Showing papers on "Small hairpin RNA published in 2019"

Effective breast cancer combination therapy targeting BACH1 and mitochondrial metabolism.

Abstract: Mitochondrial metabolism is an attractive target for cancer therapy1,2. Reprogramming metabolic pathways could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options, such as triple-negative breast cancer (TNBC)1,3. Here we show that BTB and CNC homology1 (BACH1)4, a haem-binding transcription factor that is increased in expression in tumours from patients with TNBC, targets mitochondrial metabolism. BACH1 decreases glucose utilization in the tricarboxylic acid cycle and negatively regulates transcription of electron transport chain (ETC) genes. BACH1 depletion by shRNA or degradation by hemin sensitizes cells to ETC inhibitors such as metformin5,6, suppressing growth of both cell line and patient-derived tumour xenografts. Expression of a haem-resistant BACH1 mutant in cells that express a short hairpin RNA for BACH1 rescues the BACH1 phenotype and restores metformin resistance in hemin-treated cells and tumours7. Finally, BACH1 gene expression inversely correlates with ETC gene expression in tumours from patients with breast cancer and in other tumour types, which highlights the clinical relevance of our findings. This study demonstrates that mitochondrial metabolism can be exploited by targeting BACH1 to sensitize breast cancer and potentially other tumour tissues to mitochondrial inhibitors. The transcription factor BACH1, which targets mitochondrial metabolism, is expressed at high levels in several types of cancer; reducing its expression in tumours makes them more susceptible to treatment with mitochondrial inhibitors.

Circular RNA circAGO2 drives cancer progression through facilitating HuR-repressed functions of AGO2-miRNA complexes.

Abstract: Argonaute 2 (AGO2), the core component of microRNA (miRNA)-induced silencing complex, plays a compelling role in tumorigenesis and aggressiveness. However, the mechanisms regulating the functions of AGO2 in cancer still remain elusive. Herein, we indentify one intronic circular RNA (circRNA) generated from AGO2 gene (circAGO2) as a novel regulator of AGO2-miRNA complexes and cancer progression. CircAGO2 is up-regulated in gastric cancer, colon cancer, prostate cancer, and neuroblastoma, and is associated with poor prognosis of patients. CircAGO2 promotes the growth, invasion, and metastasis of cancer cells in vitro and in vivo. Mechanistic studies reveal that circAGO2 physically interacts with human antigen R (HuR) protein to facilitate its activation and enrichment on the 3’-untranslated region of target genes, resulting in reduction of AGO2 binding and repression of AGO2/miRNA-mediated gene silencing associated with cancer progression. Pre-clinically, administration of lentivirus-mediated short hairpin RNA targeting circAGO2 inhibits the expression of downstream target genes, and suppresses the tumorigenesis and aggressiveness of xenografts in nude mice. In addition, blocking the interaction between circAGO2 and HuR by cell-penetrating inhibitory peptide represses the tumorigenesis and aggressiveness of cancer cells. Taken together, these results indicate that oncogenic circAGO2 drives cancer progression through facilitating HuR-repressed functions of AGO2-miRNA complexes.

CircRNA-5692 inhibits the progression of hepatocellular carcinoma by sponging miR-328-5p to enhance DAB2IP expression.

Abstract: Circular RNAs (circRNAs), one kind of noncoding RNAs, can interact with miRNA and transcription factors to regulate gene expression. However, little is known on which circRNA is crucial for the pathogenesis of hepatocellular carcinoma (HCC). CircRNA expression profile was analyzed by a microarray. Regulatory gene targets were predicted by bioinformatics analysis and validated by luciferase assay. Their expression was determined by qRT-PCR and Western blotting. DNA methylation was determined by methylation-specific PCR. Gene knockdown and overexpression were mediated by lentivirus-mediated shRNA and transfection with plasmids for cDNA expression, respectively. MTT assay, wound-healing assay, transwell invasion assay, and flow cytometry were used to determine malignant behaviors of HCC cells. HCC xenograft mouse model was used to determine the in vivo effects of circRNA-5692. CircRNA-5692 expression was downregulated in HCC tissues, and circRNA-5692 overexpression attenuated the malignant behaviors of HCC cells. Bioinformatics predicted that circRNA-5692 interacted with miR-328-5p, which targeted the DAB2IP mRNA. Actually, miR-328-5p promoted the malignant behaviors of HCC cells, while DAB2IP had opposite effects. Moreover, circRNA-5692 overexpression inhibited the growth of xenograft HCC tumors in vivo by decreasing miR-328-5p expression to enhance DAB2IP expression. In conclusion, the circRNA-5692–miR-328-5p–DAB2IP regulatory pathway inhibits the progression of HCC. Our findings may provide potential new targets for the diagnosis and therapy of HCC.

Cis-Acting circ-CTNNB1 Promotes β-Catenin Signaling and Cancer Progression via DDX3-Mediated Transactivation of YY1.

Abstract: Circular RNAs (circRNA), a subclass of noncoding RNA characterized by covalently closed continuous loops, play emerging roles in tumorigenesis and aggressiveness. However, the functions and underlying mechanisms of circRNA in regulating Wnt/β-catenin signaling and cancer progression remain elusive. Here, we screen cis-acting circRNA generated by β-catenin (CTNNB1)/transcription factor 7-like 2 genes and identify one intronic circRNA derived from CTNNB1 (circ-CTNNB1) as a novel driver of cancer progression. Circ-CTNNB1 was predominantly expressed in the nucleus, upregulated in cancer tissues and cell lines, and associated with unfavorable outcomes in patients with cancer. Circ-CTNNB1 promoted β-catenin activation, growth, invasion, and metastasis in cancer cells. Circ-CTNNB1 bound DEAD-box polypeptide 3 (DDX3) to facilitate its physical interaction with transcription factor Yin Yang 1 (YY1), resulting in the transactivation of YY1 and transcriptional alteration of downstream genes associated with β-catenin activation and cancer progression. Preclinically, administration of lentivirus-mediated short hairpin RNA targeting circ-CTNNB1 or a cell-penetrating inhibitory peptide blocking the circ-CTNNB1-DDX3 interaction inhibited downstream gene expression, tumorigenesis, and aggressiveness in cancer cells. Taken together, these results demonstrate cis-acting circ-CTNNB1 as a mediator of β-catenin signaling and cancer progression through DDX3-mediated transactivation of YY1. SIGNIFICANCE: These findings reveal the oncogenic functions of a cis-acting circular RNA in β-catenin activation and cancer progression, with potential value as a therapeutic target for human cancers.

Epstein−Barr virus-encoded EBNA2 alters immune checkpoint PD-L1 expression by downregulating miR-34a in B-cell lymphomas

Abstract: Cancer cells subvert host immune surveillance by altering immune checkpoint (IC) proteins. Some Epstein−Barr virus (EBV)-associated tumors have higher Programmed Cell Death Ligand, PD-L1 expression. However, it is not known how EBV alters ICs in the context of its preferred host, the B lymphocyte and in derived lymphomas. Here, we found that latency III-expressing Burkitt lymphoma (BL), diffuse large B-cell lymphomas (DLBCL) or their EBNA2-transfected derivatives express high PD-L1. In a DLBCL model, EBNA2 but not LMP1 is sufficient to induce PD-L1. Latency III-expressing DLBCL biopsies showed high levels of PD-L1. The PD-L1 targeting oncosuppressor microRNA miR-34a was downregulated in EBNA2-transfected lymphoma cells. We identified early B-cell factor 1 (EBF1) as a repressor of miR-34a transcription. Short hairpin RNA (shRNA)-mediated knockdown of EBF1 was sufficient to induce miR-34a transcription, which in turn reduced PD-L1. MiR-34a reconstitution in EBNA2-transfected DLBCL reduced PD-L1 expression and increased its immunogenicity in mixed lymphocyte reactions (MLR) and in three-dimensional biomimetic microfluidic chips. Given the importance of PD-L1 inhibition in immunotherapy and miR-34a dysregulation in cancers, our findings may have important implications for combinatorial immunotherapy, which include IC inhibiting antibodies and miR-34a, for EBV-associated cancers.

Circular RNA FOXP1 promotes tumor progression and Warburg effect in gallbladder cancer by regulating PKLR expression

Abstract: Circular RNAs (circRNAs) have recently been identified as potential functional modulators of the cellular physiology processes. The study aims to uncover the potential clinical value and driving molecular mechanisms of circRNAs in gallbladder cancer (GBC). We performed RNA sequencing from four GBC and paired adjacent normal tissues to analyze the circRNA candidates. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to measure the circFOXP1 expression from 40 patient tissue samples. Short hairpin RNA mediated knockdown or exogenous expression of circFOXP1 combined with in vitro and in vivo assays were performed to prove the functional significance of circFOXP1. Double luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were also performed. By performing RNA sequencing from GBC and paired adjacent normal tissues to analyze the circRNA candidates, we identified that circFOXP1 (hsa_circ_0008234) expression was significantly upregulated in GBC tissues and positively associated with lymph node metastasis, advanced TNM stage and poor prognosis in patients. Short hairpin RNA mediated knockdown or exogenous expression of circFOXP1 combined with in vitro assays demonstrated that circFOXP1 has pleiotropic effects, including promotion of cell proliferation, migration, invasion, and inhibition of cell apoptosis in GBC. In vivo, circFOXP1 promoted tumor growth. Mechanistically, double luciferase reporter, RNA immunoprecipitation (RIP) and biotin-labeled RNA pull-down assays clarified that circFOXP1 interacted with PTBP1 that could bind to the 3’UTR region and coding region (CDS) of enzyme pyruvate kinase, liver and RBC (PKLR) mRNA (UCUU binding bites) to protect PKLR mRNA from decay. Additionally, circFOXP1 acted as the sponge of miR-370 to regulate PKLR, resulting in promoting Warburg effect in GBC progression. These results demonstrated that circFOXP1 serve as a prognostic biomarker and critical regulator in GBC progression and Warburg effect, suggesting a potential target for GBC treatment.

RNA m 6 A Methyltransferase METTL3 Promotes The Growth Of Prostate Cancer By Regulating Hedgehog Pathway

Abstract: Purpose N6-methyladenosine (m6A) is the most abundant internal modification on eukaryotic mRNA and gained increasing attention recently. More and more evidence suggest that m6A methylation plays crucial role in tumor genesis and development. However, its role in prostate cancer remains largely unknown. Methods METTL3 expression status in prostate cancer was analyzed by using TCGA database and Western blotting. m6A content was analyzed by using RNA Methylation Quantification Kit. The role of METTL3 in prostate cancer cells was determined by proliferation, survival, colony formation, and invasion assays. The m6A level of GLI1 RNA was detected by methylated RNA immunoprecipitation (MeRIP) assay. In vivo role of METTL3 was studied on xenograft models. Results We found that m6A methyltransferase METTL3 was overexpressed in prostate cancer cell lines, together with increased m6A content. Functionally, silencing of METTL3 by shRNA in prostate cancer cell lines resulted in decreased m6A content, cell proliferation, survival, colony formation, and invasion. Interestingly, overexpression of wild-type METTL3 abrogated the repression effect of METTL3 depletion on m6A content, cell proliferation, survival, colony formation, and invasion, while the overexpression of m6A catalytic site mutant METTL3 was unable to rescue the inhibitory effect caused by METTL3 depletion. Further mechanism analysis demonstrated that METTL3 silence decreased the m6A modification and expression of GLI1, an important component of hedgehog pathway, which led to cell apoptosis. Moreover, depletion of METTL3 inhibited tumor growth in vivo. Conclusion Our results suggested that the m6A methyltransferase METTL3 promotes the growth and motility of prostate cancer cells by regulating hedgehog pathway.

miR-644-5p carried by bone mesenchymal stem cell-derived exosomes targets regulation of p53 to inhibit ovarian granulosa cell apoptosis

Abstract: This article aims to reveal the therapeutic effects and potential mechanisms of bone mesenchymal stem cell (BMSC)-derived exosomes on premature ovarian failure (POF). Exosomes were collected from BMSCs and were used to treat cisplatin-induced POF mouse models. Pathological changes of ovarian tissue were detected by using HE staining and by Western blot that detected the expression of apoptosis-related proteins. In cisplatin-induced primary granulosa cell injury, exosomes were co-cultured with the granulosa cells. The apoptosis or viability of granulosa cells was analyzed by flow cytometry or MTT, respectively. In Target scan and microT-CDS databases, an intersection of miRNAs targeting to p53 was found. The expressions of miRNAs in BMSC-derived exosomes were detected by qRT-PCR. Besides, miR-664-5p targeted to p53 of cells was verified by dual-luciferase reporter assay. BMSC-derived exosomes improved the follicular morphology of POF mice and inhibited the expression of apoptosis-related protein. By co-culture of exosomes and primary granulosa cells, BMSC-derived exosomes repressed cisplatin-induced granulosa cells apoptosis and increased cells viability, while these effects were abrogated after the exosome-containing RNA was degraded by RNase. By Target scan, microT-CDS and qRT-PCR, miR-664-5p was regarded as the dominated RNA in BMSC-derived exosomes. By dual-luciferase reporter assay, miR-664-5p negatively regulated p53 luciferase activity. After shRNA interfering miR-664-5p of BMSC, BMSC-derived exosomes exerted no protective effect on cisplatin-induced granulosa cell apoptosis. Our results indicated that miR-644-5p carried by BMSC-derived exosomes inhibited the apoptosis of ovarian granulosa cell by targeting p53 of cells, suggesting that miR-644-5p had the potential to treat POF and restore ovarian function.

METTL3 Promotes the Proliferation and Mobility of Gastric Cancer Cells.

Abstract: Methyltransferase-like 3 (METTL3) was originally known to be responsible for N6-methyladenosine (m6A) modification of mRNA. Recent studies have found that METTL3 plays important roles in a variety of tumors by regulating the translation of oncogenes. However, the functional and regulating mechanisms of METTL3 in human gastric cancer have not yet been understood. Here we knocked down METTL3 in human gastric cancer cell lines, AGS and MKN45, by using shRNA transfection. RT-qPCR assay and western blotting verified the effectiveness of RNA interference on mRNA and protein levels, respectively. Then we found that METTL3 knockdown inhibited cell proliferation, migration and invasion in AGS and MKN45 cells. Moreover, METTL3 knockdown decreased Bcl2 and increased Bax and active Caspase-3 in gastric cancer cells, which suggested the apoptotic pathway was activated. Mechanistic investigation suggested that METTL3 led to inactivation of the AKT signaling pathway in human gastric cancer cells, including decreased phosphorylation levels of AKT and expression of down-stream effectors p70S6K and Cyclin D1. In conclusion, our study reveals that down-regulation of METTL3 inhibits the proliferation and mobility of human gastric cancer cells and leads to inactivation of the AKT signaling pathway, suggesting that METTL3 may be a potential target for the treatment of human gastric cancer.

Knockdown of lncRNA NEAT1 inhibits Th17/CD4+ T cell differentiation through reducing the STAT3 protein level

Abstract: CD4+ T cells differentiated into Th17 cells are a main cause for occurrence and development of rheumatoid arthritis (RA). This study aims to define the role of long noncoding RNA nuclear-enriched abundant transcript 1 (lncRNA NEAT1) and its downstream molecule in Th17 cell differentiation. Determination of lncRNA NEAT1 expression in the peripheral blood mononuclear cells (PBMCs) of patients with RA and in Th17 cells induced differentiation in vitro used quantitative real-time polymerase chain reaction. Lentivirus-constructed short hairpin RNA interference for NEAT1 (Lenti-siRNA-NEAT1) was pretransfected into CD4+ T cells before inducing treatment of Th17 cell differentiation. NEAT1 targets STAT3 protein was proved by RNA pull down. Lenti-siRNA-NEAT1 was injected into the joint of the mice arthritis model to verify the function of NEAT1 knockdown. Our results showed that NEAT1 is significantly upregulated in the PBMCs of RA patients, as well as in Th17 cells in vitro induced from CD4+ T cells. The knockdown of NEAT1 restrains CD4+ T cells differentiate into Th17 cells. STAT3 protein, a critical molecule for Th17 cell differentiation, is a downstream molecule for NEAT and its cellular level can be positively targeted and regulated by NEAT via reducing the ubiquitination level. Moreover, the cotreatment of NEAT1 knockdown and STAT3 overexpression promotes Th17 cell differentiation compared with NEAT1 knockdown alone. Knockdown of Th17 by in vivo injection of lenti-siRNA-NEAT1 relieves arthritis degree in II type collagen induced mice arthritis model. These data concluded that NEAT1 is auxo-active molecule for CD4+ T cells differentiating into Th17 cells and knockdown of NEAT1 positively inhibits Th17/CD4+ T cell differentiation through reducing the STAT3 protein level.

Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia

Abstract: The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We find that the small molecule Ro 08-2750 (Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model. Thus, we identify a small molecule that targets MSI's oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.

Long Non-coding RNA MALAT1 Inhibits Neuron Apoptosis and Neuroinflammation While Stimulates Neurite Outgrowth and Its Correlation With MiR-125b Mediates PTGS2, CDK5 and FOXQ1 in Alzheimer's Disease.

Abstract: Background This study aimed to investigate the effect of long noncoding ribonucleic acids (RNAs) metastasis-associated lung adenocarcinoma transcript 1 (lnc-MALAT1) on regulating neuron apoptosis, neurite outgrowth and inflammation, and further explore its molecule mechanism in Alzheimer's disease (AD). Methods Control overexpression, lnc-MALAT1 overexpression, control shRNA, and lnc-MALAT1 shRNA were transfected into NGF-stimulated PC12 cellular AD model and cellular AD model from primary cerebral cortex neurons of rat embryo, which were established by Aβ1-42 insult. Rescue experiments were performed by transferring lnc-MALAT1 overexpression and lnc-MALAT1 overexpression & miR-125b overexpression plasmids. Neuron apoptosis, neurite outgrowth and inflammation were detected by Hoechst-PI/apoptosis marker expressions, and observations were made using microscope and RT-qPCR/Western blot assays. PTGS2, CDK5 and FOXQ1 expressions in rescue experiments were also determined. Results In two AD models, lnc-MALAT1 overexpression inhibited neuron apoptosis, promoted neurite outgrowth, reduced IL-6 and TNF-α levels, and increased IL-10 level compared to control overexpression, while lnc-MALAT1 knockdown promoted neuron apoptosis, repressed neurite outgrowth, elevated IL-6 and TNF-α levels, but reduced IL-10 level compared to control shRNA. Additionally, lnc- MALAT1 reversely regulated miR-125b expression, while miR-125b did not influence the lnc- MALAT1 expression. Subsequently, rescue experiments revealed that miR-125b induced neuron apoptosis, inhibited neurite outgrowth and promoted inflammation, also increased PTGS2 and CDK5 expressions but decreased FOXQ1 expression in lnc-MALAT1 overexpression treated AD models. Conclusion Lnc-MALAT1 might interact with miR-125b to inhibit neuron apoptosis and inflammation while promote neurite outgrowth in AD.

Exercise Reduces Insulin Resistance in Type 2 Diabetes Mellitus via Mediating the lncRNA MALAT1/MicroRNA-382-3p/Resistin Axis.

Abstract: Insulin resistance (IR) is the primary pathological mechanism underlying type 2 diabetes mellitus (T2DM). Here, the study aimed to ascertain whether and how exercise mediates IR in T2DM. An in vivo mouse model of high-fat diet-induced IR and an in vitro high-glucose-induced IR model were constructed. High long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) expression was detected in T2MD and was positively correlated with HOMA-IR and resistin levels. Then, short hairpin RNA targeting MALAT1 (sh-MALAT1) or pcDNA-MALAT1 was delivered into human umbilical vein endothelial cells (HUVECs) to knock down or upregulate its expression, respectively. Silencing of MALAT1 resulted in reduced levels of resistin, Ang II, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), endothelin-1 (ET-1), and p-insulin receptor substrate-1 (p-IRS)/ISR-1, and decreased cell migration, as well as enhanced glucose uptake and levels of nitric oxide (NO) and p-Akt/Akt. In the IR mouse model, exercise was observed to downregulate MALAT1 to reduce resistin, whereby exercise reduced homeostatic model assessment-insulin resistance (HOMA-IR). Besides, exercise also elevated microRNA-382-3p (miR-382-3p) expression in the serum of IR mice. Dual-luciferase reporter and RNA binding protein immunoprecipitation (RIP) assays identified that MALAT1 could bind to miR-382-3p to upregulate resistin. Collectively, the key observations of the study provide evidence that inhibition of MALAT1 elevates miR-382-3p to repress resistin, which consequently underlies the mechanism of exercise protecting against IR, highlighting a direction for T2DM therapy development.

TRAF6 inhibits colorectal cancer metastasis through regulating selective autophagic CTNNB1/β-catenin degradation and is targeted for GSK3B/GSK3β-mediated phosphorylation and degradation

Abstract: Aberrant CTNNB1 signaling is one of the fundamental processes in cancers, especially colorectal cancer (CRC). Here, we reported that TRAF6, an E3 ubiquitin ligase important for inflammatory signaling, inhibited epithelial-mesenchymal transition (EMT) and CRC metastasis through driving a selective autophagic CTNNB1 degradation machinery. Mechanistically, TRAF6 interacted with MAP1LC3B/LC3B through its LC3-interacting region 'YxxL' and catalyzed K63-linked polyubiquitination of LC3B. The K63-linked ubiquitination of LC3B promoted the formation of the LC3B-ATG7 complex and was critical to the subsequent recognition of CTNNB1 by LC3B for the selective autophagic degradation. However, TRAF6 was phosphorylated at Thr266 by GSK3B in most clinical CRC, which triggered K48-linked polyubiquitination and degradation of TRAF6 and thereby attenuated its inhibitory activity towards the autophagy-dependent CTNNB1 signaling. Clinically, decreased expression of TRAF6 was associated with elevated GSK3B protein levels and activity and reduced overall survival in CRC patients. Pharmacological inhibition of GSK3B activity stabilized the TRAF6 protein, promoted CTNNB1 degradation, and effectively suppressed EMT and CRC metastasis. Thus, targeting TRAF6 and its pathway may be meaningful for treating advanced CRC. Abbreviations: AMBRA1: autophagy and beclin 1 regulator 1; AOM: azoxymethane; ATG5: autophagy related 5; ATG7: autophagy related 7; Baf A1: bafilomycin A1; BECN1: beclin 1; CoIP: co-immunoprecipitation; CQ: chloroquine; CRC: colorectal cancer; CTNNB1/β-catenin: catenin beta 1; DSS: dextran sodium sulfate; EMT: epithelial-mesenchymal transition; FBS: fetal bovine serum; GFP: green fluorescent protein; GSK3B/GSK3β: glycogen synthase kinase 3 beta; IgG: Immunoglobulin G; IHC: immunohistochemistry; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; RFP: red fluorescent protein; RT: room temperature; shRNA: short hairpin RNA; siRNA: small interfering RNA; TRAF6: TNF receptor-associated factor 6; WT: wild-type; ZEB1: zinc finger E-box binding homeobox 1.

PD-1 silencing impairs the anti-tumor function of chimeric antigen receptor modified T cells by inhibiting proliferation activity

Abstract: Blocking programmed death-1 (PD-1) is considered to be a promising strategy to improve T cell function, and this is being explored in many ongoing clinical trials. In fact, our knowledge about PD-1 is primarily based on the results of short-term experiments or observations, but how long-lasting PD-1 blockade can affect T cell function remains unclear. We planned to use shRNA-based gene knockdown technology to mimic long-lasting PD-1 blockade. We constructed PD-1 steadily blocked chimeric antigen receptor modified T (CAR-T) cells, and with these cells we can clearly study the effects of PD-1 knockdown on T cell function. The anti-tumor function, proliferation ability and differentiation status of PD-1 silenced CAR-T cells were studied by in vitro and animal experiments. According to short-term in vitro results, it was reconfirmed that the resistance to programmed death-ligand 1 (PD-L1)-mediated immunosuppression could be enhanced by PD-1 blockade. However, better anti-tumor function was not presented by PD-1 blocked CAR-T cells in vitro or in vivo experiments. It was found that PD-1 knockdownmight impair the anti-tumor potential of CAR-T cells because it inhibited T cells’ proliferation activity. In addition, we observed that PD-1 blockade would accelerate T cells’ early differentiation and prevent effector T cells from differentiating into effect memory T cells, and this might be the reason for the limited proliferation of PD-1 silenced CAR-T cells. These results suggest that PD-1 might play an important role in maintaining the proper proliferation and differentiation of T cells, and PD-1 silencing would impair T cells’ anti-tumor function by inhibiting their proliferation activity.

SREBP1 promotes the invasion of colorectal cancer accompanied upregulation of MMP7 expression and NF-κB pathway activation

Abstract: Sterol-regulatory element binding protein 1 (SREBP1), an intracellular cholesterol sensor located in the endoplasmic reticulum, regulates the intracellular cholesterol by the Insig-Srebp-Scap pathway. Over-expression of SREBP1 can cause dyslipidemia. SREBP1 can regulate the metabolic pathway, and then promote the proliferation of tumor cells. However, there is no relevant research of metastasis and invasion in the field of colorectal cancer (CRC). Expression of SREBP1 was manipulated in CRC cell lines with low and high level SREBP1 expression by transfectiong with plasmids containing the SREBP1 gene, or by shRNA. The effect of SREBP1 on cell migration was assayed. The expression of SREBP1, p65 and MMP7 were detected by western blot. Human umbilical vein endothelial cell was used for detection of angiogenesis by adding the culture supernatant from HT29 and SW620. The level of reactive oxygen species (ROS) was detected by Dihydroethidium (DHE) staining. NF-κB inhibitor SN50 was used to test the relationship of SREBP1, NF-κB pathway and MMP7. We found that the expression of SREBP1 in colon adenocarcinoma was significantly higher than that in noncancerous tissues, especially in the invasive tumor front including tumor budding. In vitro, SREBP1 over-expressed in colon cancer cell lines HT29 promoted angiogenesis in endothelial cells, increased ROS levels, phosphorylation of NF-κB-p65 and increases MMP7 expression. The effect of SREBP1 on expression of MMP7 was lost following treatment with the NF-κB inhibitor SN50. Our results suggest that SREBP1 can promote the invasion and metastasis of CRC cells by means of promoting the expression of MMP7 related to phosphorylation of p65.

RNA m6A methyltransferase METTL3 regulates invasiveness of melanoma cells by matrix metallopeptidase 2.

Abstract: The development of immunotherapy has improved the treatment of melanoma; however, resistance and frequent recurrence persist and remain a major problem. N-methyladenosine (mA) is the most abundant epitranscriptomic mark on mRNA and is essential for various physiological processes; however, its role in melanoma is unknown. Utilizing human normal melanocyte and melanoma cell lines, we analyzed the expression of METTL3 by quantitative RT-PCR. We inhibited the METTL3 expression by shRNA and analyzed the effects on melanoma cell proliferation, colony formation ability, and invasion. Finally, we assessed the role of METTL3 by using wild-type and mA catalytic site mutant METTL3. Melanoma cell lines express higher levels of METTL3, as compared with normal melanocytes. Interestingly, silencing of METTL3 gene expression in melanoma cells resulted in decreased mA activity, colony formation and invasiveness, while its overexpression led to increased mA activity, colony formation and invasion. METTL3 overexpression promotes accumulation of MMP2 and N-cadherin in melanoma cells. Strikingly, the overexpression of mA catalytic site mutant METTL3 was unable to produce a similar increase in MMP2 expression, suggesting that mA activity of METTL3 is important for melanoma cell invasiveness. Our results for the first time uncover the role of mA modification in melanoma cell biology. We show that METTL3 is upregulated in human melanoma and plays a role in invasion/migration through MMP2. These findings provide the framework for the development and use of METTL3 inhibitors in melanoma treatment.

Long noncoding RNA UCA1 targets miR-582-5p and contributes to the progression and drug resistance of bladder cancer cells through ATG7-mediated autophagy inhibition

Abstract: Background Rently, the incidence of bladder cancer has been on the rise. Accumulating researches have been conducted to clarify the molecular mechanisms and potential therapeutic targets of bladder cancer. The present study aims to explore the regulatory mechanism of the urothelial carcinoma-associated 1 (UCA1)-miR-582-5p-ATG7 axis in bladder cancer. Methods Quantitative real-time polymerase chain reaction was used to detect mRNA level. Relative protein expression was detected by western blot. wound healing assay and transwell were used to determine migration and invasion of cells. in addtion, luciferase reporter assay and immunohistochemistry were performed. Results UCA1 expression was upregulated in bladder cancer tissues and cells, while the depletion of UCA1 by shRNA resulted in the suppression of cell proliferation, invasion, migration, and drug resistance. Further studies demonstrated that UCA1 could directly interact with miR-582-5p, and that there was an inverse correlation between miR-582-5p and UCA1. In addition, we found that ATG7 is a target of miR-582-5p and can be downregulated by either miR-582-5p overexpression or UCA1 knockdown. In particular, the autophagy is reduced when UCA1 shRNA is introduced. Moreover, the in vivo experiment further demonstrated the contribution of UCA1 in bladder cancer including tumor growth, invasion, and migration, and UCA1 knockdown can inhibit the aforementioned activities. Conclusion These results provided evidence for a novel UCA1 interaction regulatory network in bladder cancer, that is, UCA1-miR-582-5p-ATG7-autophagy axis. Our study provides a new insight into the treatment of bladder cancer.

Long noncoding RNA NEAT1 suppresses sorafenib sensitivity of hepatocellular carcinoma cells via regulating miR-335-c-Met.

Abstract: OBJECTIVES To investigate the role of long noncoding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) in regulating sorafenib (Sora) sensitivity of hepatocellular carcinoma (HCC) cells and possible signaling pathways. METHODS HCC cell lines and tumor tissue were quantified for NEAT1 expression by quantitative polymerase chain reaction (qPCR). Following shRNA (short hairpin RNA) knockdown of NEAT1, cell viability, apoptosis, and related protein expression were measured after drug treatment. The downstream target of NEAT1, including miR-335 and c-Met was studied using a combination of luciferase binding assay, gene knockdown/overexpression, western blot analysis, and cell viability/apoptosis assay. Cancer cells with NEAT1 knockdown were transplanted onto nude mice for in vivo tumorigenesis assay. RESULTS Silencing of NEAT1 in HCC cells facilitated Sora sensitivity by enhancing drug-induced apoptosis, and led to smaller tumor size on nude mice. Mechanistic study suggested that miR-335 was negatively regulated by NEAT1, and miR-335 further suppressed c-Met-Akt pathway, whose activation caused drug resistance of HCC cells. The knockdown of miR-335, or overexpression of c-Met, all remarkably abolished the proapoptotic effect of NEAT1 knockdown in HCC cells. CONCLUSION lncRNA NEAT1 mediates Sora resistance of HCC cells by suppressing miR-335 expression, and disinhibition on c-Met-Akt signaling pathway. Our results provide potency of NEAT1 as the biomarker for drug resistant HCC and possible treating targets.

Circular RNA circRHOT1 is upregulated and promotes cell proliferation and invasion in pancreatic cancer.

Abstract: Aim We aimed to identify the roles of circRHOT1 in pancreatic cancer. Materials & methods The circRHOT1 was acquired from our previous study followed by quantitative real-time PCR and fluorescence in situ hybridization validation in pancreatic cancer. We used siRNA and shRNA to explore the function of circRHOT1 in pancreatic cancer cells. Bioinformatic analyses were applied to study the potential mechanism of circRHOT1. Results The circRHOT1 was upregulated in pancreatic cancer and predominantly located in the cytoplasm. Reducing the circRHOT1 expression may inhibit the pancreatic cancer cell proliferation, invasion and migration. The circRHOT1 may play a role in pancreatic cancer through binding miR-26b, miR-125a, miR-330 and miR-382 to regulate multiple tumor-associated pathways. Conclusion This study demonstrated that circRHOT1 may serve as an oncogenic circRNA that promotes tumor progression.

LncRNA MALAT1 Promotes Lung Cancer Proliferation and Gefitinib Resistance by Acting as a miR-200a Sponge

Abstract: Introduction Lung cancer is a major public health problem, as the second causes of cancer related death worldwide, with relatively low survival rates, and accessible drug resistance. Long non-coding RNAs (LncRNAs) have been identified as activator in lung cancer with elusive mechanisms. We aimed to detect the regulation of LncRNA MALAT1 in the proliferation and gefitinib resistance in lung cancer cells. Methods MALAT1 in A549 and HCC 1299 human lung adenocarcinoma cell lines was silenced by shRNA or overexpressed using plasmid, and the cell viability and cell proliferation were evaluated by MTT assay and soft agar colony formation assay. RNA levels were detected by RT-PCR, and the protein expression was measured by western blot. The binding between MALAT1 and miR-200a was validated by luciferase reporter assays using pSi-Chech 2 vectors. Results The cell viability and proliferation of A549 cells transfected with MALAT1 shRNA were significantly lower than the control. The MALAT1 expression in gefitinib resistant A549 cells was upregulated. miR-200a significantly inhibited the fluorescence of pSi-Check 2 vector with MALAT1 gene, suggesting the direct binding between MALAT1 and miR-200a. In addition, LncRNA MALAT1 promotes ZEB1 expression in A549 cells. Conclusion Our study showed that MALAT1 promoted the proliferation and gefitinib resistance of lung cancer cells by sponging miR-200a, which regulates expression of ZEB1 in the A549 cells. This MALAT1/miR-200a axis could serve as new therapeutic target for lung cancer treatment.

Antitumor and Anti-Invasive Effect of Apigenin on Human Breast Carcinoma through Suppression of IL-6 Expression.

Abstract: Interleukin (IL)-6 plays a crucial role in the progression, invasion, and metastasis of breast cancer. Triple-negative breast cancer (TNBC) cell line MDA-MB-231 is known for its aggressive metastasis. Epithelial to mesenchymal transition (EMT) is a critical process in cancer metastasis. The positive correlation between IL-6 and EMT in tumor microenvironment is reported. We found significantly upregulated IL-6 expression in MDA-MB-231 cells. A blockade of IL-6 expression decreased levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT3), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), phosphorylated protein kinase B (pAkt), and cell cycle-related molecules, including cyclin-dependent kinases (CDKs) and cyclins in MDA-MB-231 cells. A short-hairpin RNA (shRNA)-mediated blockade of IL-6 expression inhibited migration and N-cadherin expression and induced E-cadherin expression in MDA-MB-231 cells. Growth rate was slower for the tumors derived from IL-6 shRNA-treated MDA-MB-231 cells than for those derived from control shRNA-treated MDA-MB-231 cells. The expression of pSTAT3, phosphorylated extracellular signal-regulated kinase (pERK), PI3K, pAkt, snail, vimentin, and N-cadherin was significantly lower in tumors from IL-6 shRNA-treated MDA-MB cells. In addition, apigenin treatment significantly inhibited the growth of MDA-MB-231-derived xenograft tumors along with the protein expressions of pSTAT3, pERK, IL-6, PI3K, pAkt, and N-cadherin. Our results demonstrate that the anti-invasive effect of apigenin in MDA-MB-231-derived xenograft tumors is mediated by the inhibition of IL-6-linked downstream signaling pathway.

Silence of long intergenic noncoding RNA HOTAIR ameliorates oxidative stress and inflammation response in ox-LDL-treated human macrophages by upregulating miR-330-5p.

Abstract: Evidence of the involvement of long noncoding RNAs (lncRNAs) in atherosclerosis is growing but still not well characterized. Here, we concentrated on the biological roles of lncRNA HOX transcription antisense RNA (HOTAIR) in atherosclerosis. In our study, we found that oxidized low-density lipoprotein (ox-LDL) induced human macrophages THP-1 cells apoptosis dose dependently and time dependently. Meanwhile, HOTAIR was significantly increased in THP-1 cells treated with ox-LDL. Then, HOTAIR was modulated by infection of LV-short hairpin RNA (shRNA) and LV-HOTAIR into THP-1 cells. As displayed, CD36, Oil Red O staining levels, total cholesterol, triglyceride levels and dil-ox-LDL uptake rate were greatly repressed by the silence of HOTAIR while triggered by overexpression of HOTAIR. Moreover, knockdown of HOTAIR suppressed reactive oxygen species, malondialdehyde levels, increased superoxide dismutase activity and cell apoptosis were also restrained. Reversely, overexpression of HOTAIR exhibited an opposite phenomenon. In addition, interleukin 6 (IL-6), IL-1β, cyclo-oxygenase 2, and tumor necrosis factor α protein levels were significantly depressed by LV-shRNA) of HOTAIR while increased by upregulation of HOTAIR in THP-1 cells. By carrying out bioinformatics analysis, miR-330-5p was predicted as a target of HOTAIR and the correlation between them was validated in our current study. MiR-330-5p was greatly decreased in THP-1 cells incubated with ox-LDL and overexpression of miR-330-5p was able to inhibit oxidative stress and inflammation process. Taken together, it was implied that HOTAIR contributed to atherosclerosis development by downregulating miR-330-5p in human macrophages.

p62-mediated Selective Autophagy Endows Virus-Transformed Cells With Insusceptibility to DNA Damage Under Oxidative Stress

Abstract: DNA damage response (DDR) and selective autophagy both can be activated by reactive oxygen/nitrogen species (ROS/RNS), and both are of paramount importance in cancer development. The selective autophagy receptor and ubiquitin (Ub) sensor p62 plays a key role in their crosstalk. ROS production has been well documented in latent infection of oncogenic viruses including Epstein-Barr Virus (EBV). However, p62-mediated selective autophagy and its interplay with DDR have not been investigated in these settings. In this study, we provide evidence that considerable levels of p62-mediated selective autophagy are spontaneously induced, and correlate with ROS-Keap1-NRF2 pathway activity, in virus-transformed cells. Inhibition of autophagy results in p62 accumulation in the nucleus, and promotes ROS-induced DNA damage and cell death, as well as downregulates the DNA repair proteins CHK1 and RAD51. In contrast, MG132-mediated proteasome inhibition, which induces rigorous autophagy, promotes p62 degradation but accumulation of the DNA repair proteins CHK1 and RAD51. However, pretreatment with an autophagy inhibitor offsets the effects of MG132 on CHK1 and RAD51 levels. These findings imply that p62 accumulation in the nucleus in response to autophagy inhibition promotes proteasome-mediated CHK1 and RAD51 protein instability. This claim is further supported by the findings that transient expression of a p62 mutant, which is constitutively localized in the nucleus, in B cell lines with low endogenous p62 levels recaptures the effects of autophagy inhibition on CHK1 and RAD51 protein stability. These results indicate that proteasomal degradation of RAD51 and CHK1 is dependent on p62 accumulation in the nucleus. However, small hairpin RNA (shRNA)-mediated p62 depletion in EBV-transformed lymphoblastic cell lines (LCLs) had no apparent effects on the protein levels of CHK1 and RAD51, likely due to the constitutive localization of p62 in the cytoplasm and incomplete knockdown is insufficient to manifest its nuclear effects on these proteins. Rather, shRNA-mediated p62 depletion in EBV-transformed LCLs results in significant increases of endogenous RNF168-γH2AX damage foci and chromatin ubiquitination, indicative of activation of RNF168-mediated DNA repair mechanisms. Our results have unveiled a pivotal role for p62-mediated selective autophagy that governs DDR in the setting of oncogenic virus latent infection, and provide a novel insight into virus-mediated oncogenesis.

AIM2 promotes non-small-cell lung cancer cell growth through inflammasome-dependent pathway

Abstract: The human absent in melanoma 2 (AIM2) is considered as a DNA recognizer. AIM2 has been described as a tumor suppressor gene in the early years. But recent studies suggested that it functions as an oncogene in several cancers. However, its roles in non-small-cell lung cancer (NSCLC) remain unclear. Here we reported that AIM2 highly expressed in NSCLC cells and exhibited a tumor-promoting property both in vitro and in vivo. Besides, AIM2 short hairpin RNA (shRNA)-mediated suppression of cell proliferation was triggered by the accumulation of cells at the G2/M phase. Knockdown of AIM2 reduced the inflammasome formation, while overexpression of AIM2 or stimulation by poly(dA:dT) induced the inflammasome formation. Interestingly, blockade of the inflammasome by caspase-1 inhibitor VX-765 or ASC small interfering RNA (siRNA) abolished the effects brought by AIM2 shRNA and AIM2 plasmid. In summary, our results revealed that AIM2 functioned as an oncogene in NSCLC in an inflammasome-dependent way.