LINC00629, a HOXB4-downregulated long noncoding RNA, inhibits glycolysis and ovarian cancer progression by destabilizing c-Myc.

Ovarian cancer (OC) cells typically reprogram their metabolism to promote rapid proliferation. However, the role of long noncoding RNAs (lncRNAs) in the metabolic reprogramming of ovarian cancer, especially in glucose metabolic reprogramming, remains largely unknown. LINC00629 has been reported in our previous study to promote osteosarcoma progression. Upregulated LINC00629 was found to enhance the growth-suppressive effect of apigenin on oral squamous cell carcinoma. However, the precise function of LINC00629 in ovarian cancer development remains poorly understood. In this study, we found that LINC00629 was significantly downregulated in OC tissues and that low LINC00629 expression was associated with poor survival. Inhibition of LINC00629 was required for increased glycolysis activity and cell proliferation in ovarian cancer. In vivo, overexpression of LINC00629 dramatically inhibited tumor growth and lung metastasis. Mechanistically, LINC00629 interacted with and destabilized c-Myc, leading to its ubiquitination and proteasome degradation, further resulting in increased expression of downstream glycolysis-related genes and glucose metabolic reprogramming in OC. Interestingly, HOXB4 bound to the LINC00629 promoter and inhibited its transcription, indicating that LINC00629 is a transcriptional target of HOXB4. Collectively, these findings establish a direct role for LINC00629 in suppressing glucose metabolism, and HOXB4/LINC00629/c-Myc might serve as a potential biomarker and an effective therapeutic strategy for OC cancer treatment.

SNHG1, a KLF4-upregulated gene, promotes glioma cell survival and tumorigenesis under endoplasmic reticulum stress by upregulating BIRC3 expression.

Increasing evidence indicates that long noncoding RNAs (lncRNAs) play crucial roles in the resistance to endoplasmic reticulum (ER) stress in many cancers. However, ER stress-regulated lncRNAs are still unknown in glioma. In the present study, we investigated the altered lncRNAs upon ER stress in glioma and found that small nucleolar RNA host gene 1 (SNHG1) was markedly increased in response to ER stress. Increased SNHG1 suppressed ER stress-induced apoptosis and promoted tumorigenesis in vitro and in vivo. Further mechanistic studies indicated that SNHG1 elevated BIRC3 mRNA stability and enhanced BIRC3 expression. We also found that KLF4 transcriptionally upregulated SNHG1 expression and contributed to the ER stress-induced SNHG1 increase. Collectively, the present findings indicated that SNHG1 is a KLF4-regulated lncRNA that suppresses ER stress-induced apoptosis and facilitates gliomagenesis by elevating BIRC3 expression.

ZBTB7A, a miR-144-3p targeted gene, accelerates bladder cancer progression via downregulating HIC1 expression.

BACKGROUND: Zinc finger and BTB domain-containing 7A (ZBTB7A) is a member of the POK family of transcription factors that plays an oncogenic or tumor-suppressive role in different cancers depending on the type and genetic context of cancer. However, the function and molecular mechanism of ZBTB7A in bladder cancer (BC) remain elusive. METHODS: The role of ZBTB7A in bladder cancer was detected by colony formation, transwell, and tumor formation assays. The expression levels of ZBTB7A, HIC1, and miR-144-3p were analyzed by qRT-PCR and Western blot. Bioinformatics analysis and a dual-luciferase reporter assay were used to assess the effect of ZBTB7A on the promoter activity of HIC1. RESULTS: The present study revealed that knockdown of ZBTB7A suppressed BC cell growth and migration, as indicated by an approximately 50% reduction in the number of colonies and an approximately 70% reduction in the number of migrated cells. Loss of ZBTB7A inhibited tumor growth in vivo, resulting in a 75% decrease in tumor volume and an 80% decrease in tumor weight. Further mechanistic studies revealed that ZBTB7A bound to the hypermethylated in cancer 1 (HIC1) promoter and downregulated HIC1 expression, accelerating the malignant behavior of BC. Increased expression of ZBTB7A in BC tissues was negatively corrected with the expression of HIC1. Moreover, ZBTB7A was a target of miR-144-3p, which decreased ZBTB7A expression in BC. CONCLUSION: Our data demonstrate that ZBTB7A, a targeted gene of miR-144-3p, promoted tumorigenesis of BC through downregulating HIC1 expression.

Combinatorial readout of unmodified H3R2 and acetylated H3K14 by the tandem PHD finger of MOZ reveals a regulatory mechanism for HOXA9 transcription.

Histone acetylation is a hallmark for gene transcription. As a histone acetyltransferase, MOZ (monocytic leukemia zinc finger protein) is important for HOX gene expression as well as embryo and postnatal development. In vivo, MOZ forms a tetrameric complex with other subunits, including several chromatin-binding modules with regulatory functions. Here we report the solution structure of the tandem PHD (plant homeodomain) finger (PHD12) of human MOZ in a free state and the 1.47 Å crystal structure in complex with H3K14ac peptide, which reveals the structural basis for the recognition of unmodified R2 and acetylated K14 on histone H3. Moreover, the results of chromatin immunoprecipitation (ChIP) and RT-PCR assays indicate that PHD12 facilitates the localization of MOZ onto the promoter locus of the HOXA9 gene, thereby promoting the H3 acetylation around the promoter region and further up-regulating the HOXA9 mRNA level. Taken together, our findings suggest that the combinatorial readout of the H3R2/K14ac by PHD12 might represent an important epigenetic regulatory mechanism that governs transcription and also provide a clue of cross-talk between the MOZ complex and histone H3 modifications.

Long non-coding RNA AC023115.3 suppresses chemoresistance of glioblastoma by reducing autophagy.

Malignant glioma is an aggressive brain cancer that responds poorly to chemotherapy. However, the molecular mechanism underlying the development of chemoresistance in glioma is not well-understood. In this study, we show that long non-coding RNA AC023115.3 is induced by cisplatin in human glioblastoma cells and that elevated AC023115.3 promotes cisplatin-induced apoptosis by inhibiting autophagy. Further mechanistic studies revealed that AC023115.3 acts as a competing endogenous RNA for miR-26a and attenuates the inhibitory effect of miR-26a on GSK3ß, a proline-directed serine-threonine kinase that promotes the degradation of Mcl1, leading to an increase in GSK3ß and a decrease in autophagy. Additionally, we discovered that AC023115.3 improves chemosensitivity of glioma cells to cisplatin by regulating the miR-26a-GSK3ß-Mcl1 pathway. Thus, these data indicate that the AC023115.3-miR-26a-GSK3ß signalling axis plays an important role in reducing the chemoresistance of glioma.

KLF4, a miR-32-5p targeted gene, promotes cisplatin-induced apoptosis by upregulating BIK expression in prostate cancer.

BACKGROUND: Chemotherapeutic insensitivity remains a big challenge in prostate cancer treatment. Recently, increasing evidence has indicated that KLF4 plays a key role in prostate cancer. However, the potential biological role of KLF4 in Chemotherapeutic insensitivity of prostate cancer is still unknown. METHODS: The role of KLF4 in cisplatin-induced apoptosis was detected by western blotting and a cell counting kit (CCK8). The potential molecular mechanism of KLF4 in regulating prostate cancer chemosensitivity was investigated by RNA sequencing analysis, q-RT-PCR, western blotting and chromatin immunoprecipitation (ChIP). The expression level of KLF4 mediated by miR-32-5p was confirmed by bioinformatic analysis and luciferase assays. RESULTS: Here, we found that KLF4 was induced by cisplatin in prostate cancer cells and that the increase in KLF4 promoted cell apoptosis. Further mechanistic studies revealed that KLF4 directly bound to the promoter of BIK, facilitating its transcription. Additionally, we also found that the gene encoding KLF4 was a direct target of miR-32-5p. The downregulation of miR-32-5p in response to cisplatin treatment promoted KLF4 expression, which resulted in a increase in the chemosensitivity of prostate cancer. CONCLUSION: Thus, our data revealed that KLF4 is an essential regulator in cisplatin-induced apoptosis, and the miR-32-5p-KLF4-BIK signalling axis plays an important role in prostate cancer chemosensitivity.

Structural insights on mouse L-threonine dehydrogenase: A regulatory role of Arg180 in catalysis.

Mouse L-threonine dehydrogenase (mTDH), which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and mediates threonine catabolism, plays pivotal roles in both powerful biosynthesis and signaling in mouse stem cells and has a regulatory residue Arg180. Here we determined three crystal structures of mTDH: wild-type (WT) in the apo form; in complex with NAD(+) and a substrate analog, glycerol, or with only NAD(+); as well as the R180K variant with NAD(+). This is the first description of a structure for mammalian SDR-type TDH. Structural comparison revealed the structural basis for SDR-type TDH catalysis remains strictly conserved in bacteria and mammals. Kinetic enzyme assays, and isothermal titration calorimetry (ITC) measurements indicated the R180K mutation has little effect on NAD(+) binding affinity, whereas affects the substrate's affinity for the enzyme. The crystal structure of R180K with NAD(+), biochemical and spectroscopic studies suggested that the R180K mutant should bind NAD(+) in a similar way and have a similar folding to the WT. However, the R180K variant may have difficulty adopting the closed form due to reduced interaction of residue 180 with a loop which connects a key position for mTDH switching between the closed and open forms in mTDH catalysis, and thereby exhibited a significantly decreased kcat/Km value toward the substrate, L-Thr. In sum, our results suggest that activity of GalE-like TDH can be regulated by remote interaction, such as hydrogen bonding and hydrophobic interaction around the Arg180 of mTDH.

Sialyltransferase7A, a Klf4-responsive gene, promotes cardiomyocyte apoptosis during myocardial infarction.

Myocardial infarction (MI) is one major cause of heart failure through its induction of cardiomyocyte death. However, the molecular mechanisms associated with MI-induced cardiomyocyte apoptosis in the context of sialylation of heart are not yet understood. In this study, we found that sialyltransferase7A (Siat7A), one of the members of sialyltransferase family, was significantly increased in the ischemic myocardium, as well as in the human cardiomyocyte cell line AC16 under hypoxic condition. The Sialyl-Tn antigen (Neu5Acα2-6GalNAc-O-Ser/Thr) synthesized by Siat7A also increased in the AC16 cardiomyocytes following hypoxic stimulus. Increased Siat7A promoted cardiomyocyte apoptosis. The knockdown of Siat7A expression reduced cardiomyocyte apoptosis in both of vivo and vitro. Furthermore, the decreased extracellular signal-regulated kinase ERK1 and ERK2 (ERK1/2) activity was involved in the Siat7A-induced cardiomyocyte apoptosis. Notably, we showed that Krüppel-like factor 4 (Klf4), one of the transcription factors, specifically bound to the Siat7A promoter by ChIP assays. Deletion and mutagenesis analysis identified that Klf4 could transactivate the Siat7A promoter region (nt -655 to -636 bp). The upregulated Siat7A expression, which was paralleled by the increased Klf4 in the ischemic myocardium, contributed to cardiomyocyte apoptosis. Our study suggests Siat7A could be a valuable target for developing treatments for MI patients.

Regulation of L-threonine dehydrogenase in somatic cell reprogramming.

Increasing evidence suggests that metabolic remodeling plays an important role in the regulation of somatic cell reprogramming. Threonine catabolism mediated by L-threonine dehydrogenase (TDH) has been recognized as a specific metabolic trait of mouse embryonic stem cells. However, it remains unknown whether TDH-mediated threonine catabolism could regulate reprogramming. Here, we report TDH as a novel regulator of somatic cell reprogramming. Knockdown of TDH inhibits, whereas induction of TDH enhances reprogramming efficiency. Moreover, microRNA-9 post-transcriptionally regulates the expression of TDH and thereby inhibits reprogramming efficiency. Furthermore, protein arginine methyltransferase (PRMT5) interacts with TDH and mediates its post-translational arginine methylation. PRMT5 appears to regulate TDH enzyme activity through both methyltransferase-dependent and -independent mechanisms. Functionally, TDH-facilitated reprogramming efficiency is further enhanced by PRMT5. These results suggest that TDH-mediated threonine catabolism controls somatic cell reprogramming and indicate the importance of post-transcriptional and post-translational regulation of TDH.

KLF4 suppresses anticancer effects of brusatol via transcriptional upregulating NCK2 expression in melanoma.

Brusatol (Bru), a main extract from traditional Chinese medicine Brucea javanica, has been reported to exist antitumor effect in many tumors including melanoma. However, the underlying mechanism in its anti-melanoma effect still need further exploration. Here, we reported that the protein expression of KLF4 in melanoma cells were significantly downregulated in response to brusatol treatment. Overexpression of KLF4 suppressed brusatol-induced melanoma cell apoptosis; while knockdown of KLF4 enhanced antitumor effects of brusatol on melanoma cells not only in vitro but also in vivo. Further studies on the mechanism revealed that KLF4 bound to the promoter of NCK2 directly and facilitated NCK2 transcription, which suppressed the antitumor effect of brusatol on melanoma. Furthermore, our findings showed that miR-150-3p was dramatically upregulated under brusatol treatment which resulted in the downregulation of KLF4. Our results suggested that the miR-150-3p/KLF4/NCK2 axis might play an important role in the antitumour effects of brusatol in melanoma.

HOXB9 promotes osteosarcoma cell survival and malignancy under glucose starvation via upregulating SPP1 expression.

Homeobox B9 (HOXB9) has been shown to play a critical role in several tumors. However, the precise biological mechanisms and functions of HOXB9 in osteosarcoma remain largely unknown. In this study, we found that HOXB9 was increased upon glucose starvation. Elevated HOXB9 suppressed osteosarcoma cell death and supported cell growth and migration under glucose starvation. Further mechanistic studies demonstrated that HOXB9 directly bound to the promoter of secreted phosphoprotein 1 (SPP1) and transcriptionally upregulated SPP1 expression which then led cell death decrease and cell growth increase under glucose deprivation environment. Clinically, HOXB9 was significantly upregulated in osteosarcoma compared with normal tissues and increase of HOXB9 expression was positively associated with the elevation of SPP1 in osteosarcoma. Overall, our study illustrates that HOXB9 contributes to malignancy in osteosarcoma and inhibits cell death through transcriptional upregulating SPP1 under glucose starvation.

NUCB2 inhibition antagonizes osteosarcoma progression and promotes anti-tumor immunity through inactivating NUCKS1/CXCL8 axis.

The oncogenic properties of Nucleobindin2 (NUCB2) have been observed in various cancer types. Nevertheless, the precise understanding of the biological functions and regulatory mechanisms of NUCB2 in osteosarcoma remains limited. This investigation reported that NUCB2 was significantly increased upon glucose deprivation-induced metabolic stress. Elevated NUCB2 suppressed glucose deprivation-induced cell death and reactive oxygen species (ROS) increase. Depletion of NUCB2 resulted in a reduction in osteosarcoma cell proliferation as well as metastatic potential in vitro and in vivo. Mechanically, NUCB2 ablation suppressed C-X-C Motif Chemokine Ligand 8 (CXCL8) expression which then reduced programmed cell death 1 ligand 1 (PD-L1) expression and stimulated anti-tumor immunity mediated through cytotoxic T cells. Importantly, a combination of NUCB2 depletion with anti-PD-L1 treatment improved anti-tumor T-cell immunity in vivo. Moreover, we further demonstrated that NUCB2 interacted with NUCKS1 to inhibit its degradation, which is responsible for the transcriptional regulation of CXCL8 expression. Altogether, the outcome emphasizes the function of NUCB2 in osteosarcoma and indicates that NUCB2 elevates osteosarcoma progression and immunosuppressive microenvironment through the NUCKS1/CXCL8 pathway.

p53-facilitated miR-199a-3p regulates somatic cell reprogramming.

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by ectopic expression of defined transcriptional factors. The efficiency of this process, however, is extremely low. Although inactivation of p53 has been recently shown to greatly enhance reprogramming efficiency, the underlying molecular mechanisms still remain largely unknown. Here, we report that miR-199a-3p is upregulated by p53 at the post-transcriptional level. Induction of miR-199a-3p significantly decreases reprogramming efficiency, whereas miR-199a-3p inhibition greatly enhances it. Mechanistically, miR-199a-3p overexpression inhibits cell proliferation by imposing G1 cell cycle arrest. Conversely, miR-199a-3p inhibition results in a pronounced increase in cell proliferation. Furthermore, the enhancement in reprogramming of p53 knockdown cells is almost completely reversed with replacement of miR-199a-3p. Also, miR-199a-3p inhibition partially rescues iPS generation impaired by p53. These findings suggest miR-199a-3p as a novel p53 target that negatively regulates somatic cell reprogramming.

Erratum: KLF4 suppresses the migration of hepatocellular carcinoma by transcriptionally upregulating monoglyceride lipase.

[This corrects the article on p. 1019 in vol. 8, PMID: 30034939.].

NUCKS1, a LINC00629-upregulated gene, facilitated osteosarcoma progression and metastasis by elevating asparagine synthesis.

Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) has been reported to play an oncogenic role in several cancers. However, the biological functions and regulatory mechanism of NUCKS1 in osteosarcoma have not been fully understood. In this study, we reported that NUCKS1 was significantly increased in osteosarcoma. Depletion of NUCKS1 decreased osteosarcoma cell proliferation and metastasis in vivo and in vitro. Overexpression of NUCKS1 accelerated osteosarcoma cell aggressiveness. Mechanistically, NUCKS1 facilitated asparagine (Asn) synthesis by transcriptionally upregulating asparagine synthetase (ASNS) expression and elevating the levels of Asn in osteosarcoma cells, leading to increased cell growth and metastasis. Inhibition of ASNS or reduction of Asn decreased osteosarcoma cell aggressiveness and impaired the promoting effects of NUCKS1 on tumorigenesis and metastasis. Furthermore, we also found that by acting as a sponge for miR-4768-3p, LINC00629 promoted NUCKS1 expression. Collectively, our findings highlight the role of NUCKS1 in regulating asparagine metabolism and reveal that LINC00629 is an important regulator of NUCKS1 that contributes to NUCKS1 upregulation in osteosarcoma.

HOXB9 a miR-122-5p regulated gene, suppressed the anticancer effects of brusatol by upregulating SCD1 expression in melanoma.

Brusatol (Bru), a Chinese medicine Brucea javanica extract, has a variety of antitumour effects. However, its role and underlying mechanism in melanoma have not been fully elucidated. In this study, we found that brusatol inhibited melanoma cell proliferation and migration and promoted cell apoptosis in vitro, in addition to suppressing melanoma cell tumorigenesis in vivo. Further studies on the mechanism revealed that brusatol significantly downregulated the expression of stearoyl-CoA desaturase 1 (SCD1). Increased SCD1 expression could impair the antitumour effects of brusatol on melanoma cells. Subsequently, we found that HOXB9, an important transcription factor, was directly bound to the promoter of SCD1, facilitating its transcription. Overexpression of HOXB9 inhibited brusatol-induced SCD1 reduction and promoted cell survival. Furthermore, our results revealed that miR-122-5p was significantly increased in response to brusatol treatment and led to a decrease in HOXB9 in melanoma. Collectively, our data suggested that the miR-122-5p/HOXB9/SCD1 axis might play an important role in the antitumour effects of brusatol and that brusatol might have potential clinical implications in melanoma therapy.

LINC00629, a KLF10-responsive lncRNA, promotes the anticancer effects of apigenin by decreasing Mcl1 stability in oral squamous cell carcinoma.

Apigenin, a naturally occurring flavonoid, is known to exhibit antitumor activity in many cancers. However, the regulatory mechanism of apigenin and the long noncoding RNAs (lncRNAs) altered upon apigenin treatment in oral squamous cell carcinoma (OSCC) remain unclear. In this study, we found that LINC00629 was significantly upregulated in response to apigenin treatment. Upregulated LINC00629 enhanced the growth-suppressive and proapoptotic effects of apigenin on OSCC cells by interacting with Mcl1 and facilitating its degradation. Subsequently, our data indicated that KLF10, an important transcription factor, directly bound to the promoter of LINC00629, facilitating its transcription and contributing to apigenin-induced LINC00629 expression. Collectively, these results suggest that the KLF10-LINC00629-Mcl1 axis plays an important role in the anticancer effects of apigenin.

LINC00629 protects osteosarcoma cell from ER stress-induced apoptosis and facilitates tumour progression by elevating KLF4 stability.

BACKGROUND: Escaping from ER stress-induced apoptosis plays an important role in the progression of many tumours. However, its molecular mechanism in osteosarcoma remains incompletely understood. METHODS: The molecular mechanism was investigated using RNA sequencing, qRT-PCR and Western blot assays. The relationship between LINC00629 and KLF4 was investigated using RNA pulldown and ubiquitylation assays. The transcriptional regulation of laminin subunit alpha 4 (LAMA4) by KLF4 was identified using bioinformatic analysis, a luciferase assay, and a chromatin immunoprecipitation assay. RESULTS: Here, we demonstrated that LINC00629 was increased under ER stress treatment. Elevated LINC00629 inhibited ER stress-induced osteosarcoma cell apoptosis and promoted clonogenicity and migration in vitro and in vivo. Further mechanistic studies indicated that LINC00629 interacted with KLF4 and suppressed its degradation, which led to a KLF4 increase in osteosarcoma. In addition, we also found that KLF4 upregulated LAMA4 expression by directly binding to its promoter and that LINC00629 inhibited ER stress-induced apoptosis and facilitated osteosarcoma cell clonogenicity and metastasis by activating the KLF4-LAMA4 pathway. CONCLUSION: Collectively, our data indicate that LINC00629 is a critical long noncoding RNA (lncRNA) induced by ER stress and plays an oncogenic role in osteosarcoma cell by activating the KLF4-LAMA4 axis.

[Role of dysregulation of Bim in resistance of melanoma cells to endoplasmic reticulum stress-induced apoptosis].

OBJECTIVE: To establish a model of ER stress-induced apoptosis with tunicamycin and to examine whether Bim is dysregulated and its potential role in resistance of melanoma cells to apoptosis under endoplasmic reticulum (ER) stress. METHODS: A model of ER stress-induced apoptosis was established with tunicamycin. Apoptotic cells were quantitated using the annexin V/propidium iodide method by flow cytometry. Hoechst staining was also used to confirm the apoptotic cell death. Western blotting was used to measure the activation of caspase-3 and -9, and the expression of Bim, GRP78, CHOP, and Foxo1 at the protein level. The expression of Bim, CHOP and Foxo1 at the mRNA level was quantitated by qPCR. The siRNA technique was used to inhibit the expression of Bim. RESULTS: Treatment of the melanoma cells with tunicamycin did not induce significant apoptosis and activation of caspase cascade, whereas it caused marked activation of caspase-3 and -9, and apoptosis in HEK293 cells which were used as a control. With exposure to tunicamycin (3 µmol/L) for 12, 24, 36 hours the Bim protein levels were not increased in Mel-RM and MM200 cells. Its mRNA levels were 0.37 ± 0.05, 0.13 ± 0.02 and 0.02 ± 0.01 in Mel-RM cells, while 0.41 ± 0.06, 0.16 ± 0.04 and 0.21 ± 0.03 in MM200 cells, respectively. The expression of Bim mRNA was significantly reduced compared with that in the control groups of the two cell lines (P < 0.01). siRNA knockdown of Bim protected HEK293 cells against activation of caspase-3. The cell apoptosis of Bim siRNA group was (5.69 ± 0.38)%, significantly lower than that of the siRNA control group (40.32 ± 1.64)% and blank control group (35.46 ± 2.01)% (P < 0.01). In the melanoma cells after exposure to tunicamycin (3 µmol/L) for 6, 12, 24, and 36 hours the transcription factor CHOP at mRNA level were significantly increased and the expressions at protein level were also up-regulated. The expressions of another transcription factor Foxo1 at mRNA level significantly decreased and the expressions at protein level were down-regulated, too. CONCLUSIONS: The lack of Bim up-regulation contributes to the resistance of melanoma cells to ER stress-induced apoptosis and may be a mechanism by which melanoma cells adapt to ER stress conditions. Transcription factors CHOP and Foxo1 may be responsible for the dysregulation of Bim in melanoma cells upon ER stress.