Long non-coding RNA ARHGAP5-AS1 inhibits migration of breast cancer cell via stabilizing SMAD7 protein.

PURPOSE: Tumor metastasis is the main cause of death from breast cancer patients and cell migration plays a critical role in cancer metastasis. Recent studies have shown long non-coding RNAs (lncRNAs) play an essential role in the initiation and progression of cancer. In the present study, the role of an LncRNA, Rho GTPase Activating Protein 5- Antisense 1 (ARHGAP5-AS1) in breast cancer was investigated. METHODS: RNA sequencing was performed to find out dysregulated LncRNAs in MDA-MB-231-LM2 cells. Transwell migration assays and F-actin staining were utilized to estimate cell migration ability. RNA pulldown assays and RNA immunoprecipitation were used to prove the interaction between ARHGAP5-AS1 and SMAD7. Western blot and immunofluorescence imaging were used to examine the protein levels. Dual luciferase reporter assays were performed to evaluate the activation of TGF-ß signaling. RESULTS: We analyzed the RNA-seq data of MDA-MB-231 and its highly metastatic derivative MDA-MB-231-LM2 cell lines (referred to as LM2) and identified a novel lncRNA (NR_027263) named as ARHGAP5-AS1, which expression was significantly downregulated in LM2 cells. Further functional investigation showed ARHGAP5-AS1 could inhibit cell migration via suppression of stress fibers in breast cancer cell lines. Afterwards, SMAD7 was further identified to interact with ARHGAP5-AS1 by its PY motif and thus its ubiquitination and degradation was blocked due to reduced interaction with E3 ligase SMURF1 and SMURF2. Moreover, ARHGAP5-AS1 could inhibit TGF-ß signaling pathway due to its inhibitory role on SMAD7. CONCLUSION: ARHGAP5-AS1 inhibits breast cancer cell migration via stabilization of SMAD7 protein and could serve as a novel biomarker and a potential target for breast cancer in the future.

Hypoxia activated long non-coding RNA HABON regulates the growth and proliferation of hepatocarcinoma cells by binding to and antagonizing HIF-1 alpha.

The adaptation of tumour cells to hypoxic microenvironment is one of the most significant characteristics of many malignant tumour diseases including hepatocarcinoma. Recently, long non-coding RNAs (lncRNAs) have been reported to play important roles in the various levels of gene regulation thus functioning in growth and survival of tumour cells. Here, new hypoxia-related lncRNAs in hepatocarcinoma cells were screened and validated by lncRNA chip-array as well as real-time RT-PCR. Among them, a hypoxia-activated lncRNA that we identified and termed Hypoxia-Activated BNIP3 Overlapping Non-coding RNA (HABON), was not only regulated by hypoxic-induced factor-1α (HIF-1α) but its expression increased significantly under hypoxia in tumour cells. We deciphered the biological characteristics of HABON including its cell localization, genomic location, as well as its full-length sequence, and proved HABON could promote growth, proliferation and clone-formation of hepatocarcinoma cells under hypoxia. Then, we revealed that HABON was transcriptionally activated by HIF-1α in hypoxic cells, furthermore, it could interact with HIF-1α and promote its protein degradation, thus affecting transcription of HIF-1α's target genes to exert its effects on cells. Besides, the elevated expression of HABON under hypoxia could promote the transcriptional activation of BNIP3 through HIF-1α, and increasing the expression level of BNIP3. This research provides a novel clue for the adaptive survival and growth mechanism of tumour under hypoxia, and gives a way to reveal the nature of tumour cells' resistance characteristics to harsh microenvironment.

MicroRNA-630 inhibits breast cancer progression by directly targeting BMI1.

MicroRNAs (miRNAs) play critical roles in breast cancer cell biological processes, including proliferation and apoptosis by inhibiting the expression of their target genes. Herein, we reported that miR-630 overexpression initiates apoptosis, blocks cell cycle progression and suppresses cell proliferation in breast cancer cells. Furthermore, BMI1, a member of polycomb group family, was identified as a direct target of miR-630, and there was a negative correlation between the expression levels of BMI1 and miR-630 in human breast cancer samples. With a series of biology approaches, subsequently, we proved that BMI1 was a functional downstream target of miR-630 and mediated the property of miR-630-dependent inhibition of breast cancer progression. Taken together, these findings provide further evidence on the tumor-suppression function of miR-630 in breast cancer, and clarify BMI1 as a novel functional target gene of miR-630.

MiR-124 targets Slug to regulate epithelial-mesenchymal transition and metastasis of breast cancer.

MicroRNAs (miRNAs or miR) have been integrated into tumorigenic programs as either oncogenes or tumor suppressor genes. The miR-124 was reported to be attenuated in several tumors, such as glioma, medulloblastoma and hepatocellular carcinoma. However, its role in cancer remains greatly elusive. In this study, we show that the miR-124 expression is significantly suppressed in human breast cancer specimens, which is reversely correlated to histological grade of the cancer. More intriguingly, ectopic expression of miR-124 in aggressive breast cancer cell lines MDA-MB-231 and BT-549 strongly inhibits cell motility and invasive capacity, as well as the epithelial-mesenchymal transition process. Also, lentivirus-delivered miR-124 endows MDA-MB-231 cells with the ability to suppress cell colony formation in vitro and pulmonary metastasis in vivo. Further studies have identified the E-cadherin transcription repressor Slug as a direct target gene of miR-124; its downregulation by miR-124 increases the expression of E-cadherin, a hallmark of epithelial cells and a repressor of cell invasion and metastasis. Moreover, knockdown of Slug notably impairs the motility of MDA-MB-231 cells, whereas re-expression of Slug abrogates the reduction of motility and invasion ability induced by miR-124 in MDA-MB-231 cells. These findings highlight an important role for miR-124 in the regulation of invasive and metastatic potential of breast cancer and suggest a potential application of miR-124 in cancer treatment.

[The down-regulation of miR-129 in breast cancer and its effect on breast cancer migration and motility].

To search the microRNAs (miRNA) which suppress metastasis of breast cancer, we utilize three well known micoRNA target prediction programs, Targetscan, Pictar and miRanda, to select the microRNAs that target the genes related to tumor metastasis. We chose MDA-MB-231 with high metastasis ability as the model to evaluate the effect of miRNAs on cell motility through Transwell migration assay. After the first round of screening, miR-129 is found to significantly inhibit the migration of MDA-MB-231 both in Transwell migration assay and wound healing assay. Furthermore, miR-129 also shows great suppressive ability to cell mobility and migration in another two breast cancer cell lines BT549 and MDA-MB-435s. Most importantly, miR-129 is down-regulated both in breast cancer tissues compared with the paired adjacent normal breast tissues, and in breast cancer cell lines compared with normal breast epithelial cell MCF10A (P < 0.05). These results indicate that over-expression of miR-129 could inhibit breast cancer motility and migration, and the down-regulation of miR-129 may participate in the breast cancer migration and metastasis.

Pathologically decreased miR-26a antagonizes apoptosis and facilitates carcinogenesis by targeting MTDH and EZH2 in breast cancer.

The role of miR-26a in carcinogenesis appears to be a complicated one, in the sense that both oncogenic and tumor suppressive effects were reported in cancers such as glioblastoma and hepatocellular carcinoma, respectively. Here, we report for the first time that miR-26a is downregulated in breast cancer specimens and cell lines and its transient transfection initiates apoptosis of breast cancer cell line MCF7 cells. Furthermore, retrovirus-delivered miR-26a impairs the in vitro colony forming and in vivo tumor-loading ability of MCF7 cells. Subsequently, MTDH and EZH2 are identified as two direct targets of miR-26a and they are significantly upregulated in breast cancer. MCF7 xenografts with exogenous miR-26a show that a decrease in expression of both MTDH and EZH2 is accompanied by an increase in apoptosis. Moreover, knockdown of MTDH causes apoptosis while reexpression of MTDH partially reverses the proapoptotic effect of miR-26a in MCF7 cells. Our findings suggest that miR-26a functionally antagonizes human breast carcinogenesis by targeting MTDH and EZH2.

Nuclear translocation of dihydrofolate reductase is not a pre-requisite for DNA damage induced apoptosis.

Dihydrofolate reductase (DHFR) is a key enzyme for the synthesis of thymidylate, and therefore, of DNA. By applying subcellular proteomic analysis, we identified that the DHFR protein was translocated from cytoplasm into the nucleus when apoptosis was induced by NSC606985, a camptothecin analogue. The nuclear translocation of DHFR protein during apoptosis was independent of the cellular context, but it was more sensitive in cell death induction by DNA damaging agents such as doxorubicin, etoposide and ultraviolent radiation than endoplasmic reticulum stressors (brefeldin-A and tunicamycin) and anti-microtubule agents (paclitaxel and nocodozole). The addition of methotrexate almost completely blocked the nuclear translocation of DHFR protein. Further investigations showed that the nuclear translocation of DHFR was not a pre-requisite for DNA damage induced apoptosis. Therefore, its potential biological significance remains to be further explored.

MiR-133b targets Sox9 to control pathogenesis and metastasis of breast cancer.

The miR-133b, a commonly recognized muscle-specific miRNA, was reported to be deregulated in many kinds of cancers. However, its potential roles in tumorigenesis remain greatly elusive. Herein, we demonstrate that miR-133b is significantly suppressed in human breast cancer specimens, which is reversely correlated to histological grade of the cancer. Ectopic expression of miR-133b suppresses clonogenic ability and metastasis-relevant traits in vitro, as well as carcinogenesis and pulmonary metastasis in vivo. Further studies have identified Sox9, c-MET, and WAVE2 as direct targets of miR-133b, in which Sox9 contributes to all miR-133b-endowed effects including cell proliferation, colony formation, as well as cell migration and invasion in vitro. Moreover, re-expression of Sox9 reverses miR-133b-mediated metastasis suppression in vivo. Taken together, these findings highlight an important role for miR-133b in the regulation of tumorigenesis and metastatic potential of breast cancer and suggest a potential application of miR-133b in cancer treatment.

MicroRNA-494 inhibits breast cancer progression by directly targeting PAK1.

MicroRNA (miRNA) is involved in the progression and metastasis of diverse human cancers, including breast cancer, as strong evidence has been found that miRNAs can act as oncogenes or tumor suppressor genes. Here, we show that miR-494 is decreased in human breast cancer specimens and breast cancer cell lines. Ectopic expression of miR-494 in basal-like breast cancer cell lines MDA-MB-231-LUC-D2H3LN and BT-549 inhibits clonogenic ability and metastasis-relevant traits in vitro. Moreover, ectopic expression of miR-494 suppresses neoplasm initiation as well as pulmonary metastasis in vivo. Further studies have identified PAK1, as a direct target gene of miR-494, contributes to the functions of miR-494. Remarkably, the expression of PAK1 is inversely correlated with the level of miR-494 in human breast cancer samples. Furthermore, re-expression of PAK1 partially reverses miR-494-mediated proliferative and clonogenic inhibition as well as migration and invasion suppression in breast cancer cells. Taken together, these findings highlight an important role for miR-494 in the regulation of progression and metastatic potential of breast cancer and suggest a potential application of miR-494 in breast cancer treatment.

MiR-630 suppresses breast cancer progression by targeting metadherin.

MicroRNAs have been integrated into tumorigenic programs as either oncogenes or tumor suppressor genes. The miR-630 was reported to be deregulated and involved in tumor progression of several human malignancies. However, its expression regulation shows diversity in different kinds of cancers and its potential roles remain greatly elusive. Herein, we demonstrate that miR-630 is significantly suppressed in human breast cancer specimens, as well as in various breast cancer cell lines. In aggressive MDA-MB-231-luc and BT549 breast cancer cells, ectopic expression of miR-630 strongly inhibits cell motility and invasive capacity in vitro. Moreover, lentivirus delivered miR-630 bestows MDA-MB-231-luc cells with the ability to suppress cell colony formation in vitro and pulmonary metastasis in vivo. Further studies identify metadherin (MTDH) as a direct target gene of miR-630. Functional studies shows that MTDH contributes to miR-630-endowed effects including cell migration and invasion as well as colony formation in vitro. Taken together, these findings highlight an important role for miR-630 in the regulation of metastatic potential of breast cancer and suggest a potential application of miR-630 in breast cancer treatment.

MicroRNA-26b is underexpressed in human breast cancer and induces cell apoptosis by targeting SLC7A11.

MicroRNAs are widely dysregulated in various cancers and integrated into tumorigenic programs as either oncogenes or tumor suppressor genes. Here, we show that miR-26b, which is down-regulated in human breast cancer specimens and cell lines, impairs viability and triggers apoptosis of human breast cancer MCF7 cells. SLC7A11 is identified as a direct target of miR-26b and its expression is remarkably increased in both breast cancer cell lines and clinical samples. Furthermore, SLC7A11 silence mimics miR-26b-aroused viability impairment and apoptosis in MCF7 cells. Our studies reveal a protective role of miR-26b in the molecular etiology of human breast cancer by promoting apoptosis.