MicroRNA-200c Enhances Radiosensitivity Of Human Breast Cancer Cells

[Purpose]Breast cancer is one of the most common malignancies in women. And in somedeveloped countries, its incidence is the highest among all the women-related cancers. Theincidence is lower in our country, however, it upgrades significantly,which is becomingthe major cause of cancer-related deaths in women．Radiation therapy, one of the threeprimary modalities, is used in breast cancer treatment and its benefits have been studiedextensively during the last decades. Clinical studies have demonstrated that whole-breastirradiation in women with breast cancer who undergo breast-conserving surgery, whichcould reduce the risk of local recurrence and improve survival outcomes. Due to individualradiosensitivity, cancer cells intrinsic radiosensitivity and various influence factors inclinical therapy, the result of radiotherapy is not good. So the assistant-aids to improve theclinical efficacy of radiation is desirable, which would sensitize malignant cells toradiation and increase tumor response while minimizing normal tissue toxicity by loweringeffective therapeutic doses. The agents of improvement malignant cells to radiosensitivityhave been studied for many years, but we still cannot find the safe and effective drugs inthe treatment of radiotherapy. Along with gradually increased understanding ofmicroRNAs (miRNAs), it has a key role in the progression and development of humanmalignant tumors.MicroRNAs (miRNAs) are small non-coding RNA gene products about22nt longthat are widely found in diverse organisms, including animals and plants. MiRNAsregulate gene expression by targeting mRNAs through base pairing at partially or fullycomplementary sites for cleavage or translational repression. And several miRNAs havebeen found to play key roles in breast cancer progression, including let-7family,miR-34,miR-125a and125b,miR-200family,miR-205,miR-10,miR-21and miR-17-92familyet al. Some miRNAs could enhances cancer cells sensitivity to iron radiation (IR), such asmiR145,miR-7and miR-34et al. Our lab has found that miR-200c was downregulated incancer tissue induced by IR, compared with the normal tissue. And overexpression ofmiR-200c could induce DNA damage. It is reported that miR-200c plays a tumorsuppressor role in cancer progression, and is considered as an important gene in regulatingEMT (epithelial to mesenchymal transition) and invasion. MiR-200expression regulates sensitivity to chemotherapeutic agents and is associated with cancer prognosis. AlthoughmiR-200c presents an attractive target for cancer therapy, its potential radiosensitizingeffects is unknown.The objective of this study is to investigate whether miR-200c could regulate breastcancer cell radiosensitivity, and the possible molecular mechanism.[Methods]The paper would be divided into three parts to investigate the effect and molecularmechanism of miR-200c on radiosensitivity in two breast cancer cell lines.Part Ⅰ: MiR-200c and radiosensitivity in breast cancer1. Real-time-PCR (RT-PCR) for miRNA was performed to measure the expression ofmiR-200c of the two breast cancer cell lines. U6RNA was used as a miRNA internalcontrol. Then the expressions of miR-200c of the cells transfected with miR-200c mimicsor miR-200c inhibitor were measured. Finally, cell clonogenic assay were performed toobserve the two breast cancer cell radiosensitivity.2. RT-PCR for miRNA was performed to measure the expression of miR-200c, whilethe breast cancer cells were exposure to different dose of IR.3. RT-PCR for miRNA was performed to measure the expression of miR-200c, whilethe breast cancer cells were exposure to5-aza-2'-deoxycytidine.Part Ⅱ: The effect of miR-200c on radiosensitivity in breast cancer cells1. CCK8assay and cell clonogenic assay were performed to detect the effect ofmiR-200c overexpression and IR on cell generation.2. FACS was used to detect the effect of miR-200c overexpression and IR onapoptotic index.3. A double-strand break (DSB) assay was done to detect the effect of miR-200coverexpression and IR by counting the colocalized phospho-γH2AX foci.4. CCK8assay and cell clonogenic assay were performed to detect the effect ofmiR-200c knockdown and IR on cell generation.Part Ⅲ: Molecular mechanism of miR-200c on radiosensitivity in breast cancer cells1. Bioinformatics was used to predict the target of miR-200c.2. A luciferase activity assay was performed with the dual luciferase reporter assaysystem to testify whether miR-200c targeted TBK13'UTR.3. CCK8assay and cell clonogenic assay were performed to detect the effect of TBK1 knockdown and IR on cell generation.4. FACS was used to detect the effect of TBK1overexpression on radiosensitivityinduced by miR-200c.[Results]Part Ⅰ: MiR-200c and radiosensitivity1. The expression of miR-200c was obviously higher in the MCF-7breast cancer cellsthan the MDA-MB-231cells. In contrast, MCF-7breast cancer cells (SF2,0.5±0.03)showed higher radiosensitivity, compared with MDA-MB-231cells (SF2,0.8±0.53).2. The expression of miR-200c in the MCF-7and MDA-MB-231breast cancer cellswere induced by IR and the induction was dose dependent.3. We found that miR-200c knockdown partially inhibited IR-induced death in breastcancer cells, compared with the control transfected with inh-NC.Part Ⅱ: The effect of miR-200c on radiosensitivity in breast cancer cells1. We found that overexpression of miR-200c significantly enhanced IR-induceddeath in breast cancer cells, compared with the control transfected with miR-NC (MCF-7cells: control,0.75±0.01; miR-NC,0.78±0.02; miR-200c,0.62±0.01; MDA-MB-231cells:control,0.90±0.01; miR-NC,0.90±0.03; miR-200c,0.72±0.01). Notably, miR-200c alsoincreased cell death in unirradiated group (p<0.05). overexpression of miR-200ccooperated with IR to reduce clonogenic growth of MCF-7and MDA-MB-231cells, ascompared with the control transfected with miR-NC (MCF-7cells: control,0.069±0.003;miR-NC,0.069±0.002; miR-200c,0.027±0.003; MDA-MB-231cells: control,0.27±0.023;miR-NC,0.29±0.035; miR-200c,0.19±0.048).2. Using Annexin V/PI staining, we noticed that at8Gy, overexpression of miR-200csignificantly enhanced IR-induced apoptosis in both cell lines compared with the controltransfected with miR-NC. Detailed analyses revealed that>30%apoptotic cells weredetected in the miR-200c population compared with11%cells in the miR-NC populationin MDA-MB-231cells. Notably, miR-200c also increased cell apoptosis in unirradiatedgroup。3. We observed that more foci formed in overexpression of miR-200c cells comparedwith the control transfected with miR-NC cells. Detailed analyses in MCF-7cells revealedthat about90%of the control cells didnot contain any foci, and whereas about45%did notcontain any foci and33%have1-10foci in transfected miR-200c cells. After IR, about 43%of the control cells did not contain any foci, and whereas about17%did not containany foci, about37%have more than10foci in transfected miR-200c cells. A uniform resultin MDA-MB-231breast cancer cells was revealed.Part Ⅲ: Molecular mechanism of miR-200c on radiosensitivity in breast cancer cells1. The expression of miR-200c was upregulated by5-aza, whether irradiation orunirradiation (0Gy:3.2±0.35；4Gy:4.7±0.50).2. As predicted by several methods for target gene prediction, we found miR-200cwas identified as a candidate target gene of TBK1. And the TBK1-encoded mRNAcontained a3'UTR element, which was partially complementary to miR-200c.MiR-200c significantly reduced the luciferase activity of the WT construct of theTBK13'UTR compared with the negative control, whereas such a suppressive effect wasnot observed in the Mut construct of TBK13'UTR cells.3. Knockdown TBK1significantly enhanced IR-induced death in breast cancer cells,compared with the control transfected with siRNA.4. Our results showed that overexpression of miR-200c could reduce TBK1expression compared with the control transfected with miR-NC in breast cancer cells, andon the contrary, the inhibition of miR-200c increased the TBK1expression. And TBK1andmiR-200c simultaneous expression markedly inhibited miR-200c-induced apoptosis. ZEB1or ZEB2and miR-200c coexpression prevented miR-200c-induced apoptosis.[Conclusion]In summary, the present work showed for the first time that miR-200c exhibitedpotentially synergistic activity in combination with radiotherapy against breast cancer cells.The possible underlying mechanisms seem to be multifaceted because of many targets ofmiR-200c. And we found that not all of miR-200c-effect on radiosensitivity could beattributed to targeting of TBK1, ZEB1or ZEB2. We further proved thatmiR-200c-mediated repression of TBK1was also involved in enhancing radiosensitivity.Our results suggested that miR-200c overexpression may represent a therapeutic target inradiotherapy.