The combined restoration of miR-424-5p and miR-142-3p effectively inhibits MCF-7 breast cancer cell line via modulating apoptosis,proliferation, colony formation,cell cycle and autophagy

Background

The combined restoration of tumor-suppressive microRNAs (miRs) has been identified as a promising approach for inhibiting breast cancer development. This study investigated the effect of the combined restoration of miR-424-5p and miR-142-3p on MCF-7 cells and compared the efficacy of the combined therapy with the monotherapies with miR-424-5p and miR-142-3p.

Methods

After transfection of miR-424-5p and miR-142-3p mimics into MCF-7 cells in the combined and separated manner, the proliferation of tumoral cells was assessed by the MTT assay. Also, the apoptosis, autophagy, and cell cycle of the cells were analyzed by flow cytometry. Western blot and qRT-PCR were used to study the expression levels of c-Myc, Bcl-2, Bax, STAT-3, Oct-3, and Beclin-1.

Results

Our results have demonstrated that the combined restoration of miR-424-5p and miR-142-3p is more effective in inhibiting tumor proliferation via upregulating Bax and Beclin-1 and downregulating Bcl-2 and c-Myc. Besides, the combined therapy has arrested the cell cycle in the sub-G1 and G2 phases and has suppressed the clonogenicity via downregulating STAT-3 and Oct-3, respectively.

Conclusion

The combined restoration of miR-424-5p and miR-142-3p is more effective in inhibiting MCF-7 breast cancer development than monotherapies with miR-424-5p and miR-142-3p.

     

miR-142-3p is a tumor suppressor that inhibits estrogen receptor expression in ER-positive breast cancer

Estrogen receptors (ERs) are involved in the development of many types of malignant tumors, in particular, breast cancer. Among others, ERs affect cell growth, proliferation, and differentiation. The microRNA (miRNA) miR-142-3p has been shown to inhibit carcinogenesis by regulating various cellular processes, including cell cycle progression, cell migration, apoptosis, and invasion. It does so via targeting molecules involved in a range of signaling pathways. We surgically collected 20 ER-positive breast cancer samples, each with matched adjacent normal breast tissue, and measured the expression of miR-142-3p via quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics methods, luciferase reporter assay, qRT-PCR, and western blot analysis were used to assess whether miR-142-3p could target ESR1, which encodes the estrogen receptor, in ER-positive breast cancer cells and patient samples. We also restored miRNA expression and performed cell viability, cytotoxicity, and colony formation assays. Western blot analysis and qRT-PCR were used to study the expression of apoptosis and stemness markers. We found that miR-142-3p is downregulated in ER-positive breast cancers. Restoration of miR-142-3p expression in ER-positive breast cancer cells reduced cell viability, induced apoptosis via the intrinsic pathway and decreased both colony formation and the expression of stem cell markers. Bioinformatic analysis predicted miR-142-3p could bind to 3′-untranslated region ESR1 messenger RNA (mRNA). Consistently, we demonstrated that miR-142-3p reduced luciferase activity in ER-positive breast cancer cells, and decreased ESR1 expression in both mRNA and protein levels. The results revealed miR-142-3p and ESR1 expression correlated negatively in ER-positive breast cancer samples. The results suggest miR-142-3p acts as a tumor suppressor via multiple mechanisms. Thus, restoration of miR-142-3p expression, for example, via miRNA replacement therapy, may represent an effective strategy for the treatment of ER-positive breast cancer patients.     

A loop involving NRF2, miR-29b-1-5p and AKT,regulates cell fate of MDA-MB-231 triple-negative breast cancer cells

The present study shows that nuclear factor erythroid 2-related factor 2 (NRF2) and miR-29b-1-5p are two opposite forces which could regulate the fate of MDA-MB-231 cells, the most studied triple-negative breast cancer (TNBC) cell line. We show that NRF2 activation stimulates cell growth and markedly reduces reactive oxygen species (ROS) generation, whereas miR-29b-1-5p overexpression increases ROS generation and reduces cell proliferation. Moreover, NRF2 downregulates miR-29b-1-5p expression, whereas miR-29b-1-5p overexpression decreases p-AKT and p-NRF2. Furthermore, miR-29b-1-5p overexpression induces both inhibition of DNA N-methyltransferases (DNMT1, DNMT3A, and DNMT3B) expression and re-expression of HIN1, RASSF1A and CCND2. Conversely, NRF2 activation induces opposite effects. We also show that parthenolide, a naturally occurring small molecule, induces the expression of miR-29b-1-5p which could suppress NRF2 activation via AKT inhibition. Overall, this study uncovers a novel NRF2/miR-29b-1-5p/AKT regulatory loop that can regulate the fate (life/death) of MDA-MB-231 cells and suggests this loop as therapeutic target for TNBC.     

Curcumin inhibits proteasome activity in triple-negative breast cancer cells through regulating p300/miR-142–3p/PSMB5 axis

BackgroundCurcumin functions as a proteasome inhibitor. However, the molecular mechanisms behind this action need more detailed explanations.PurposeThis study aimed to investigate the inhibitory effect of curcumin on 20S proteasome activity and to elucidate its exact mechanism in triple-negative breast cancer (TNBC) MDA-MB-231 cells.MethodsProteasomal peptidase activities were assayed using synthetic fluorogenic peptide substrates. Knockdown or overexpression of microRNA (miRNA or miR) or protein was used to investigate its functional effect on downstream cellular processes. BrdU (5‑bromo‑2′-deoxyuridine) assay was performed to identify cell proliferation. Western blot and quantitative real-time PCR(qRT-PCR) were carried out to determine protein abundance and miRNA expression, respectively. Correlations between protein expressions, miRNA levels, and proteasome activities were analyzed in TNBC tissues. Xenograft tumor model was performed to observe the in vivo effect of curcumin on 20S proteasome activity.ResultsCurcumin significantly reduced PSMB5 protein levels, accompanied with a reduction in the chymotrypsin-like (CT-l) activity of proteasome 20S core. Loss of PSMB5 markedly inhibited the CT-l activity of 20S proteasome. Furthermore, curcumin treatment significantly elevated miR-142–3p expression. PSMB5 was a direct target of miR-142–3p and its protein levels were negatively regulated by miR-142–3p. Moreover, histone acetyltransferase p300 suppressed miR-142–3p expression. Overexpression of p300 mitigated the promotive effect of curcumin on miR-142–3p expression. The correlations among p300 abundances, miR-142–3p levels, PSMB5 expressions, and the CT-l activities of 20S proteasome were evidenced in TNBC tissues. In addition, loss of p300 and PSMB5 reduced cell proliferation. Inhibition of miR-142–3p significantly attenuated the inhibitory impact of curcumin on cell proliferation. These curcumin-induced changes on p300, miR-142–3p, PSMB5, and 20S proteasome activity were further confirmed in in vivo solid tumor model.ConclusionThese findings demonstrated that curcumin suppressed p300/miR-142–3p/PSMB5 axis leading to the inhibition of the CT-l activity of 20S proteasome. These results provide a novel and alternative explanation for the inhibitory effect of curcumin on proteasome activity and also raised potential therapeutic targets for TNBC treatment.     

Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression

It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients’ shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases.     

Hsa_circ_0069094 positively regulates the expression of oncogenic ZNF217 by competitively targeting miR-758–3p to promote the development of breast cancer

To investigate the functions and potential mechanisms of hsa_circ_0069094 in this cancer. The expression of hsa_circ_0069094, zinc finger protein 217 (ZNF217) and microRNA-758–3p (miR-758–3p) was detected by quantitative polymerase chain reaction (qPCR), and the protein level of ZNF217 was detected by western blot. Cell proliferation was assessed using cell counting kit-8 (CCK-8) assay and colony formation assay. Cell cycle progression and cell apoptosis were determined using flow cytometry assay. Cell invasion and cell migration were monitored using transwell assay and wound healing assay. The protein levels of apoptosis-related proteins were quantified by western blot. The putative relationship between miR-758–3p and hsa_circ_0069094 and ZNF217 was confirmed using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Xenograft model was constructed in mice to explore the role of hsa_circ_0069094 on solid tumor growth.Hsa_circ_0069094 and ZNF217 were highly expressed, while miR-758–3p was poorly expressed in tissues and cells of breast cancer. Hsa_circ_0069094 knockdown or ZNF217 knockdown inhibited cell proliferation, invasion and migration and induced cell apoptosis and cell cycle arrest in breast cancer cells. The inhibitory effects of hsa_circ_0069094 knockdown on cell malignant behaviors were abolished by ZNF217 overexpression. Hsa_circ_0069094 competed with ZNF217 for the binding site of miR-758–3p, and hsa_circ_0069094 positively regulated ZNF217 expression by competitively binding to miR-758–3p. Hsa_circ_0069094 knockdown also blocked solid tumor growth in mice. Collectively, Hsa_circ_0069094 played oncogenic effects in breast cancer by activating the expression of ZNF217 via competitively binding to miR-758–3p, which might be a novel strategy for breast cancer suppression.     

miR-139-5p is a regulator of metastatic pathways in breast cancer

Metastasis is a complex, multistep process involved in the progression of cancer from a localized primary tissue to distant sites, often characteristic of the more aggressive forms of this disease. Despite being studied in great detail in recent years, the mechanisms that govern this process remain poorly understood. In this study, we identify a novel role for miR-139-5p in the inhibition of breast cancer progression. We highlight its clinical relevance by reviewing miR-139-5p expression across a wide variety of breast cancer subtypes using in-house generated and online data sets to show that it is most frequently lost in invasive tumors. A biotin pull-down approach was then used to identify the mRNA targets of miR-139-5p in the breast cancer cell line MCF7. Functional enrichment analysis of the pulled-down targets showed significant enrichment of genes in pathways previously implicated in breast cancer metastasis (P < 0.05). Further bioinformatic analysis revealed a predicted disruption to the TGFβ, Wnt, Rho, and MAPK/PI3K signaling cascades, implying a potential role for miR-139-5p in regulating the ability of cells to invade and migrate. To corroborate this finding, using the MDA-MB-231 breast cancer cell line, we show that overexpression of miR-139-5p results in suppression of these cellular phenotypes. Furthermore, we validate the interaction between miR-139-5p and predicted targets involved in these pathways. Collectively, these results suggest a significant functional role for miR-139-5p in breast cancer cell motility and invasion and its potential to be used as a prognostic marker for the aggressive forms of breast cancer.     

MicroRNAs function as cis- and trans-acting modulators of peripheral circadian clocks

Based on their extracellular expression and targeting of the clock gene Bmal1, miR-142-3p and miR-494 were analyzed for evidence of vesicle-mediated communication between cells and intracellular functional activity. Our studies demonstrate that: miR-142-3p + miR-494 overexpression decreases endogenous BMAL1 levels, increases the period of Per2 oscillations, and increases extracellular miR-142-3p/miR-494 abundance in conditioned medium; miRNA-enriched medium increases intracellular expression of miR-142-3p and represses Bmal1 3′-UTR activity in naïve cells; and inhibitors of vesicular trafficking modulate intercellular communication of these miRNAs and ensemble Per2 rhythms. Thus, miR-142-3p and miR-494 may function as cis- and trans-acting signals contributing to local temporal coordination of cell-autonomous circadian clocks.     

Metastatic breast tumors downregulate miR-145 regulating the hypoxia-induced vasculogenic mimicry

Tumor cells grow in three-dimensional (3D) channels-like structures denoted as vasculogenic mimicry (VM), which provides a route for nutrients and oxygen acquisition. VM is activated by hypoxia and associated with metastasis and poor prognosis. MetastamiRs are microRNAs regulating metastasis, however, if they control VM in breast cancer remains poorly understood. The aim of this study was to evaluate the expression of VM-associated microRNAs in tumors of metastatic breast cancer patients. Firstly, we constructed microRNAs/mRNAs coregulation networks using expression data from TCGA databases. Dozens of microRNAs regulating genes involved in VM and metastasis were found. Of these, we selected 10 microRNAs for further characterization. The presence of VM in histological samples from patients with or without metastasis was evaluated using CD31-/PAS+ immunophenotyping. Remarkably, data showed that VM was significantly increased in tumors from patients with metastasis in comparison with no-metastatic group. Gene expression analysis indicated that miR-145, miR-142-3p, miR-31, miR-148a, miR-200b-3p and miR-526b were downregulated in primary tumors from patients with metastatic disease and positive for VM. Moreover, modulated microRNAs showed a predictive clinical value in overall survival in a cohort (n=1262) of breast cancer patients. Of these, we evaluated the role of miR-145 in formation of hypoxia-induced 3D channels-like using an in vitro model that recapitulates the early stages of VM. Data showed that miR-145 mimics was able to abolish the VM development in both metastatic Hs578t and MDA-MB-231 breast cancer cells. In conclusion, manipulation of miR-145 levels may represent a therapeutic approach in metastatic breast cancer patients that developed VM.     

Extracellular vesicles extracted from bone marrow mesenchymal stem cells carrying MicroRNA-342-3p inhibit the INHBA/IL13Rα2 axis to suppress the growth and metastasis of breast cancer

Increasing focus has come to the role of extracellular vesicles (EVs) in various cancers. Hence, we designed this study to explore the mechanism whereby microRNA-342-3p (miR-342-3p)-containing EVs derived from BMSCs might affect breast cancer. MCF-7 breast cancer cell line was co-incubated with the EVs isolated from rat BMSCs, followed by alteration of miR-342-3p and INHBA expression. Microarray-based analyses predicted a possible regulatory mechanism involving miR-342-3p, INHBA, and IL13Rα2 in breast cancer, which was verified by luciferase reporter, RNA pull-down, and RIP assays. Besides, in order to evaluate the effects of miR-342-3p on the biological features of breast cancer cells in vitro and in vivo, we employed the scratch assay, Transwell assay, CCK-8 assay, and nude mouse tumorigenicity assay. miR-342-3p carried by BMSC-EVs was transferred into breast cancer cells through co-culture, which inhibited the proliferation and metastasis of breast cancer cells in vitro. miR-342-3p downregulated the expression of INHBA, which further repressed the expression of IL13Rα2. Finally, the in vivo experimental results revealed the inhibitory role of miR-342-3p in tumor growth and metastasis in nude mice. To sum up, BMSC-EVs carrying miR-342-3p could prevent breast cancer growth and metastasis by downregulating the INHBA/IL13Rα2 axis, highlighting a potential target for anti-cancer treatment for breast cancer.     

Up-regulated circBACH2 contributes to cell proliferation,invasion, and migration of triple-negative breast cancer

An increasing amount of evidence has proven the vital role of circular RNAs (circRNAs) in cancer progression. However, there remains a dearth of knowledge on the function of circRNAs in triple-negative breast cancer (TNBC). Utilizing a circRNA microarray dataset, four circRNAs were identified to be abnormally expressed in TNBC. Among them, circBACH2 was most significantly elevated in TNBC cancerous tissues and its high expression was positively correlated to the malignant progression of TNBC patients. In normal human mammary gland cell line, the overexpression of circBACH2 facilitated epithelial to mesenchymal transition and cell proliferation. In TNBC cell lines, circBACH2 knockdown suppressed the malignant progression of TNBC cells. Mechanistically, circBACH2 sponged miR-186-5p and miR-548c-3p, thus releasing the C-X-C chemokine receptor type 4 (CXCR4) expression. The interference of miR-186-5p/miR-548c-3p efficiently promoted the cell proliferation, migration, and invasion suppressed by circBACH2 knockdown in the TNBC cell lines. Finally, circBACH2 knockdown repressed the growth and lung metastasis of TNBC xenografts in nude mice. In summary, circBACH2 functions as an oncogenic circRNA in TNBC through a novel miR-186-5p/miR-548c-3p/CXCR4 axis.Subject terms: Cancer, Cell biology     

The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.     

基于生物信息学分析研究miR-182-5p通过靶向调控EP300促进乳腺癌细胞的侵袭和转移

近期研究表明,miR-182-5p对多种癌症的侵袭和转移具有重要作用,但其在乳腺癌侵袭转移中的研究相对较少。本研究通过网上在线microRNA分析工具下载乳腺癌组织及正常乳腺组织表达比较的数据集,分析发现在GSE4589、GSE38167、GSE61438等3个数据库中,在乳腺癌组织中存在26个相同的microRNA,其中8个上调,而我们实验验证发现hsa-miR-182在8例病理组织中的表达上调差异最显著(P=0.001),选定目的基因hsa-miR-182;qRT-PCR检测细胞中miR-182-5p的表达,结果显示,与MCF-10A相比,miR-182-5p在MDA-MB-231、T47D、MDA-MB-453、MCF-7中表达上调(P<0.05);转染miR-182-5p干扰质粒,qRT-PCR检测细胞中miR-182-5p的表达情况。结果显示,miR-182-5p表达显著降低(P=0.003),提示转染成功;Transwell侵袭结果显示,MDAMB-231细胞敲低miR-182-5p,与对照组相比,体外侵袭能力明显降低(P=0.002);Western印迹检测转染miR-182-5p干扰质粒时,MDA-MB-231中上皮-间质转化(epithelial-mesenchymal transition,EMT)相关标志物的表达情况,结果显示,与对照组相比,敲低miR-182-5p使细胞中上皮-钙黏着蛋白(E-cadherin)表达上调,神经-钙黏着蛋白(N-cadherin)、波形蛋白(vimentin)表达下调。为研究探讨miR-182-5p的靶蛋白,采用在线预测软件预测可能与miR-182-5p结合的靶蛋白,cytoscape构建蛋白质互作网络图并筛选出hub基因;双荧光素酶结果证实,miR-182-5p可与EP300靶向结合(P=0.001);采用qRT-PCR、Western印迹检测转染miR-182-5p干扰质粒后EP300在mRNA及蛋白质水平的表达,结果显示,与对照组相比,在敲低miR-182-5p组中EP300在mRNA及蛋白质的表达上调(P=0.001)。综上所述,miR-182-5p可靶向调节EP300,促进乳腺癌细胞的侵袭与转移。     

MiR-101 Is Involved in Human Breast Carcinogenesis by Targeting Stathmin1

Background

MicroRNA-101 (miR-101) expression is negatively associated with tumor growth and blood vessel formation in several solid epithelial cancers. However, the role of miR-101 in human breast cancer remains elusive.

Results

MiR-101 was significantly decreased in different subtypes of human breast cancer tissues compared with that in adjacent normal breast tissues (P<0.01). Up-regulation of miR-101 inhibited cell proliferation, migration and invasion, and promoted cell apoptosis in ER alpha-positive and ER alpha-negative breast cancer cells and normal breast cells. Down-regulation of miR-101 displayed opposite effects on cell growth and metastasis. Further investigation revealed a significant inverse correlation between the expression of miR-101 and Stathmin1 (Stmn1), and miR-101 could bind to the 3′-untranslated region (UTR) of Stmn1 to inhibit Stmn1 translation. The inhibition of cell growth and metastasis induced by up-regulation of miR-101 was partially restored by overexpresson of Stmn1. Knockdown of Stmn1 attenuates the down-regulation of miR-101-mediated enhancement of cell growth and metastasis. More importantly, in vivo analysis found that Stmn1 mRNA and protein level in different subtypes of human breast cancer tissues, contrary to the down-regulation of miR-101, were significantly elevated.

Conclusions

This study demonstrates that down-regulation of miR-101 in different subtypes of human breast cancer tissues is linked to the increase of cellular proliferation and invasiveness via targeting Stmn1, which highlights novel regulatory mechanism in breast cancer and may provide valuable clues for the future clinical diagnosis of breast cancer.     

Wnt pathway targeting reduces triple-negative breast cancer aggressiveness through miRNA regulation in vitro and in vivo

Triple-negative breast cancer, devoid of estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER-2) expression, is deprived of commonly used targeted therapies. MicroRNAs (miRNAs) are undergoing a revolution in terms of potentially diagnostic or therapeutic elements. Combining computational approaches, we enriched miRNA binding motifs of Wnt pathway-associated upregulated genes. Our in-depth bioinformatics, in vitro and in vivo analyses indicated that miR-381 targets main genes of the Wnt signaling pathway including CTNNB1, RhoA, ROCK1, and c-MYC genes. The expression level of miR-381 and target genes was assessed by quantitative real-time polymerase chain reaction (RT-qPCR) in MCF-7, MDA-MB-231, and MCF-10A as well as 20 breast cancer samples and normal tissues. Luciferase reporter assay was performed. Lentiviral particles containing miR-381 were used to evaluate the effect of miR-381 restoration on cell proliferation, migration, and invasion of the invasive triple-negative MDA-MB-231 cell line and also in a mouse model of breast cancer. The expression of miR-381 was lower than that of normal cells, especially in TNBC cell line and breast tissues. Luciferase assay results confirmed that miR-381 targets all the predicted 3′-untranslated regions (3′-UTRs). Upon miR-381 overexpression, the expression of target genes declined, and the migration and invasion potential of miR-381-receiving MDA-MB-231 cells decreased. In a mouse model of triple-negative breast cancer, miR-381 re-expression inhibited the invasion of cancer cells to lung and liver and prolonged the survival time of cancer cell-bearing mice. Therefore, miR-381 is a regulator of Wnt signaling and its re-expression provides a potentially effective strategy for inhibition of TNBC.     

Homoharringtonine inhibited breast cancer cells growth via miR-18a-3p/AKT/mTOR signaling pathway

Homoharringtonine (HHT), a natural alkaloid derived from the cephalotaxus, exhibited its anti-cancer effects in hematological malignancies clinically. However, its pesticide effects and mechanisms in treating solid tumors remain unclear. In this study, we found that HHT was capable of inhibiting tumor growth after 5-days treatment of breast cancer cells, MCF-7, in vivo. Furthemore, HHT also significantly inhibited the cancer cell growth and induced cell apoptosis in vitro. miRNA sequencing proved miR-18a-3p was noticeably downregulated in the cells after HHT treatment. Moreover, downregulating miR-18a-3p increased HHT-induced cell apoptosis; our data supported that HHT suppressed miR-18a-3p expression and inhibited tumorigenesis might via AKT-mTOR signaling pathway. In conclusion: our study proved that HHT suppressed breast cancer cell growth and promoted apoptosis mediated by regulating of the miR-18a-3p-AKT-mTOR signaling pathway, HHT may be a promising antitumor agent in breast cancer treatment.     

Long noncoding RNA SNHG15 promotes human breast cancer proliferation,migration and invasion by sponging miR-211-3p

Long non-coding RNAs (lncRNA) have been demonstrated to act as essential regulators in the development and progression of breast cancer. In our study, we found that long noncoding RNA SNHG15 was highly expressed in breast cancer tissues and cell lines. And the expression of SNHG15 was correlated with TNM stage, lymphnode metastasis and survival in breast cancer patients. SNHG15 knockdown significantly inhibited the proliferation and induced apoptosis in breast cancer cells in vitro and in vivo. Besides, SNHG15 downregulation suppressed cell migration and invasion in MCF-7 and BT-20 cells, and inhibited epithelial-mesenchymal transition (EMT). In mechanism, we found that SNHG15 acted as a competing endogenous RNA to sponge miR-211-3p, which was downregulated in breast cancers and inhibited cell proliferation and migration. Our results showed that there was a negative correlation between SNHG15 and miR-211-3p expression in breast cancer patients. Collectively, we, for the first time, revealed the functions of SNHG15 and miR-211-3p in breast cancer.     

Induced expression of miR-1250-5p exerts tumor suppressive role in triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and it has a prevalence rate of 15%–20% among all breast cancer cases in younger women. Still, the underlying molecular mechanisms of its pathogenesis are not entirely understood. In the previous study, we identified that microRNA (miR)-1250-5p is significantly down-expressed in TNBC cells. Thus, in the present study, we explore the functional anticancer role of miR‑1250‑5p in the transient mimic transfected TNBC cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to examine the effect of miR-1250-5p on cell viability of TNBC (MDA-MB-231 and MDA-MB-453) cells. The confocal microscopy, quantitative real-time polymerase chain reaction, and western blot analysis techniques were used to assess the effect of miR-1250-5p on cancer hallmarks in test cells. Induced miR‑1250-5p expression in MDA-MB-231 and MDA-MB-453 cells decreased cell viability in a time-dependent manner. Increased miR‑1250-5p expression levels significantly decreased cell cycle G1/S phase transition markers (Cyclin D1 and CDK4) at messenger RNA (mRNA) and protein levels in TNBC cells compared to scrambled sequence transfected cells. Transient transfection of TNBC cells with miR-1250-5p mimic increased apoptosis in TNBC cells by increasing the level of active caspase (Caspase 8 and Caspase 3) of the intrinsic pathway. Apoptosis-related morphological changes were also observed in the test cells. Further, the induced expression of miR-1250-5p significantly decreased epithelial-mesenchymal transition (EMT) by altering the mRNA and protein levels of E-cadherin and Vimentin. Moreover, results of confocal microscopy revealed increased reactive oxygen species generation, and decreased mitochondria membrane potential in miR-1250-5p mimic transient transfected TNBC cells. In conclusion, miR‑1250-5p acts as tumor suppressor in TNBC cells and its induction by therapeutics might be a novel strategy for the disease treatment.