connecting estrogen a and Piwi12. It can accept Piwi12 and estrogen a to inhibit or stimulate signals, and then effectively regulate the EMT of breast cancer cells. In addition, ER signal can also participate in the expression of Piwi12 and antagonize Piwi12 to promote EMT.


Keywords


PIWIL2; Breast Cancer Stem Cells; EMT Mechanism

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References


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DOI: http://dx.doi.org/10.18686/aem.v10i3.202

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