CircTMTC1 contributes to nasopharyngeal carcinoma progression through targeting miR-495-MET-eIF4G1 translational regulation axis

Nasopharyngeal carcinoma (NPC) is the most common primary malignancy arising from the epithelial cells of nasopharynx. CircTMTC1 is upregulated in NPC patients, but its role and molecular mechanism in NPC are unknown. Normal nasopharyngeal epithelium and tumor tissues were collected. The expression of circTMTC1, miR-495, MET/eIF4G1 pathway-related molecules were examined. Colony formation and transwell assays were used to assess cell proliferation, migration, and invasion. Cell apoptosis was analyzed by annexin V and propidium iodide (PI) staining. Gene interaction was examined by RNA immunoprecipitation (RIP) and luciferase activity assays. Subcutaneous and intravenous xenograft mouse models were established to analyze NPC growth and metastasis in vivo. CircTMTC1 was highly expressed and miR-495 was downregulated in NPC, which were associated with poor prognosis of NPC. Both circTMTC1 knockdown and miR-495 overexpression inhibited NPC cell proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT) and promoted cell apoptosis. CircTMTC1 directly targeted miR-495 to promote the expression of its downstream target gene MET. miR-495 knockdown enhanced the expression of c-Myc, Cyclin D1, and survivin and accelerated NPC cell proliferation, migration, invasion, and EMT through targeting MET and activating the MET-eIF4G1 axis. CircTMTC1 silence inhibited NPC growth and lung metastasis by targeting the miR-495-MET-eIF4G1 translational regulation axis in vivo. CircTMTC1 accelerates NPC progression through targeting miR-495 and consequently activating the MET-eIF4G1 translational regulation axis, suggesting potential therapeutic targets for NPC treatment.Subject terms: Cancer, Diseases     

LncRNA SNHG12 promotes proliferation and epithelial mesenchymal transition in hepatocellular carcinoma through targeting HEG1 via miR-516a-5p

Hepatocellular carcinoma (HCC) is the most common cancer and its prognosis is poor due to metastasis and recurrence. EMT is associated with metastasis. A deep understanding of regulatory mechanism of EMT is critical. LncRNA is involved in regulation of various biological processes including EMT. This study aimed to investigate the regulatory signal axis among lncRNA SNHG12, miR-516a-5p and the target gene HEG1 during EMT. Cell cycle and apoptosis were analyzed by flow cytometry. Tumorigenesis was analyzed by clone formation assay. Wound healing assay and transwell assay was performed to detect migration and invasion, respectively. Interaction among SNHG12, miR-516a-5p and HEG1 were analyzed by dual luciferase assay and RIP assay. We also detected expression of RNA and protein by QPCR and western blotting. Finally, tumor growth was analyzed by tumorigenesis assay in vivo. Ki-67 and HEG1 level in tumor tissues was analyzed by IHC. SNHG12 and HEG1 were upregulated, miR-516a-5p was downregulated in HCC cell lines. SNHG12 could interact with and inhibit miR-516a-5p. MiR-516a-5p could interact with HEG1 and inhibit HEG1 expression. Knock down SNHG12 inhibited proliferation, migration, invasion, EMT and promoted apoptosis of HCC cells. Such effects were antagonized by inhibiting miR-516a-5p. SNHG12 overexpression lead to opposite results. Similar results were observed in mice. SNHG12 could promote EMT in HCC through targeting and inhibiting miR-516a-5p, which eventually upregulated HEG1 expression, in both cell and mice.     

MiR-192-5p inhibits proliferation,migration, and invasion in papillary thyroid carcinoma cells by regulation of SH3RF3

Background: The decreased level of miR-192-5p has been reported in several kinds of cancers, including bladder, colon, ovarian, and non-small cell lung cancer. However, the expression and function of miR-192-5p in papillary thyroid carcinoma/cancer (PTC) remains unknown.Objective: The present study aimed to explore the function and underlying mechanism of miR-192-5p in PTC development.Methods: PTC tissues and relative normal controls from PTC patients were collected. qRT-PCR analysis was performed to measure miR-192-5p and SH3RF3 mRNA level in PTC tissues and cell lines. CCK-8 method and FCM assay were used to test cell proliferation and apoptosis in TPC-1 cells, respectively. The abilities of cell migration and invasion were detected by wound healing and transwell assays, respectively. The protein expression was evaluated by Western blot. The interaction between miR-192-5p and Src homology 3 (SH3) domain containing ring finger 3 (SH3RF3) were confirmed by dual-luciferase reporter assay.Results: MiR-192-5p level was obviously decreased in PTC tissues and cell lines. Overexpression of miR-192-5p suppressed proliferation, migration, invasion, and EMT process, while induced apoptosis in TPC-1 cells. In addition, miR-192-5p negatively modulated SH3RF3 expression by binding to its 3′-untranslated region (3′UTR). Silencing SH3RF3 inhibited the migration, invasion, and EMT of TPC-1 cells. In the meantime, matrine, an alkaloid extracted from herb, exerted its anti-cancer effects in PTC cells dependent on increase in miR-192-5p expression and decrease in SH3RF3 expression.Conclusion: We firstly declared that miR-192-5p played a tumor suppressive role in PTC via targeting SH3RF3. Moreover, matrine exerted its anti-cancer effects in PTC via regulating miR-192-5p/SH3RF3 pathway.     

Circ_0000052/miR-382-3p axis induces PD-L1 expression and regulates cell proliferation and immune evasion in head and neck squamous cell carcinoma

A better understanding of the mechanisms underlying PD-L1 aberrant expression in head and neck squamous cell carcinoma (HNSCC) will help reveal predictive biomarkers and overcome resistance to treatment. In this study, the prognostic significance of PD-L1 in forty-five HNSCC archival samples was determined by qRT-PCR. The biological function associated with malignant behaviour was assessed by PD-L1 depletion, miR-382-3p re-expression and regulation of circ_0000052. The interactions of PD-L1-miRNA and miRNA-circRNA were determined by qRT-PCR, Western blot analysis, dual-luciferase reporter assays and RNA immunoprecipitation assays. PD-L1 was highly expressed in patient samples and cancer cell lines. Higher levels of PD-L1 were associated with patient recurrences and play a pivotal role in regulating cell proliferation, migration, invasion, clonogenicity and apoptosis. In addition to demonstrating that the IFN-γ/JAK2/STAT1 signalling pathway can induce PD-L1 overexpression in HNSCC, a novel mechanism by which upregulated circ_0000052 mediates PD-L1 overexpression was also demonstrated. To do this, circ_0000052 competitively binds to miR-382-3p and alleviates its repression of PD-L1. This leads to overexpression of PD-L1, causing the aggressiveness of the cells. Our data demonstrate that circ_0000052 is oncogenic, and the circ_0000052/miR-382-3p/PD-L1 axis is critical in HNSCC progression. The manipulation of circRNAs/miRNAs in combination with anti-PD-L1 therapy may improve personalized disease management.     

Re-expression of miR-21 contributes to migration and invasion by inducing epithelial-mesenchymal transition consistent with cancer stem cell characteristics in MCF-7 cells

MiR-21 is known to play an important role in the development and progression, including migration and invasion, of many malignancies including breast cancer. Accumulating evidence suggest that the induction of epithelial-mesenchymal transition (EMT) phenotype and acquisition of cancer stem cell (CSC) characteristics are highly interrelated, and contribute to tumorigenesis, tumor progression, metastasis, and relapse. The molecular mechanisms underlying EMT and CSC characteristics during miR-21 contributes to cell migration and invasion of breast cancer are poorly understood. Therefore, we established miR-21 re-expressing breast cancer MCF-7 (MCF-7/miR-21) cells, which showed increasing cell growth, migration and invasion, self-renewal and clonogenicity. Our data showed that re-expression of miR-21 induced the acquisition of EMT phenotype by activation of mesenchymal cell markers (N-cadherin, Vimentin, α-SMA) and inhibition of epithelial cell marker (E-cadherin) in MCF-7/miR-21 cells, which consistent with increased cell subpopulation expressing CSC surface markers (ALDH1(+) and CD44(+)/CD24(-/low)) and the capacity of sphereforming (mammospheres). Our results demonstrated that re-expression of miR-21 is responsible for migration and invasion by activating the EMT process and enhancing the characteristics of CSCs in MCF-7 cells.     

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.     

miR-377-5p inhibits lung cancer cell proliferation,invasion, and cell cycle progression by targeting AKT1 signaling

Lung carcinoma is the most common type of malignant tumors globally, and its molecular mechanisms remained unclear. With the aim to investigate the effects of microRNA (miR)-377-5p on the cell development, invasion, metastasis, and cycle of lung carcinoma, this study was performed. We evaluated miR-377-5p expression levels in lung cancer tissues and cell models. Cell viability, proliferation, migration, invasion abilities, and cell cycle distribution were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, crystal violet, transwell, and flow cytometry assay. Furthermore, expression levels of protein kinase B α subunit (AKT1) and proteins related to cell cycle and epithelial-mesenchymal transition (EMT) were assessed using Western blot analysis and quantitative real-time polymerase chain reaction. These results suggested that miR-377-5p was downregulated in vivo and in cell models, and miR-377-5p overexpression inhibited cell viability, proliferation, migration, invasion, and induced cell-cycle arrest. In addition, as a target of miR-377-5p, AKT1 alleviated the decreases of cell viability, proliferation, migration, invasion, the S-phase cells, the expression of cyclin D1, fibronectin, and vimentin, as well as the increases of the G0/G1-phase cells, the expression of Foxo1, p27 ^kip1, p21 ^Cip1 and E-cadherin when miR-377-5p overexpressed. In conclusion, miR-377-5p inhibited cell development and regulated cell cycle distribution and EMT by targeting AKT1, which provided a theoretical basis for further study of lung carcinoma therapeutics.     

Salvianolic acid B suppresses EMT and apoptosis to lessen drug resistance through AKT/mTOR in gastric cancer cells

The drug resistance of tumor cells greatly reduces the efficacy of chemotherapy drugs in gastric cancer. Salvianolic acid B (Sal-B) is considered as a chemopreventive agent which suppresses oxidative stress and apoptosis. Therefore, the study aims to clarify the mechanism of Sal-B in drug-resistant gastric cancer cells. CCK8 assay analyzed cell viabilities after GES1, AGS and AGS/DDP cells were respectively treated by Sal-B of different concentration or after AGS/DDP cells were disposed by cisplatin (DDP) in different concentration. The colony formation, ROS generation, apoptosis, migration, invasion and EMT marker proteins were respectively analyzed through formation assay, ROS kits, TUNNEL staining, Wound healing, Transwell assays and Western blot. The results demonstrated that Sal-B acted alone or in synergy with DDP to reduce cell viabilities, initiate ROS generation, promote cell apoptosis, as well as decrease migration, invasion and EMT in AGS and AGS/DDP cells. AKT activator and mTOR activator significantly reversed the above effects of Sal-B. Collectively, Sal-B regulated proliferation, EMT and apoptosis to reduce the resistance to DDP via AKT/mTOR pathway in DDP-resistant gastric cancer cells. Sal-B could be a potential anti-drug resistance agent to chemotherapy in gastric cancer.     

EGFR activation induced Snail-dependent EMT and myc-dependent PD-L1 in human salivary adenoid cystic carcinoma cells

Epithelial-to-mesenchymal transition (EMT) confers cancer cells the ability of invasion and metastasis. However, how does EMT contribute to evasion of immune surveillance is unclear, especially in salivary adenoid cystic carcinoma (SACC). In this study, we investigated the molecular link between EGF-induced EMT and the immune checkpoint ligand programmed death-ligand 1 (PD-L1) by immunoprecipitation (IP) and Westernblot analysis. Cell migration and invasion activity was assayed by transwell assay. Immunohistochemical (IHC) staining analysis was performed for measurement of EMT markers and PD-L1 expression levels in tumor tissues. We found that EGF-induced EGFR activation stabilized Snail expression and induced EMT in SACC. Interestingly, EGFR activation induced simultaneously both EMT and PD-L1 in SACC. Importantly, knockdown of Snail greatly suppressed EGF-induced EMT, but not EGF-induced PD-L1 expression; whereas knockdown of c-Myc strongly repressed PD-L1 expression, but not snail expression and EMT. The molecular link is strongly supported by robust correlations between the EMT markers and PD-L1 expression in human cancer samples.These results suggest that EGFR activated EMT and PD-L1 via two distinct mechanisms. EGFR activation induced EMT and PD-L1 expression in SACC. Snail is required for EGF-induced EMT, but not PD-L1 expression; whereas c-Myc is required for EGFR-mediated PD-L1 upregulation but not EMT. Thus, targeting activated EGFR may inhibit both EMT and PD-L1, which may potentiate the therapeutic effect of PD-L1-based immunotherapy, especially in the malignant subgroups of SACC patients with activated EGFR.     

miR-219-5p targets TBXT and inhibits breast cancer cell EMT and cell migration and invasion

miR-219-5p has been reported to act as either a tumor suppressor or a tumor promoter in different cancers by targeting different genes. In the present study, we demonstrated that miR-219-5p negatively regulated the expression of TBXT, a known epithelial–mesenchymal transition (EMT) inducer, by directly binding to TBXT 3′-untranslated region. As a result of its inhibition on TBXT expression, miR-219-5p suppressed EMT and cell migration and invasion in breast cancer cells. The re-introduction of TBXT in miR-219-5p overexpressing cells decreased the inhibitory effects of miR-219 on EMT and cell migration and invasion. Moreover, miR-219-5p decreased breast cancer stem cell (CSC) marker genes expression and reduced the mammosphere forming capability of cells. Overall, our study highlighted that TBXT is a novel target of miR-219-5p. By suppressing TBXT, miR-219-5p plays an important role in EMT and cell migration and invasion of breast cancer cells.     

MiR-126 acts as a tumor suppressor in pancreatic cancer cells via the regulation of ADAM9

The epithelial-mesenchymal transition (EMT) is a critical step for pancreatic cancer cells as an entry of metastatic disease. Wide variety of cytokines and signaling pathways are involved in this complex process while the entire picture is still cryptic. Recently, miRNA was found to regulate cellular function including EMT by targeting multiple mRNAs. We conducted comprehensive analysis of miRNA expression profiles in invasive ductal adenocarcinoma (IDA), intraductal papillary mucinous adenoma, intraductal papillary mucinous carcinoma, and human pancreatic cancer cell line to elucidate essential miRNAs which regulate invasive growth of pancreatic cancer cells. Along with higher expression of miR-21 which has been shown to be highly expressed in IDA, reduced expression of miR-126 in IDA and pancreatic cancer cell line was detected. The miR-126 was found to target ADAM9 (disintegrin and metalloproteinase domain-containing protein 9) which is highly expressed in pancreatic cancer. The direct interaction between miR-126 and ADAM9 mRNA was confirmed by 3' untranslated region assay. Reexpression of miR-126 and siRNA-based knockdown of ADAM9 in pancreatic cancer cells resulted in reduced cellular migration, invasion, and induction of epithelial marker E-cadherin. We showed for the first time that the miR-126/ADAM9 axis plays essential role in the inhibition of invasive growth of pancreatic cancer cells.     

SNF5, a core subunit of SWI/SNF complex,regulates melanoma cancer cell growth,metastasis, and immune escape in response to matrix stiffness

Increased stiffness of the extracellular matrix is an important hallmark of melanoma development and progression, but its regulatory role and related mechanisms remain unclear. We adapted polydimethylsiloxane (PDMS)-micropillar-based matrix platform and investigated the effect of matrix stiffness on the proliferation, epithelial-mesenchymal transition (EMT), and immune escape of melanoma cells. We observed a stiff matrix enhanced cell proliferation, EMT, and immune escape of A375 cells. Furthermore, the expression of SNF5 on the stiffer matrix was higher than that on the softer matrix. Next, we investigated whether SNF5 is an important transducer in response to matrix stiffness. Our results revealed that knockdown of SNF5 significantly decreased stiff matrix-induced activation of cell proliferation, EMT and immune escape. Meanwhile, the overexpression of SNF5 showed its ability to increase cell proliferation, invasion and immune escape by activating the STAT-3 pathway in vitro. Furthermore, SNF5 deficiency elevated the level of tumor-infiltrating CD8⁺T cells and decreased the number of PD-L1 positive cells in vivo. Together, our findings suggested that stiffer substrate enhanced melanoma development by upregulating SNF5 expression, and SNF5 is a key mediator of stiffer matrix-induced immune evasion of melanoma cancer cells.     

MEG3 interacted with miR-494 to repress bladder cancer progression through targeting PTEN

The long noncoding RNA MEG3 is a significant tumor-suppressive gene in various tumors. But its biological role in bladder cancer remains uninvestigated. Herein, the biological mechanism of MEG3 in bladder cancer pathogenesis was explored. First, the expression of MEG3 in bladder cancer cells was examined, and we found that it was significantly reduced. In addition, in bladder cancer cells, we observed htat miR-494 was increased. Then, MEG3 was overexpressed in UMUC3 and SW780 cells and it could negatively modulate miR-494 expression. Bladder cancer cell proliferation was repressed, cell apoptosis was triggered and meanwhile, the cell cycle was remarkably arrested by the overexpression of MEG3. Moreover, the increase of MEG3 suppressed bladder cancer cell migration and invasion capacity. MEG3 can sponge miR-494 and the binding sites between them were confirmed by carrying out a series of functional assays. Furthermore, PTEN was speculated as a putative target of miR-494. Meanwhile, we found that miR-494 inhibitors induced PTEN. Finally, in vivo assays were conducted to prove that MEG3 can restrain bladder tumor growth by modulating miR-494 and PTEN. In conclusion, it was suggested MEG3 can interact with miR-494 to regulate PTEN in bladder cancer development.