MKRN2 knockout causes male infertility through decreasing STAT1, SIX4, and TNC expression.

Makorin-2 (Mkrn2) is an evolutionarily conserved gene whose biological functions are not fully known. Although recent studies have shed insights on the potential causes of male infertility, its underlining mechanisms still remain to be elucidated. We developed a Mrkn2 knockout mice model to study this gene and found that deletion of Mkrn2 in mice led to male infertility. Interestingly, the expression level of signal transducer and activator of the transcription (STAT)1 was significantly decreased in MKRN2 knockout testis and MEF cells. Co-IP assay showed an interaction between MKRN2 and STAT1. Moreover, our results further indicated that MKRN2 regulated the expression level of SIX4 and tenascin C (TNC) via the EBF transcription factor 2 (EBF2) in mice. The results of our study will provide insights into a new mechanism of male infertility.

TBX15/miR-152/KIF2C pathway regulates breast cancer doxorubicin resistance via promoting PKM2 ubiquitination.

BACKGROUND: Chemoresistance is a critical risk problem for breast cancer treatment. However, mechanisms by which chemoresistance arises remains to be elucidated. The expression of T-box transcription factor 15 (TBX-15) was found downregulated in some cancer tissues. However, role and mechanism of TBX15 in breast cancer chemoresistance is unknown. Here we aimed to identify the effects and mechanisms of TBX15 in doxorubicin resistance in breast cancer. METHODS: As measures of Drug sensitivity analysis, MTT and IC50 assays were used in DOX-resistant breast cancer cells. ECAR and OCR assays were used to analyze the glycolysis level, while Immunoblotting and Immunofluorescence assays were used to analyze the autophagy levels in vitro. By using online prediction software, luciferase reporter assays, co-Immunoprecipitation, Western blotting analysis and experimental animals models, we further elucidated the mechanisms. RESULTS: We found TBX15 expression levels were decreased in Doxorubicin (DOX)-resistant breast cancer cells. Overexpression of TBX15 reversed the DOX resistance by inducing microRNA-152 (miR-152) expression. We found that KIF2C levels were highly expressed in DOX-resistant breast cancer tissues and cells, and KIF2C was a potential target of miR-152. TBX15 and miR-152 overexpression suppressed autophagy and glycolysis in breast cancer cells, while KIF2C overexpression reversed the process. Overexpression of KIF2C increased DOX resistance in cancer cells. Furthermore, KIF2C directly binds with PKM2 for inducing the DOX resistance. KIF2C can prevent the ubiquitination of PKM2 and increase its protein stability. In addition, we further identified that Domain-2 of KIF2C played a major role in the binding with PKM2 and preventing PKM2 ubiquitination, which enhanced DOX resistance by promoting autophagy and glycolysis. CONCLUSIONS: Our data identify a new mechanism by which TBX15 abolishes DOX chemoresistance in breast cancer, and suggest that TBX15/miR-152/KIF2C axis is a novel signaling pathway for mediating DOX resistance in breast cancer through regulating PKM2 ubiquitination and decreasing PKM2 stability. This finding suggests new therapeutic target and/or novel strategy development for cancer treatment to overcome drug resistance in the future.

Effects of combination of pargyline and doxorubicin on proliferation and migration of tripple negative breast cancer 4T-1 cells / 中国药理学通报

Aim To study the combination of lysinespecifc demethylase 1 (lysine-specifc demethylase 1, LSD1) inhibitor pargyline and the chemotherapy drug doxorubicin on the proliferation, migration and invasion of murine triple negative breast cancer 4T-1 cells. Methods In vitro, the effect on the proliferation, invasion and migration of 4T-1 cells of the combination of these two drugs were detected with CCK-8 method, lactate dehydrogenase release test, Chou-Talay method, Scratch test, Transwell assay, Western blot and etc. Tumor-bearing mice were used to investigate the combined effect of these two drugs on the proliferation of 4T-1 cells in vivo. Results The combination of pargyline and doxorubicin effectively inhibited the proliferation, migration and invasion of 4T-1 cells. Compared with single drug group, the combination of these two drugs could significantly inhibit the proliferation of breast cancer and prolong the survival time of mice with triple negative breast cancer. Conclusions The combined application of pargyline and doxorubicin has a synergistic inhibitory effect on the proliferation, migration and invasion of mouse breast cancer 4T-1 cells, and has potential value for clinical treatment on triplenegative breast cancer.

HB-EGF Activates the EGFR/HIF-1α Pathway to Induce Proliferation of Arsenic-Transformed Cells and Tumor Growth.

Arsenic was recently identified as a pollutant that is a major cause of lung cancer. Since heparin-binding EGF-like growth factor (HB-EGF) was reported to be a promising therapeutic target for lung cancer, we investigated the role and mechanism of HB-EGF during arsenic-induced carcinogenesis and development of lung cancer. HB-EGF expression were upregulated in As-T cells, lung cancer cell lines, and in most lung cancer tissue samples; and HB-EGF activated the EGFR/p-ERK/HIF-1α pathway and induced VEGF by regulating HIF-1α transcription. HIF-1α transcriptional stimulation by HB-EGF was facilitated by PKM2 and played an important role in HB-EGF's effect on cells. An HB-EGF inhibitor(CRM197, cross-reacting material 197) slowed cell proliferation and inhibited migration of As-T and A549 cells, and inhibited tumor growth. PKM2 also played an important role in the proliferation and migration in As-T cells. The positive staining ratios of EGFR phosphorylation (Y1068) and PKM2 were significantly higher in most cases of lung cancer than in paired normal tumor-adjacent lung tissues; and HB-EGF expression levels strongly correlated with p-EGFR expression levels. Thus, HB-EGF drives arsenic-induced carcinogenesis, tumor growth, and lung cancer development via the EGFR/PKM2/HIF-1α pathway.

Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis.

The apoptosis that occurs in the immature testis under physiological conditions is necessary for male germ cell development, whereas improper activation of apoptosis can impair spermatogenesis and cause defects in reproduction. We previously demonstrated that in mice, the makorin-2 (Mkrn 2) gene is expressed exclusively in the testis and its deletion leads to male infertility. To understand the potential molecular mechanism, in this study, we found that levels of apoptosis in the testis were abnormally high in the absence of Mkrn 2. To identify specific gene(s) involved, we performed digital gene expression profiling (DGE) and pathway analysis via gene set enrichment analysis (GSEA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and we found that MKRN2 inhibits p53 apoptosis effector related to PMP22 (PERP) expression and that levels of the protein in sperm samples have an inverse correlation with infertility levels. GSEA additionally indicated that PERP is a negative regulator of spermatogenesis and that its ectopic expression induces male infertility. Further, Gene Expression Omnibus (GEO) dataset analysis showed that p53, upstream of PERP, was upregulated in oligoasthenoteratozoospermia (OAT). These observations suggest that Mkrn 2 is crucial for protecting germ cells from excessive apoptosis and implicate Mkrn 2-based suppression of the p53/PERP signaling pathway in spermatogenesis and male fertility.

Estrogen-induced miR-196a elevation promotes tumor growth and metastasis via targeting SPRED1 in breast cancer.

BACKGROUND: Estrogen plays a critical role in breast cancer (BC) progression through estrogen receptor (ER)-mediated gene regulation. Emerging studies suggest that the malignant progress of BC cells is influenced by the cross talk between microRNAs (miRNAs) and ER-α signaling. However, the mechanism and functional linkage between estrogen and miRNAs remain unclear. METHODS: The expression levels of miR-196a and SPRED1 in BC were tested by qRT-PCR in 46 paired BC and adjacent tissues and by the GEO datasets. The role of miR-196a in estrogen-induced BC development was examined by CCK-8 assay, wound healing assay, Matrigel invasion assay and tumorigenicity assay in nude mice. The binding site of ER-α in miR-196a promoter region was analyzed by ChIP-seq, ChIP assay and luciferase reporter assay. The potential targets of miR-196a in BC cells were explored using the luciferase reporter assay and western blot analysis, and the correlation between miR-196a and SPRED1 was analyzed by Spearman's correlation analysis in BC specimens and GEO dataset. TCGA BRCA data was used to characterize the ESR1 signatures according to MSigDB gene set. RESULTS: The expression levels of miR-196a were higher in ER-positive (ER+) breast tumors compared to ER-negative (ER-) tumor tissue samples. Besides, miR-196a was involved in estrogen-induced BC cell proliferation, migration and invasion. Notably, the up-regulation of miR-196a was mediated by a direct interaction with estrogen receptor α (ER-α) but not estrogen receptor ß (ER-ß) in its promoter region, and miR-196a expression levels were positively correlated to ER-α signature scores. Furthermore, SPRED1 was a new direct target of miR-196a which participated in miR-196a-promoted BC development and was suppressed by ligand-activated ER-α signal pathway. Finally, forced expression of miR-196a induced tumor growth of MCF7 cells, while inhibition of miR-196a significantly suppressed the tumor progress in vivo. CONCLUSIONS: Overall, the identification of estrogen/miR-196a/SPRED1 cascade will shed light on new molecular mechanism of estrogen signaling in BC development and therapy.

Deficiency of Mkrn2 causes abnormal spermiogenesis and spermiation, and impairs male fertility.

Although recent studies have shed insights on some of the potential causes of male infertility, new underlining molecular mechanisms still remain to be elucidated. Makorin-2 (Mkrn2) is an evolutionarily conserved gene whose biological functions are not fully known. We developed an Mrkn2 knockout mouse model to study the role of this gene, and found that deletion of Mkrn2 in mice led to male infertility. Mkrn2 knockout mice produced abnormal sperms characterized by low number, poor motility, and aberrant morphology. Disruption of Mkrn2 also caused failure of sperm release (spermiation failure) and misarrangement of ectoplasmic specialization (ES) in testes, thus impairing spermiogenesis and spermiation. To understand the molecular mechanism, we found that expression of Odf2, a vital protein in spermatogenesis, was significantly decreased. In addition, we found that expression levels of Odf2 were decreased in Mkrn2 knockout mice. We also found that MKRN2 was prominently expressed in the sperm of normal men, but was significantly reduced in infertile men. This result indicates that our finding is clinically relevant. The results of our study provided insights into a new mechanism of male infertility caused by the MKRN2 downregulation.

Estrogen regulates miRNA expression: implication of estrogen receptor and miR-124/AKT2 in tumor growth and angiogenesis.

It is currently known that estrogen plays an important role in breast cancer (BC) development, but the underlying molecular mechanism remains to be elucidated. Accumulating evidence has revealed important roles of microRNAs in various kinds of human cancers, including BC. In this study, we found that among the microRNAs regulated by estrogen, miR-124 was the most prominent downregulated miRNA. miR-124 was downregulated by estradiol (E2) treatment in estrogen receptor (ER) positive BC cells, miR-124 overexpression suppressed cell proliferation, migration and invasion in BC cells; while the suppression of miR-124 using Anti-miR-124 inhibitor had opposite cellular functions. Under the E2 treatment, miR-124 had stronger effect to inhibit cellular functions in MCF7 cells than that in MDA-MB-231 cells. In addition, we identified that ERα, but not ERß, was required for E2-induced miR-124 downregulation. Furthermore, AKT2, a known oncogene, was a novel direct target of miR-124. AKT2 expression levels were inversely correlated with miR-124 expression levels in human breast cancer specimens. AKT2 was overexpressed in BC specimens, and its expression levels were much higher in ERα positive cancer tissues than those ERα negative cancer tissues. Consistent with miR-124 suppression, E2 treatment increased AKT2 expression levels in MCF7 cells via ERα. Finally, overexpression of miR-124 in MCF7 cells significantly suppressed tumor growth and angiogenesis by targeting AKT2. Our results provide a mechanistic insight into a functional role of new ERα/miR-124/AKT2 signaling pathway in BC development. miR-124 and AKT2 may be used as biomarkers for ERα positive BC and therapeutic effect in the future.