Imprinted lncRNA KCNQ1OT1 regulates CDKN1C expression through promoter binding and chromatin folding in pigs.

Long noncoding RNAs (lncRNAs) are implicated in a number of regulatory functions in eukaryotic genomes. In humans, KCNQ1OT1 is a 91 kb imprinted lncRNA that inhibits multiple surrounding genes in cis. Among them, CDKN1C is closely related to KCNQ1OT1 and is involved in multiple epigenetic disorders. Here, we found that pigs also had a relatively conserved paternal allele expressing KCNQ1OT1 and had a shorter 5' end (∼27 kb) compared to human KCNQ1OT1. Knockdown of KCNQ1OT1 using antisense oligonucleotides (ASO) showed that upregulation of CDKN1C expression in pigs. However, porcine KCNQ1OT1 did not affect the DNA methylation status of the CpG islands in the promoters of KCNQ1OT1 and CDKN1C. Inhibition of DNA methyltransferase using Decitabine treatment resulted in a significant increase in both KCNQ1OT1 and CDKN1C expression, suggesting that the regulation between KCNQ1OT1 and CDKN1C may not be dependent on RNA interference. Further use of chromosome conformation capture and reverse transcription-associated trap detection in the region where CDKN1C was located revealed that KCNQ1OT1 bound to the CDKN1C promoter and affected chromosome folding. Phenotypically, inhibition of KCNQ1OT1 at the cumulus-oocyte complex promoted cumulus cell transformation, and to upregulated the expression of ALPL at the early stage of osteogenic differentiation of porcine bone marrow mesenchymal stem cells. Our results confirm that the expression of KCNQ1OT1 imprinting in pigs as well as porcine KCNQ1OT1 regulates the expression of CDKN1C through direct promoter binding and chromatin folding alteration. And this regulatory mechanism played an important role in cell differentiation.

DNA methylation regulates RNA m6 A modification through transcription factor SP1 during the development of porcine somatic cell nuclear transfer embryos.

Epigenetic modifications play critical roles during somatic cell nuclear transfer (SCNT) embryo development. Whether RNA N6-methyladenosine (m6 A) affects the developmental competency of SCNT embryos remains unclear. Here, we showed that porcine bone marrow mesenchymal stem cells (pBMSCs) presented higher RNA m6 A levels than those of porcine embryonic fibroblasts (pEFs). SCNT embryos derived from pBMSCs had higher RNA m6 A levels, cleavage, and blastocyst rates than those from pEFs. Compared with pEFs, the promoter region of METTL14 presented a hypomethylation status in pBMSCs. Mechanistically, DNA methylation regulated METTL14 expression by affecting the accessibility of transcription factor SP1 binding, highlighting the role of the DNA methylation/SP1/METTL14 pathway in donor cells. Inhibiting the DNA methylation level in donor cells increased the RNA m6 A level and improved the development efficiency of SCNT embryos. Overexpression of METTL14 significantly increased the RNA m6 A level in donor cells and the development efficiency of SCNT embryos, whereas knockdown of METTL14 suggested the opposite result. Moreover, we revealed that RNA m6 A-regulated TOP2B mRNA stability, translation level, and DNA damage during SCNT embryo development. Collectively, our results highlight the crosstalk between RNA m6 A and DNA methylation, and the crucial role of RNA m6 A during nuclear reprogramming in SCNT embryo development.

ß-nicotinamide mononucleotide rescues the quality of aged oocyte and improves subsequent embryo development in pigs.

Oocyte senescence alters the shape and function, thereby weakening the fertilization potential. Nicotinamide mononucleotide (NMN) reverses age-related dysfunctions in various organs. Studies had shown long-term administration of NMN reduced the physiological decline associated in aged mice and reversed the aging of the ovaries. However, the protective effect of NMN on aged porcine oocytes is still unclear. In this study, we investigated the effects of NMN on aging porcine oocytes and subsequent embryonic development. We established a model of senescence of porcine oocytes after ovulation by extending the culture time in vitro. NMN supplementation significantly reduced reactive oxygen species (ROS) levels in senescence oocytes and increased the mRNA levels of antioxidant genes SOD1 and Cat. The mitochondrial membrane potential of aged oocytes treated with NMN was increased compared with that of untreated oocytes. In addition, the mRNA level of apoptosis-related gene Bax was significantly decreased in senescence oocytes treated with NMN, while the mRNA level of anti-apoptosis-related gene BCL-2 was significantly increased. Furthermore, NMN supplementation enhanced the subsequent development ability of senescent oocytes during in vitro aging. Compared with untreated senescent oocytes, the blastocyst formation rate and pluripotent genes of senescent oocytes treated with NMN were significantly increased. Taken together, these results suggest that NMN is beneficial for delaying the aging process in porcine oocytes.

Disulfiram/Copper Induce Ferroptosis in Triple-Negative Breast Cancer Cell Line MDA-MB-231.

BACKGROUND: The complex formed by disulfiram (DSF) and copper (Cu) is safe and effective for the prevention and treatment of triple-negative breast cancer (TNBC). Although previous studies have shown that DSF/Cu induces ferroptosis, the mechanism remains unclear. METHODS: The mitochondrial morphology of TNBC treated with DSF/Cu was observed by transmission microscopy, and intracellular levels of iron, lipid reactive oxygen species (ROS), malondialdehyde, and glutathione were evaluated to detect the presence of ferroptosis. Target genes for the DSF/Cu-activated ferroptosis signaling pathway were examined by transcriptome sequencing analysis. Expression of the target gene, HOMX1, was detected by qRT-PCR, immunofluorescence and western blot. RESULTS: The mitochondria of TNBC cells were significantly atrophied following treatment with DSF/Cu for 24 h. Addition of DSF/Cu supplement resulted in significant up-regulation of intracellular iron, lipid ROS and malondialdehyde levels, and significant down-regulation of glutathione levels, all of which are important markers of ferroptosis. Transcriptome analysis confirmed that DSF/Cu activated the ferroptosis signaling pathway and up-regulated several ferroptosis target genes associated with redox regulation, especially heme oxygenase-1 (HMOX-1). Inhibition of ferroptosis by addition of the ROS scavenger N-acetyl-L-cysteine (NAC) significantly increased the viability of DSF/Cu-treated TNBC cells. CONCLUSIONS: These results show that DSF/Cu increases lipid peroxidation and causes a sharp increase in HMOX1 activity, thereby inducing TNBC cell death through ferroptosis. DSF/Cu is a promising therapeutic drug for TNBC and could lead to ferroptosis-mediated therapeutic strategies for human cancer.

Transcription factor ELK1 regulates the expression of histone 3 lysine 9 to affect developmental potential of porcine preimplantation embryos.

A series of changes occur in the early embryo that are critical for subsequent development, and the pig is an excellent animal model of human disease, so understanding the regulatory mechanisms of early embryonic development in the pig is of very importance. To find key transcription factors regulating pig early embryonic development, we first profiled the transcriptome of pig early embryos, and confirmed that zygotic gene activation (ZGA) in porcine embryos starts from 4 cell stage. Subsequent enrichment analysis of up-regulated gene motifs during ZGA revealed that the transcription factor ELK1 ranked first. The expression pattern of ELK1 in porcine early embryos was analyzed by immunofluorescence staining and qPCR, and the results showed that the transcript level of ELK1 reached the highest at the 8 cell stage, while the protein level reached the highest at 4 cell stage. To further investigate the effect of ELK1 on early embryo development in pigs, we silenced ELK1 in zygotes and showed that ELK1 silencing significantly reduced cleavage rate, blastocyst rate as well as blastocyst quality. A significant decrease in the expression of the pluripotency gene Oct4 was also observed in blastocysts from the ELK1 silenced group by immunofluorescence staining. Silencing of ELK1 also resulted in decreased H3K9Ac modification and increased H3K9me3 modification at 4 cell stage. To investigate the effect of ELK1 on ZGA, we analyzed transcriptome changes in 4 cell embryos after ELK1 silencing by RNA seq, which revealed that ELK1 silencing resulted in significant differences in the expression of a total of 1953 genes at the 4 cell stage compared with their normal counterparts, including 1106 genes that were significantly upregulated and 847 genes that were significantly downregulated. Through GO and KEGG enrichment, we found that the functions and pathways of down-regulated genes were concentrated in protein synthesis, processing, cell cycle regulation, etc., while the functions of up-regulated genes were focused on aerobic respiration process. In conclusion, this study demonstrates that the transcription factor ELK1 plays an important role in regulation of preimplantation embryo development of pigs and deficiency of ELK1 leads to abnormal epigenetic reprogramming as well as zygotic genome activation, thus adversely affecting embryonic development. This study will provide important reference for the regulation of transcription factors in porcine embryo development.

Naïve-like conversion of bovine induced pluripotent stem cells from Sertoli cells.

Feeder cells are essential to derive pluripotent stem cells (PSCs). Mouse embryonic fibroblasts (MEF) are widely used as feeder to generate and culture embryonic stem cells (ESCs) and induced PSCs (iPSCs) in many species. However it may not be suitable for livestock ESCs/iPSCs due to interspecies difference. Previously we derived bovine iPSCs from bovine Sertoli cells using MEF feeder. Here we compared the effects of MEF feeder and bovine embryonic fibroblasts (BEF) feeder on the maintenance of bovine iPSC pluripotency and morphology as well their contributions to the naïve-like conversion, based on a naïve medium (NM). The results showed successful conversion of the primed bovine iPSCs to naïve-like state within 3-4 days both on MEF feeder and BEF feeder in NM (termed as MNM and BNM respectively). These naïve-like iPSCs showed normal karyotype. There were more iPSC colonies under BNM condition than MNM condition. Epigenetically, histone modification H3K4 was upregulated, while H3K27 was downregulated in the naïve-like iPSCs. We further analyzed the naïve markers and differentiation potential both in vitro and in vivo of these cells, which were all reserved throughout the maintenance. Together, bovine naïve-like iPSCs can be generated both on MEF and BEF feeder in NM condition. The BNM condition is able to sustain the pluripotency and differentiation potential of the naïve-like bovine iPSCs, and improve the conversion efficiency.

Transcription factor AP-2 gamma affects porcine early embryo development by regulating epigenetic modification.

RESEARCH QUESTION: What is the role and mechanism of action of transcription factor AP-2 gamma (TFAP2C) in porcine early embryo development? DESIGN: TFAP2C siRNA were injected into porcine oocytes, which subsequently underwent IVF. Different stages of embryos were collected for RNA sequencing, quantitative polymerase chain reaction, immunofluorescence staining to explore the affects in gene expression and epigenetic modification. Porcine fetal fibroblasts were transfected with siRNA, and cells were collected for chromatin immunoprecipitation and dual luciferase reporter assays. RESULTS: The deficiency of TFAP2C led to disorders in early embryonic development; 1208 genes were downregulated and 792 genes were upregulated in TFAP2C knockdown (TFAP2C-KD) embryos. The expression of epigenetic modification enzymes KDM5B, SETD2 were significantly elevated in the TFAP2C-KD group (P < 0.001). Meanwhile, the modification levels of H3K4me3 and H3K4me2 were significantly decreased (P = 0.0021, P = 0.0029), and H3K36me3 and DNA methylation were significantly increased in TFAP2C-KD group (P = 0.0045, P = 0.0025). DNMT1 was mainly expressed in nuclei in the TFAP2C-KD group (P = 0.0103). In addition, TFAP2C could bind to the promoter region of SETD2, and the mutation of the TFAP2C binding site resulted in increased activity of SETD2 promoter (P < 0.001). CONCLUSIONS: The knockdown of TFAP2C affects early embryonic development by regulating histone modification and DNA methylation.

Profiling the transcriptomic signatures and identifying the patterns of zygotic genome activation - a comparative analysis between early porcine embryos and their counterparts in other three mammalian species.

BACKGROUND: The transcriptional changes around zygotic genome activation (ZGA) in preimplantation embryos are critical for studying mechanisms of embryonic developmental arrest and searching for key transcription factors. However, studies on the transcription profile of porcine ZGA are limited. RESULTS: In this study, we performed RNA sequencing in porcine in vivo developed (IVV) and somatic cell nuclear transfer (SCNT) embryo at different stages and compared the transcriptional activity of porcine embryos with mouse, bovine and human embryos. The results showed that the transcriptome map of the early porcine embryos was significantly changed at the 4-cell stage, and 5821 differentially expressed genes (DEGs) in SCNT embryos failed to be reprogrammed or activated during ZGA, which mainly enrichment to metabolic pathways. c-MYC was identified as the highest expressed transcription factor during ZGA. By treating with 10,058-F4, an inhibitor of c-MYC, the cleavage rate (38.33 ± 3.4%) and blastocyst rate (23.33 ± 4.3%) of porcine embryos were significantly lower than those of the control group (50.82 ± 2.7% and 34.43 ± 1.9%). Cross-species analysis of transcriptome during ZGA showed that pigs and bovines had the highest similarity coefficient in biological processes. KEGG pathway analysis indicated that there were 10 co-shared pathways in the four species. CONCLUSIONS: Our results reveal that embryos with impaired developmental competence may be arrested at an early stage of development. c-MYC helps promote ZGA and preimplantation embryonic development in pigs. Pigs and bovines have the highest coefficient of similarity in biological processes during ZGA. This study provides an important reference for further studying the reprogramming regulatory mechanism of porcine embryos during ZGA.

LncRNA affects epigenetic reprogramming of porcine embryo development by regulating global epigenetic modification and the downstream gene SIN3A.

The study of preimplantation development is of great significance to reproductive biology and regenerative medicine. With the development of high-throughput deep sequencing technology, it has been found that lncRNAs play a very important role in the regulation of embryonic development. In this study, key lncRNAs that regulate embryonic development were screened by analyzing the expression pattern of lncRNAs in porcine in vivo fertilization (IVV) embryos. By knocking down lncRNA expression in in vitro fertilization (IVF) embryos, we investigated its function and mechanism of regulating embryonic development. The results showed that the expression pattern of lncRNA was consistent with the time of gene activation. The lncRNAs were highly expressed in the 4-cell to blastocyst stage but barely expressed in the oocytes and 2-cell stage. So we speculated this part of lncRNAs may regulate gene expression. The lncRNA LOC102165808 (named lncT because the gene near this lncRNA is TFAP2C) was one of them. The knockdown (KD) of lncT inhibited embryonic development, resulting in decreased H3K4me3, H3K4me2, and H3K9me3, and increased DNA methylation. Meanwhile, RNAseq showed SIN3A was the top decreased gene in lncT-KD embryos. There was a severe blastocyst formation defect in SIN3A-KD embryos. Both lncT and SIN3A could affect NANOG and induce more cell apoptosis. In conclusion, the knockdown of lncT inhibits embryonic development by regulating H3K4me3, H3K4me2, DNA methylation, pluripotency gene, and apoptosis, and SIN3A is one of the downstream genes of lncT in regulating embryonic development.

Maternal Prepregnancy 5-Hydroxytryptamine Exposure Affects the Early Development of the Fetus.

In recent decades, the increasing incidence of depression has contributed to an increase in the use of serotonergic drugs, such as antidepressants, which predisposes humans to serotonin syndrome. Serotonin syndrome is caused by elevated serotonin levels in the central and peripheral nervous systems. It has been well documented that the development of offspring can be affected by maternal exposure to environmental challenges, such as stress, diseases, or an unhealthy diet during pregnancy. Serotonin, also called 5-hydroxytryptamine (5-HT), is widely expressed in the female reproductive system and plays an important role in the development of follicles and embryos. However, whether the suffering of the mother from serotonin syndrome before pregnancy affects fetal development is still uncertain. In the present study, to explore the effect of maternal prepregnancy 5-HT exposure on the fetus, intraperitoneal injection of 5-HT was used to change maternal prepregnancy 5-HT levels. It was found that maternal prepregnancy 5-HT exposure significantly reduced the body weight and liver weight and the levels of estrogen and progesterone in female mice. Although there was no significant difference in the cleavage rate and blastocyst rate between the 5-HT and control groups, maternal prepregnancy 5-HT exposure increased the percentage of embryo resorption, decreased placental weight, and led to placental inflammation at E13.5. Notably, 5-HT exposure caused weight loss in the offspring at 2 weeks. These results suggested that maternal prepregnancy 5-HT exposure could affect the development of the offspring, which was partly caused by reduced hormonal secretion and placental inflammation.

Effects of dimethyl sulfoxide (DMSO) on DNA methylation and histone modification in parthenogenetically activated porcine embryos.

Epigenetic mechanisms play an important role in oogenesis and early embryo development in mammals. Dimethyl sulfoxide (DMSO) is frequently used as a solvent in biological studies and as a vehicle for drug therapy. Recent studies suggest that DMSO detrimentally affects porcine embryonic development, yet the mechanism of the process in parthenogenetically activated porcine embryos has not been reported. In this study, we found that treatment of embryos with 1.5% DMSO significantly decreased the cleavage and blastocyst rates, total cell number of blastocysts and the anti-apoptotic gene BCL-2 transcription level; however, the percentage of apoptotic cells and the expression levels of the pro-apoptotic gene BAX were not changed. Treatment with DMSO significantly decreased the expression levels of DNMT1 , DNMT3a , DNMT3b , TET1 , TET2 , TET3 , KMT2C , MLL2 and SETD3 in most of the stages of embryonic development and increased 5-mC signals, while the staining intensity for 5-hmC had no change in porcine preimplantation embryos from 2-cell to the blastocyst stages. Meanwhile, DMSO decreased the level of H3K4me3 during the development of parthenogenetically activated porcine embryos. After treatment with DMSO, expression levels of the pluripotency-related genes POU5F1 and NANOG decreased significantly (P <0.01), whereas the imprinted gene H19 did not change (P >0.05). In conclusion, these results suggest that DMSO can affect genome-wide DNA methylation and histone modification by regulating the expression of epigenetic modification enzymes, and DMSO also influences the expression level of pluripotent genes. These dysregulations lead to defects in embryonic development.

Cycloleucine negatively regulates porcine oocyte maturation and embryo development by modulating N6-methyladenosine and histone modifications.

Maturation of oocytes and early embryo development are regulated precisely by numerous factors at transcriptional and posttranslational levels through precise mechanisms. N6-methyladenosine (m6A) is the most common modification in mRNA which regulates RNA metabolism and gene expression. However, the role of RNA m6A on porcine oocyte maturation and early embryogenesis is largely unknown. Here, we found that oocytes treated with cycloleucine (CL), an RNA m6A inhibitor, express impaired cumulus expansion, increased production of reactive oxygen species (ROS) in the mitochondria, and delayed maturation of oocytes by disrupting spindle organization and chromosome alignment. Also, CL halted the development of embryos at the 4-cell stage and resulted in low-quality blastocysts. Furthermore, CL treatment decreased the RNA m6A, H3K4me3, and H3K9me3 levels, but increased the acetylation level of H4K16 during parthenogenetic embryonic development in pigs. Single-cell RNA-seq (scRNA-seq) analysis further revealed that CL treatment dramatically up-regulated the expression of metabolism-related genes (SLC16A1, and MAIG3 etc.) and maternal related genes, including BTG4, WEE2, and BMP15 among others, at the blastocyst stage. Taken together, inhibition of RNA m6A by CL impaired meiosis of oocytes and early embryonic development of porcine via RNA m6A methylation, histone modifications, and altering the expression of metabolism-related genes in blastocysts.

Combination of the 6-thioguanine and disulfiram/Cu synergistically inhibits proliferation of triple-negative breast cancer cells by enhancing DNA damage and disrupting DNA damage checkpoint.

Because of the lack of specific molecular targeted therapies, triple-negative breast cancer (TNBC) has high tumour recurrence and metastasis rates. It is urgent to develop novel chemotherapeutic strategies to improve patient survival. DNA damaging agents have been shown to sensitize cancer to genotoxic chemotherapies. We first found that 6-thioguanine (6-TG) can activate the NF-кB signalling pathway. Our results showed that NF-кB signalling was reduced when cells were treated with 6-TG/disulfiram (DSF)/Cu. DSF/Cu enhanced the 6-TG-mediated inhibition of proliferation. 6-TG/DSF/Cu inhibited cell cycle progression, causing cell cycle arrest in the S phase and G2/M phase. Moreover, the combined effect of 6-TG and DSF/Cu induced apoptosis, and either agent alone was able to induce apoptosis. The accumulation of γH2A indicated that DSF/Cu increased the DNA damage induced by 6-TG. Combined treatment with 6-TG and DSF/Cu synergistically reduced the levels of both phosphorylated and total ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR), suggesting that DSF/Cu promoted 6-TG-induced DNA damage by suppressing ATR protein kinases, therefore enhancing cell apoptosis. In conclusion, we demonstrate that the combination of 6-TG and DSF/Cu exerted a significant synergistic antitumour effect on human TNBC in vitro and in vivo by enhancing DNA damage and disrupting DNA damage checkpoints. We propose that this combination therapy could be a novel strategy for the treatment of TNBC.

MiR-10b-5p inhibits tumorigenesis in gastric cancer xenograft mice model through down-regulating Tiam1.

The miR-10b-5p plays an important role in gastric cancer development but its exact effect on gastric cancer development in vivo has not been fully studied. We showed that miR-10b-5p inhibited the proliferation and migration of gastric cancer cells by down-regulating Tiam1 which was up-regulated in both gastric cancer cells and tissues. Gastric cancer xenograft experiment showed that lenti-miR-10b-5p treatment and agomir-10b-5p injection could significantly retard tumor growth and reduce tumor size and induced apoptosis. Therefore, our results elucidate the tumor suppressor role of miR-10b-5p in gastric cancer in which it acts as a negative regulator of Tiam1 and also provide a molecular mechanism for agomir-10b-5p to treat gastric cancer.

Transcriptome and lipidome profile of human mesenchymal stem cells with reduced senescence and increased trilineage differentiation ability upon drug treatment.

Human Mesenchymal stem cells (hMSCs) are multi-potential cells which are widely used in cell therapy. However, the frequently emerged senescence and decrease of differentiation capabilities limited the broad applications of MSC. Several strategies such as small molecules treatment have been widely studied and used to improve the stem characteristics bypassing the senescence but the exact mechanisms for them to reduce senescence have not been fully studied. In this study, hMSCs were treated by rapamycin, oltipraz, metformin, and vitamin C for the indicated time and these cells were subjected to senescence evaluation and trilineage differentiation. Furthermore, transcriptomics and lipidomics datasets of hMSCs after drug treatment were analyzed to interpret biological pathways responsible for their anti-senescence effects. Although four drugs exhibited significant activities in promoting MSC osteogenic differentiation, metformin is the optimal drug to promote trilineage differentiation. GO terms illustrated that the anti-aging effects of drugs were mainly associated with cellular senescence, mitotic and meiosis process. Biosynthesis of phosphatidylcholines (PC) and phosphatidylethanolamine (PE) were inhibited whereas production of phosphatidylinositols (PIs) and saturated fatty acids (SFA)/ mono-unsaturated fatty acids (MUFA) conversion was activated. Medium free fatty acids (FFA) was increased in hMSCs with different anti-aging phenotypes. Therefore, we established a comprehensive method in assessing drug intervention based on the results of transcriptomics and lipidomics. The method can be used to study different biological phenotypes upon drug intervention in MSC which will extend the clinical application of hMSCs.

TRIM28 maintains genome imprints and regulates development of porcine SCNT embryos.

Pre-implantation embryos undergo genome-wide DNA demethylation, however certain regions, like imprinted loci remain methylated. Further, the mechanisms ensuring demethylation resistance by TRIM28 in epigenetic reprogramming remain poorly understood. Here, TRIM28 was knocked down in oocytes, and its effects on porcine somatic cell nuclear transfer (SCNT) embryo development was examined. Our results showed that SCNT embryos constructed from TRIM28 knockdown oocytes had significantly lower cleavage (53.9 ± 3.4% vs 64.8 ± 2.7%) and blastocyst rates (12.1 ± 4.3% vs 19.8 ± 1.9%) than control-SCNT embryos. The DNA methylation levels at the promoter regions of the imprinting gene IGF2 and H19 were significantly decreased in the 4-cell stage, and the transcript abundance of other imprinting gene was substantially increased. We also identified an aberrant two-fold decrease in the expression of CXXC1and H3K4me3 methyltransferase (ASH2L and MLL2), and the signal intensity of H3K4me3 had a transient drop in SCNT 2-cell embryos. Our results indicated that maternal TRIM28 knockdown disrupted the genome imprints and caused epigenetic variability in H3K4me3 levels, which blocked the transcription activity of zygote genes and affected the normal developmental progression of porcine SCNT embryos.

The regulatory effect of 6-TG on lncRNA-miRNA-mRNA ceRNA network in triple-negative breast cancer cell line.

Breast cancer is one of the most prevalent and recurring cancer types that leads to deaths in women. Triple-negative breast cancer (TNBC) is difficult to treat due to the lack of therapeutic targets. Many studies have focused on identifying drugs for use as alternative treatments for breast cancer. Thioguanine (6-TG) exerts antitumor effects in cancer. Increasing evidence has demonstrated that competitive endogenous ribonucleic acids (ceRNAs) are involved in cancer processes. However, the mechanism by which 6-TG regulates lncRNA-miRNA-mRNAs has not been elucidated. We evaluated the antitumor effect of 6-TG in MDA-MB-231 cells and comprehensively analyzed the RNA-Seq data of MDA-MB-231 cells treated with 6-TG. Our results showed that most tumor pathways were blocked by 6-TG. The hub genes were FN1, FLNA, FLNB, VCL, GSN, MYH10, ACTN4, KDR and EREG, and they were all down-regulated after 6-TG treatment. The coexpression network consisted of 18 microRNAs (miRNAs), 9 long noncoding RNAs (lncRNAs) and 20 mRNAs. Hsa-mir-16-5p and Hsa-mir-335-5p targeted the greatest number of mRNAs in the network. These molecules could bind to PAX8-AS1 and eliminate the inhibition of target mRNA expression. We showed that PAX8-AS1 is the main lncRNA affected by 6-TG and that PAX8-AS1 regulates the hub genes in tumor pathways by competitively binding with miR-16-5p and miR-335-5p.

Construction and analysis of competing endogenous RNA network of MCF-7 breast cancer cells based on the inhibitory effect of 6-thioguanine on cell proliferation.

Previous research has proven that 6-thioguanine (6-TG) inhibits the growth of MCF-7 breast cancer cells. Accumulating evidence indicates that long non-coding (lnc)RNAs are involved in the development of various cancer types as competitive endogenous (ce)RNA molecules. The present study was conducted to investigate the regulatory mechanism underlying the function of lncRNAs as ceRNA molecules in MCF-7 cells and to identify more effective prognostic biomarkers for breast cancer treatment. The expression profiles of lncRNAs in untreated MCF-7 cells and 6-TG-treated MCF-7 cells were compared by RNA-seq. The regulatory associations among lncRNAs, micro (mi)RNAs and mRNAs were analyzed and verified by the TargetScan, miRDB and miRTarBas databases. The ceRNA networks were constructed by Cytoscape. The expression levels of two lncRNAs and two miRNAs in the ceRNA network were measured by reverse transcription-quantitative PCR. The OncoLnc and Kaplan-Meier plotter network databases were utilized to determine the effects of lncRNA and miRNA expression on the survival of patients with breast cancer. A ceRNA network was constructed for MCF-7 breast cancer cells treated with 6-TG, and this network may provide valuable information for further research elucidating the molecular mechanism underlying the effects of 6-TG on breast cancer. Moreover, LINC00324, MIR22HG, miR-370-3p and miR-424-5p were identified as potential prognostic and therapeutic biomarkers for breast cancer.

Thioguanine Induces Apoptosis in Triple-Negative Breast Cancer by Regulating PI3K-AKT Pathway.

Triple-negative breast cancer (TNBC) is notoriously difficult to treat due to the lack of biological targets and poor sensitivity to conventional therapies. Chemotherapy is the main clinical therapy, but the effective screening strategy for chemotherapy drugs is poorly investigated. Drug repositioning has been the center of attention in recent years attracting numerous studies. Here, we firstly found multiple common features between leukemia and TNBC by analyzing the global transcriptome profiles based on the transformed comparison data from NCI60. Therefore, we investigated the role of the classic leukemia drug thioguanine (6-TG) in TNBC cancer cells. Our results indicated that 6-TG inhibited cell proliferation and tumor cell progression by suppressing PI3K-AKT pathway via downregulating the DNA methylation level of PTEN. Moreover, apoptosis was induced via the activation of PI3K-AKT downstream TSC1 and the downregulation of methylation levels of DAXX, TNF, FADD and CASP8 etc. These findings indicated 6-TG exerts its anti-tumor effects in vitro and in vivo through regulating the DNA methylation levels of genes involved in PI3K-AKT and apoptosis pathway. Meanwhile, our study suggested that transcriptome-based drug screening has potential implications for breast cancer therapy and drug selection.