Extracellular vesicles-encapsulated let-7i shed from bone mesenchymal stem cells suppress lung cancer via KDM3A/DCLK1/FXYD3 axis

Accumulating evidence has suggested that extracellular vesicles (EVs) play a crucial role in lung cancer treatment. Thus, we aimed to investigate the modulatory role of bone marrow mesenchymal stem cell (BMSC)-EV-derived let-7i and their molecular mechanism in lung cancer progression. Microarray-based analysis was applied to predict lung cancer-related miRNAs and their downstream genes. RT-qPCR and Western blot analyses were conducted to determine Let-7i, lysine demethylase 3A (KDM3A), doublecortin-like kinase 1 (DCLK1) and FXYD domain-containing ion transport regulator 3 (FXYD3) expressions, after which dual-luciferase reporter gene assay and ChIP assay were used to identify the relationship among them. After loss- and gain-of-function assays, the effects of let-7i, KDM3A, DCLK1 and FXYD3 on the biological characteristics of lung cancer cells were assessed. Finally, tumour growth in nude mice was assessed by xenograft tumours in nude mice. Bioinformatics analysis screened out the let-7i and its downstream gene, that is KDM3A. The findings showed the presence of a high expression of KDM3A and DCLK1 and reduced expression of let-7i and FXYD3 in lung cancer. KDM3A elevated DCLK1 by removing the methylation of H3K9me2. Moreover, DCLK1 suppressed the FXYD3 expression. BMSC-EV-derived let-7i resulted in the down-regulation of KDM3A expression and reversed its promoting role in lung cancer development. Consistently, in vivo experiments in nude mice also confirmed that tumour growth was suppressed by the BMSC-EV-derived let-7i. In conclusion, our findings demonstrated that the BMSC-EV-derived let-7i possesses an inhibitory role in lung cancer progression through the KDM3A/DCLK1/FXYD3 axis, suggesting a new molecular target for lung cancer treatment.     

KDM4B Overexpression Promotes the Growth,Migration, and Invasion of Rheumatoid Arthritis Fibroblast-Like Synoviocytes by Activating STAT3 Pathway

In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) present a unique aggressive phenotype and have a passive response to the inflammatory microenvironment, which are critical for the disease’s progression. KDM4B, as a histone demethylase, functions as an oncogenic factor in many cancers and is implicated in osteoclastogenesis as well as pro-inflammatory cytokine release in inflammatory diseases. However, the effects of KDM4B on RA FLS have not been reported. To investigate this issue, our study determined the expression of KDM4B in RA FLS using RT-qPCR and western blot. The effects of KDM4B on RA FLS viability, apoptosis, migration, and invasion were detected by MTT, flow cytometry, transwell migration, and invasion assays. Furthermore, the interaction of KDM4B with STAT3 signaling was studied by western blot, MTT, flow cytometry, transwell migration, and invasion assays. The experimental results showed that KDM4B expression was upregulated in RA synovial tissues and FLS as compared to healthy control tissues and normal FLS. Knockdown of KDM4B obviously suppressed RA FLS viability, migration and invasion, and induced apoptosis. In addition, knockdown of KDM4B in RA FLS decreased the expression of p-STAT3 and MMP-9 but increased cleaved caspase-3 expression compared with the control group. Moreover, KDM4B overexpression could promote cell growth, migration and invasion, and suppress apoptosis in RA FLS by activating STAT3 signaling. Therefore, these findings provide new insight for understanding the pathogenesis of RA and indicate that KDM4B may have a potential to be an effective therapeutic target for RA.

     

Lysine-specific demethylase 2B (KDM2B)-let-7-enhancer of zester homolog 2 (EZH2) pathway regulates cell cycle progression and senescence in primary cells

Sustained expression of the histone demethylase, KDM2B (Ndy1/FBXL10/JHDM1B), bypasses cellular senescence in primary mouse embryonic fibroblasts (MEFs). Here, we show that KDM2B is a conserved regulator of lifespan in multiple primary cell types and defines a program in which this chromatin-modifying enzyme counteracts the senescence-associated down-regulation of the EZH2 histone methyltransferase. Senescence in MEFs epigenetically silences KDM2B and induces the tumor suppressor miRNAs let-7b and miR-101, which target EZH2. Forced expression of KDM2B promotes immortalization by silencing these miRNAs through locus-specific histone H3 K36me2 demethylation, leading to EZH2 up-regulation. Overexpression of let-7b down-regulates EZH2, induces premature senescence, and counteracts immortalization of MEFs driven by KDM2B. The KDM2B-let-7-EZH2 pathway also contributes to the proliferation of immortal Ink4a/Arf null fibroblasts suggesting that, beyond its anti-senescence role in primary cells, this histone-modifying enzyme functions more broadly in the regulation of cellular proliferation.     

CTHRC1 promotes growth,migration and invasion of trophoblasts via reciprocal Wnt/β-catenin regulation

Preeclampsia (PE) is a pregnancy complication that is characterized by high blood pressure and is associated with high maternal and fetal morbidities. At a mechanistic level, PE is characterized by reduced invasion ability of trophoblasts. Collagen triple helix repeat containing-1 (CTHRC1) is a well-known tumor-promoting factor in several malignant tumors, but its role in trophoblasts remains unknown. In this study, we characterized the expression of CTHRC1 in placenta tissue samples from PE pregnancies and from normal pregnancies. We used the trophoblasts cell lines HTR-8/SVneo and JEG-3 to investigate the role of CTHRC1 in cell migration, invasion and proliferation. Western blot, PCR and TOP/FOP luciferase activity assays were used to investigate the molecular mechanisms underlying these cell behaviors. Placenta tissue samples obtained from pregnant women with PE expressed lower levels of CTHRC1 than those of placenta tissues from women with normal pregnancies. Down-regulation of CTHRC1 impaired cell proliferation, migration and invasion of trophoblasts, while CTHRC1 overexpression promoted nuclear translocation of β-catenin, a result that was further confirmed by TOP/FOP luciferase activity assay. Our findings suggest that CTHRC1 promotes migration and invasion of trophoblasts via reciprocal Wnt/β-catenin signaling pathway. Down-regulation of CTHRC1 may be a potential mechanism underpinning the development of preeclampsia.     

Circ_0005615 promotes cervical cancer cell growth and metastasis by modulating the miR-138-5p/KDM2A axis

Cervical cancer (CC) is a highly fatal gynecological malignancy due to its high metastasis and recurrence rate. Circular RNA (circRNA) has been regarded as a regulator of CC. However, the underlying molecular mechanism of circ_0005615 in CC remains unclear. The levels of circ_0005615, miR-138-5p, and lysine demethylase 2A (KDM2A) were measured using qRT-PCR or western blot. Cell proliferation was assessed by Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine, and colony formation experiments. Cell invasion and migration were tested by transwell assay and wound healing assay. Flow cytometry and Caspase-Glo 3/7 Assay kit were used to analyze cell apoptosis. The expression of proliferation-related and apoptosis-related markers was detected by western blot. The binding relationships among circ_0005615, miR-138-5p, and KDM2A were verified by dual-luciferase reporter assay or RNA immunoprecipitation assay. Xenograft assay was applied to detect the effect of circ_0005615 in vivo. Circ_0005615 and KDM2A were upregulated, while miR-138-5p was downregulated in CC tissues and cells. Circ_0005615 knockdown retarded cell proliferation, migration, and invasion, while promoting apoptosis. Besides, circ_0005615 sponged miR-138-5p, and miR-138-5p could target KDM2A. miR-138-5p inhibitor reversed the regulation of circ_0005615 knockdown on CC cell growth and metastasis, and KDM2A overexpression also abolished the inhibitory effect of miR-138-5p on CC cell growth and metastasis. In addition, we also discovered that circ_0005615 silencing inhibited CC tumor growth in vivo. Circ_0005615 acted as a tumor promoter in CC by regulating the miR-138-5p/KDM2A pathway.     

Extracellular vesicle-derived microRNA-18b ameliorates preeclampsia by enhancing trophoblast proliferation and migration via Notch2/TIM3/mTORC1 axis

Preeclampsia (PE), a common disorder of pregnancy, is characterized by insufficient trophoblast migration and inadequate vascular remodelling, such that promotion of trophoblast proliferation might ameliorate PE. In the current study, we sought to study the underlying mechanism of extracellular vesicle (EV)-derived microRNA-18 (miR-18b) in PE. Human umbilical cord mesenchymal stem cells (HUCMSCs) isolated from placental tissues were verified through osteogenic, adipogenic and chondrogenic differentiation assays. Bioinformatics analyses and dual-luciferase reporter gene assay were adopted to confirm the targeting relationship between miR-18b and Notch2. The functional roles of EV-derived miR-18b and Notch2 in trophoblasts were determined using loss- and gain-of-function experiments, and trophoblast proliferation and migration were assayed using CCK-8 and Transwell tests. In vivo experiments were conducted to determine the effect of EV-derived miR-18b, Notch2 and TIM3/mTORC1 in a rat model of PE, with monitoring of blood pressure and urine proteinuria. TUNEL staining was conducted to observe the cell apoptosis of placental tissues of PE rats. We found down-regulated miR-18b expression, and elevated Notch2, TIM3 and mTORC1 levels in the placental tissues of PE patients compared with normal placenta. miR-18b was delivered to trophoblasts and targeted Notch2 and negatively its expression, whereas Notch2 positively mediated the expression of TIM3/mTORC1. EV-derived miR-18b or Notch2 down-regulation enhanced trophoblast proliferation and migration in vitro and decreased blood pressure and 24 hours urinary protein in PE rats by deactivating the TIM3/mTORC1 axis in vivo. In summary, EV-derived miR-18b promoted trophoblast proliferation and migration via down-regulation of Notch2-dependent TIM3/mTORC1.     

KDM5B promotes self-renewal of hepatocellular carcinoma cells through the microRNA-448–mediated YTHDF3/ITGA6 axis

Histone methylation plays important roles in mediating the onset and progression of various cancers, and lysine-specific demethylase 5B (KDM5B), as a histone demethylase, is reported to be an oncogene in hepatocellular carcinoma (HCC). However, the mechanism underlying its tumorigenesis remains undefined. Hence, we explored the regulatory role of KDM5B in HCC cells, aiming to identify novel therapeutic targets for HCC. Gene Expression Omnibus database and StarBase were used to predict important regulatory pathways related to HCC. Then, the expression of KDM5B and microRNA-448 (miR-448) in HCC tissues was detected by RT-qPCR and Western blot analysis. The correlation between KDM5B and miR-448 expression was analysed by Pearson's correlation coefficient and ChIP experiments, and the targeting of YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) by miR-448 was examined by luciferase assay. Additionally, the effect of KDM5B on the proliferation, migration, invasion and apoptosis as well as tumorigenicity of transfected cells was assessed using ectopic expression and depletion experiments. KDM5B was highly expressed in HCC cells and was inversely related to miR-448 expression. KDM5B demethylated H3K4me3 on the miR-448 promoter and thereby inhibited the expression of miR-448, which in turn targeted YTHDF3 and integrin subunit alpha 6 (ITGA6) to promote the malignant phenotype of HCC. Moreover, KDM5B accelerated HCC progression in nude mice via the miR-448/YTHDF3/ITGA6 axis. Our study uncovered that KDM5B regulates the YTHDF3/ITGA6 axis by inhibiting the expression of miR-448 to promote the occurrence of HCC.     

Long noncoding RNA LINC00339 promotes laryngeal squamous cell carcinoma cell proliferation and invasion via sponging miR-145

Laryngeal squamous cell carcinoma (LSCC) is a very common neoplasm of the head and neck in the world. Long noncoding RNAs play key roles in cell infiltration, fate, apoptosis, and invasion. However, the functional role and expression of LINC00339 remains unclear in LSCC. In this study, we showed that the expression level of LINC00339 was upregulated in LSCC tissues and cell lines. LINC00339 silencing suppressed the proliferation, invasion, and epithelial-mesenchymal transition (EMT) progression of LSCC cells. In addition, we showed that LINC00339 acted as a sponge of miR-145, and LINC00339 silencing promoted the expression of miR-145 in Hep2 cell. Furthermore, the expression of miR-145 was lower in LSCC tissues than in their paired normal samples and the miR-145 expression level was negatively correlated with LINC00339 expression in LSCC tissues. The knockdown of miR-145 promoted the proliferation, invasion, and EMT progression of LSCC cells. Finally, we indicated that LINC00339 silencing inhibited the proliferation, invasion, and EMT progression of LSCC cells by suppressing the miR-145 expression. These data suggested that LINC00339 acted as an oncogene in the development of LSCC, partly by regulating the miR-145 expression.     

Upregulated long noncoding RNA Linc00261 in pre-eclampsia and its effect on trophoblast invasion and migration via regulating miR-558/TIMP4 signaling pathway

Pre-eclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality but the exact underlying mechanisms of PE pathogenesis remain elusive. Accumulated data suggested that the long noncoding RNAs (lncRNAs) play important roles in the pathogenesis of PE. The present study identified the changes of lncRNA Linc00261 in PE and its effects on trophoblasts invasion and migration. Our results showed that the expression of Linc00261 was upregulated in placental tissues of PE women compared with those of healthy pregnant women. Overexpression of Linc00261 suppressed cell invasion and migration, induced cell apoptosis, and caused cell-cycle arrest at G₀/G₁ phase of HTR-8/SVneo cells; while knockdown of Linc00261 had the opposite effects on the HTR-8/SVneo cells. Mechanistic studies showed Linc00261 functioned as a competing endogenous RNA for miR-558 in HTR-8/SVneo cells, and miR-558 was negatively regulated by Linc00261. The expression level of miR-558 in the PE group was significantly lower than the control group, and the expression level of Linc00261 was negatively correlated with the expression level of miR-558 in the placental tissues of women with PE. Furthermore, miR-558 was found to negatively regulate the expression of TIMP metallopeptidase inhibitor 4 (TIMP4) via targeting the 3′ untranslated region in the HTR-8/SVneo cells. Overexpression of miR-558 increased HTR-8/SVneo cell invasion and migration, which was attenuated by TIMP4 overexpression. More importantly, both overexpression of miR-558 and knockdown of TIMP4 partially reversed the suppressive effects of Linc00261 overexpression on cell invasion and migration of HTR-8/SVneo cells. Collectively, our results for the first time showed the upregulation of Linc00261 in the placental tissues of severe PE patients. The mechanistic results indicated that Linc00261 exerted the suppressive effects on the trophoblast invasion and migration via targeting miR-558/TIMP4 axis, which may involve in the pathogenesis of PE.     

Upregulated lncRNA UCA1 inhibits trophoblast cell invasion and proliferation by downregulating JAK2

Numerous studies have suggested that urothelial cancer-associated 1 (UCA1) acts as a suppressor gene affecting cell proliferation and migration. However, the biological role and the potential mechanism of UCA1 in the progression of pre-eclampsia (PE) remains unclear. The UCA1 level was markedly upregulated in PE pregnancies relative to non-PE ones in GSE75010 and tissues. A higher body mass index (BMI), maximum systolic blood pressure (BP), and maximum diastolic BP were observed in PE pregnancies, whereas the newborn weight z-score was lower compared with those of non-PE pregnancies. Knockdown of UCA1 accelerated the proliferative migratory abilities and cell cycle progression, but inhibited apoptosis of HTR-8/SVneo and JAR cells. Then, we found that Janus kinases 2 (JAK2) was negatively correlated with UCA1. In addition, JAK2 was downregulated in the placenta of PE pregnancies and was negatively regulated by UCA1. UCA1 was mainly enriched in the nucleus. Knockdown of UCA1 reduced the occupancies of the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and H3K27me3 on the Janus kinase 2 (JAK2) promoter regions. Finally, rescue experiments found that transfection of short-hairpin JAK2 attenuated proliferative and migratory abilities of trophoblasts, which were partially reversed after UCA1 knockdown. In short, UCA1 is upregulated in the trophocytes of PE pregnancies and accelerates trophoblast cell invasion and proliferation by downregulating JAK2.     

MicroRNA let-7f inhibits tumor invasion and metastasis by targeting MYH9 in human gastric cancer

Background

MicroRNAs (miRNAs) are important regulators that play key roles in tumorigenesis and tumor progression. A previous report has shown that let-7 family members can act as tumor suppressors in many cancers. Through miRNA array, we found that let-7f was downregulated in the highly metastatic potential gastric cancer cell lines GC9811-P and SGC7901-M, when compared with their parental cell lines, GC9811 and SGC7901-NM; however, the mechanism was not clear. In this study, we investigate whether let-7f acts as a tumor suppressor to inhibit invasion and metastasis in gastric cancers.

Methodology/Principal

Real-time PCR showed decreased levels of let-7f expression in metastatic gastric cancer tissues and cell lines that are potentially highly metastatic. Cell invasion and migration were significantly impaired in GC9811-P and SGC7901-M cell lines after transfection with let-7f-mimics. Nude mice with xenograft models of gastric cancer confirmed that let-7f could inhibit gastric cancer metastasis in vivo after transfection by the lentivirus pGCsil-GFP- let-7f. Luciferase reporter assays demonstrated that let-7f directly binds to the 3′UTR of MYH9, which codes for myosin IIA, and real-time PCR and Western blotting further indicated that let-7f downregulated the expression of myosin IIA at the mRNA and protein levels.

Conclusions/Significance

Our study demonstrated that overexpression of let-7f in gastric cancer could inhibit invasion and migration of gastric cancer cells through directly targeting the tumor metastasis-associated gene MYH9. These data suggest that let-7f may be a novel therapeutic candidate for gastric cancer, given its ability to reduce cell invasion and metastasis.     

Silencing lncRNA-DGCR5 increased trophoblast cell migration,invasion and tube formation,and inhibited cell apoptosis via targeting miR-454-3p/GADD45A axis

Long noncoding RNA (lncRNA)-DGCR5 has been recognized as a potential tumor progression regulator, while its expression and specific functions in preeclampsia (PE) development remain unveiled. The expressions of miR-454-3p, lncRNA-DiGeorge syndrome critical region gene 5 (DGCR5) and growth arrest and DNA damage protein-inducible 45A (GADD45A) in placental tissues from PE patients or HTR-8/SVneo cells were assessed by Western blot or qRT-PCR. Dual-luciferase reporter assay determined the binding relations between miR-454-3p and GADD45A and between miR-454-3p and lncRNA-DGCR5. The viability, apoptosis, migration, invasiveness and tube formation of HTR-8/SVneo cell were evaluated using cell counting kit (CCK)-8, Annexin-V/Propidium iodide staining, wound healing, transwell and tube formation assays, respectively. miR-454-3p was low-expressed in PE tissue, and upregulation of miR-454-3p increased viability and promoted migration, invasion and tube formation in HTR-8/SVneo cells while inhibiting apoptosis. Then, miR-454-3p was found to directly target GADD45A which was high-expressed in PE tissues. Overexpressing GADD45A decreased the viability and inhibited the migration, invasion and tube formation of HTR-8/SVneo cells while enhancing apoptosis, and it neutralized the effect of miR-454-3p upregulation. In turn, miR-454-3p upregulation reversed the effect of GADD45A overexpression. Meanwhile, miR-454-3p could also target lncRNA-DGCR5. Silencing lncRNA-DGCR5 increased miR-454-3p expression and cell viability and promoted migration, invasion and tube formation in HTR-8/SVneo cells while inhibiting apoptosis, and it counteracted the effect of miR-454-3p downregulation. As usual, miR-454-3p downregulation reversed the effect of lncRNA-DGCR5 silencing. To conclude, silencing lncRNA-DGCR5 increased viability, promoted migration, invasion and tube formation, and inhibited apoptosis in HTR-8/SVneo cells by rescuing the inhibition of GADD45A expression caused by miR-454-3p.

Graphical abstract

     

Long non-coding RNA DIO3OS/let-7d/NF-κB2 axis regulates cells proliferation and metastasis of thyroid cancer cells

Due to the steadily rising morbidity and mortality, thyroid cancer remains the most commonly seen endocrine cancer. The present study attempted to investigate the mechanism from the perspective of long non-coding RNA (lncRNA) regulation. We identified 53 markedly increased lncRNAs in thyroid cancer samples according to TCGA data. Among them, high lncRNA DIO3OS expression was a risk factor for thyroid cancer patients’ poorer overall survival. DIO3OS showed to be considerably increased within thyroid cancer tissue samples and cells. Knocking down DIO3OS within thyroid carcinoma cells suppressed cancer cell viability, the capacity of DNA synthesis, cell invasion, as well as cell migration; besides, proliferating markers, ki-67 and PCNA, were decreased by DIO3OS knockdown. Cancer bioinformatics analysis suggested that NF-κB2 might be related to DIO3OS function in thyroid cancer carcinogenesis. NF-κB2 was positively correlated with DIO3OS, and DIO3OS knockdown decreased NF-κB2 protein levels. Knocking down NF-κB2 within thyroid carcinoma cells suppressed cancer cell viability, the capacity of DNA synthesis, cell invasion, cell migration, and the protein levels of proliferating markers. Let-7d directly targeted DIO3OS and NF-κB2; DIO3OS knockdown upregulated let-7d expression. The overexpression of let-7d suppressed cancer cell viability, the capacity of DNA synthesis, cell invasion, cell migration, as well as the protein levels of proliferating markers. Let-7d inhibition remarkably attenuated the functions of DIO3OS knockdown in NF-κB2 expression and thyroid cancer cell phenotype. In conclusion, DIO3OS/let-7d/NF-κB2 axis regulates the viability, DNA synthesis capacity, invasion, and migration of thyroid cancer cells. The clinical application of this axis needs further in vivo and clinical investigation.Electronic supplementary materialThe online version of this article (10.1007/s12079-020-00589-w) contains supplementary material, which is available to authorized users.     

PAX3 silencing inhibits prostate cancer progression through the suppression of the TGF‐β/Smad signaling axis

Multiple studies have confirmed the pro‐oncogenic effects of PAX3 in an array of cancers, but its role in prostate cancer (PCa) remains largely undefined. The aim of this study is to investigate the role of PAX3 in PCa. PAX3 expression was compared between PCa tumor tissue and nontumor tissues and PCa cell lines and normal prostate epithelial cells (PNT2) by western blot analysis and immunohistochemistry staining. MTT and immunofluorescence assays were used to detect PCa cell proliferation. Flow cytometry was used to evaluate cell apoptosis in PCa. Transwell assays were used for the determination of cell migration and PCa cell invasion. PAX3 expression was higher in PCa tissues and human PCa cell lines. Moreover, PAX3 silencing inhibited the proliferation, metastasis, and epithelial–mesenchymal transition (EMT) of PCa cells, and increased the rates of apoptosis. PAX3 silencing inhibited transforming growth factor‐β (TGF‐β)/Smad signaling in PCa cells. The effects of si‐PAX3 on the proliferation, apoptosis, metastasis, and EMT of PCa cells were alleviated by TGF‐β1 treatment. PAX3 silencing inhibits PCa progression through the inhibition of TGF‐β/Smad signaling. This reveals PAX3 as a novel biomarker and therapeutic target for future PCa treatments.     

Silencing of Paternally Expressed Gene 10 Inhibits Trophoblast Proliferation and Invasion

Paternally expressed gene 10 (PEG10) is an imprinted and monoallelic expressed gene. Previous study using a knockout mouse model revealed a crucial role of PEG10 in placental development, yet the exact function of PEG10 during placentation remains to be elucidated. In this study, denuded chorionic villi were prepared from first trimester human placentas, and transduced with PEG10 small interference RNA (siRNA) or non-targeting control sequence by lentiviral infection. Immunohistochemical staining revealed that silencing of PEG10 in the chorionic villous explants resulted in reduced immune-reactivity to CK7, Ki67 and integrin α5, implying that silencing of PEG10 impaired the proliferation of villous trophoblasts and may interfere with the activity of extravillous trophoblasts. We further investigated the role of PEG10 in the proliferation, migration and invasion of JEG-3 trophoblast cell line and the primary chorionic villous cells. PEG10-silenced JEG-3 cells and primary chorionic villous cells displayed a reduced proliferation rate and impaired invasiveness in vitro. Silencing of PEG10 in trophoblast cells led to upregulated expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) as well as downregulated expression of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, knockdown of TIMP-1 reversed the suppressed invasiveness of PEG10 siRNA-transduced JEG-3 cells. In conclusion, our study demonstrates that PEG10 plays an important role in trophoblast proliferation and promotes trophoblast invasion through TIMP-1.