| MicroRNAs (miRNAs) are small approximately 21-23 nucleotide RNA species that are expressed from specialized genes. It has been reported that miRNAs participate cell growth, cell death, apoptosis, and metastasis during the carcinogenesis and tumor progression. Recently, it was reported that miR-520b was involved in immune regulation. CD46, CD55 and CD59 are clusters of membrane-bound complement-regulatory proteins (mCRPs), which are able to inhibit the complement-dependent cytotoxicity (CDC) through inhibiting C5 convertase during the complement activation cascade. Therefore, they play important roles in protecting tumor cells from complement attack. However, the mechanism of regulation for mCRPs remains unclear. It is unclear whether miR-520b is involved in the tumor immunosuppression. In the present study, we investigated the correlation between miR-520b or miR-520e and CRP using parallel breast cancer cell lines, such as MCF-7 and LM-MCF-7 (National patent of invent, China) that have different metastatic capabilities. The investigation contains two parts as follows:Part one:miR-520b and miR-520e are involved in complement-dependent cytotoxicity in breast cancer cells by directly targeting CD46According to our previous data in microarray comparing hepatoma cells and normal liver cells, we found that miR-520b was downreguated in hepatoma cells. Furthermore, to investigate the function of miR-520b and miR-520e in immunosuppression during breast cancer progression, we firstly examined the expression levels of miR-520b or miR-520e in normal breast HBL-100 cell line and other three breast cancer cell lines, including MCF-7, LM-MCF-7, and MDA-MB-231, by quantitative real-time PCR.. The results indicated that the expression levels of miR-520b and miR-520e in breast cancer MCF-7, LM-MCF-7, and MDA-MB-231 cells were downregulated relative to HBL-100 cells. Futhermore, we also examined the expression level of miR-520b in normal breast tissue and breast tumor tissue. And we found that the expression level of miR-520b between normal breast tissue and breast tumor tissue was significangtly different. We also found that the resistant capability of the breast cancer cells to CDC was stronger than that of normal breast cell line. To further elucidate the effect of miR-520b and miR-520e on CDC of breast cancer cells, we transfected miR-520b or miR-520e mimics as well as its anti-mimics into MCF-7 or MDA-MB-231 cells. The complement-mediated cytolysis assay revealed that the overexpression of miR-520b or miR-520e mimics was able to enhance the CDC sensitivity in breast cancer cells. Conversely, the suppression of miR-520b or miR-520e in breast cancer cells could decrease the sensitivity of breast cancer cells to CDC. Thus, we conclude that miR-520b and miR-520e are involved in immunosuppressive regulation mediated by complement.Furthermore, to elucidate the mechanism of immunosuppressive regulation mediated by miR-520b and miR-520e in breast cancer cells, we identified the candidate targets of miR-520b and miR-520e using program online, TargetScan and DIANA microT3.0. We found that both miR-520b and miR-520e could target a complement cascade regulatory target, such as the membrane co-factor (MCP, CD46). Overexpression of miR-520b or miR-520e mimics could decrease the protein expression of CD46 rather than the mRNA level of CD46. Conversely, overexpression of anti-miR-520b or anti-miR-520e could increase the protein expression level of CD46. These data demonstrated that CD46 indeed acted as a target of miR-520b or miR-520e. We also indicated that miR-520b or miR-520e was able to regulate CD46 by directly targeting 3'UTR of CD46 mRNA. To further confirm the finding, we constructed a dual-luciferase reporter gene vector containing the 3'UTR targeting sites of miR-520b or miR-520e mRNA. The luciferase reporter gene assay showed that miR-520b or miR-520e was able to directly target the 3'UTR of CD46 mRNA. And the ELISA assay indicated that overexpression of miR520b or miR-520e could increase the deposition of C3b on breast cancer cells, so that it could promot the CDC effect on breast cancer cells. Thus, we conclude that both miR-520b and miR-520e are involved in CDC by directly targeting CD46.Part two:Effect of miR-520b targeting HBXIP on complement-dependent cytotoxicity in breast cancer cellsPrevious studies revealed that hepatitis B X-interacting protein (HBXIP) was involved in cancer cell growth and inhibiting apoptosis. In this study, we investigated the relationship between HBXIP and CDC in breast cancer cells. We firstly examined the expression levels of HBXIP in HBL-100, MCF-7, LM-MCF-7 and MDA-MB-231 cell lines by RT-PCR and Western blot analysis. We found that the expression levels of HBXIP in the three breast cancer cell lines were higher than that in normal breast cell line. Moreover, Western blot analysis showed that HBXIP expression was positively correlated with the expression levels of CD46, CD55 and CD59. Thus, we supposed that HBXIP might act as a regulator of mCRPs. Then, Dual-luciferase reporter gene assay revealed that the CD46 and CD59 promoter activities were increased in HBXIP stably transfected breast cancer cell line. Contrary, the promoter activities of CD46 and CD59 were decreased by RNAi targeting HBXIP mRNA in breast cancer cells. Furthermore, we showed that HBXIP upregulated the mRNA expression levels of CD46, CD55 and CD59 in breast cancer cells by real-time PCR as well as the protein expression level by Western blot. These data indicate that HBXIP is able to regulate the expression of CD46, CD55, and CD59 in breast cancer cells. We further found that HBXIP could activate NF-κB in breast cancer cells by reporter gene assay, Western blot analysis and immunofluorescence staining. The treatment with NF-κB specific inhibitor PDTC could inhibit the expression of CD46, CD55 and CD59 in breast cancer cells by luciferase reporter gene assay and Western blot analysis. Thus, we conclude that HBXIP is able upregulate the expression of CD46, CD55 and CD59 through NF-κB signaling pathway. Moreover, we found that HBXIP could increase the phosphorylated level of ERK1/2 rather than p38 and c-Jun N-terminal kinase (JNK). The activated ERK1/2 could upregulate the expression levels of CD46, CD55 and CD59. Contrarily, the specific inhibitor of MEK1 (upstream kinase of ERK1/2) PD98059 was able to decrease the expression levels of CD46, CD55 and CD59. Western blot analysis, dual-luciferase reporter gene assay and immunofluorescence staining assay showed that the activation of ERK1/2 could stimulate NF-κB translocation so that promoting the expression levels of CD46, CD55 and CD59. Therefore, we conclude that HBXIP is able to regulate the CDC sensitivity to protect the breast cancer cells from complement attack through upregulating CD46, CD55 and CD59 involving ERK1/2-NF-κB cascade signaling pathways. To investigate the upstream regulator of HBXIP, we constructed a luciferase reporter gene vector containing full-length 3'UTR of HBXIP mRNA according to the predicted bound sites of miR-520b using TargetScan program. Dual-luciferase reporter gene assay and Western blot analysis manifested that miR-520b was able to downregulate the expression of HBXIP at protein level by targeting its 3'UTR, suggesting that miR-520b targeting HBXIP is involved in the regulation of complement-dependent cytotoxicity in breast cancer cells.In summary, we found that miR-520b and miR-520e were involved in immunosuppression in breast cancer cells. Our finding showed that miR-520b and miR-520e regulated the complement attack in breast cancer cells by directly targeting CD46. Moreover, HBXIP, one of the targets of miR-520b, could upregulate the expression levels of mCRPs to protect breast cancer cells from complement attack involving ERK1/2-NF-κB cascade signaling pathways. Our data provide a novel regulatory mechanism in immunosuppression of breast cancer cells. |
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