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Microfluidics approaches have gained popularity in the field of directed cell migration, enabling control of the extracellular environment and integration with live-cell microscopy; however, technical hurdles remain. Among the challenges are the stability and predictability of the environment, which are especially critical for the observation of fibroblasts and other slow-moving cells. Such experiments require several hours and are typically plagued by the introduction of bubbles and other disturbances that naturally arise in standard microfluidics protocols. Here, we report on the development of a passive pumping strategy, driven by the high capillary pressure and evaporative capacity of paper, and its application to study fibroblast chemotaxis. The paper pumps—flowvers (flow + clover)—are inexpensive, compact, and scalable, and they allow nearly bubble-free operation, with a predictable volumetric flow rate on the order of μl/min, for several hours. To demonstrate the utility of this approach, we combined the flowver pumping strategy with a Y-junction microfluidic device to generate a chemoattractant gradient landscape that is both stable (6+ h) and predictable (by finite-element modeling calculations). Integrated with fluorescence microscopy, we were able to recapitulate previous, live-cell imaging studies of fibroblast chemotaxis to platelet derived growth factor (PDGF), with an order-of-magnitude gain in throughput. The increased throughput of single-cell analysis allowed us to more precisely define PDGF gradient conditions conducive for chemotaxis; we were also able to interpret how the orientation of signaling through the phosphoinositide 3-kinase pathway affects the cells’ sensing of and response to conducive gradients.

In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously (‘‘cells-in-gels-in-paper’’ or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, ‘‘sections’’ all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures.

Angiogenesis plays an important role in tissue development and repair, and how to regulate angiogenesis effectively is a widely studied problem in the biomedical field. In recent years, the role of autophagy in vascular endothelial cells has attracted extensive attention. Icariin (ICA) is a traditional Chinese medicine that has been proven to have outstanding protective effects on the vascular system and to regulate cellular autophagy effectively. However, at present, it has not been reported whether ICA can affect the angiogenic ability of endothelial cells by affecting autophagy. In this study, we aimed to investigate whether ICA affects the angiogenesis capacity of EA.hy926 human vascular endothelial cells through autophagy and explain the underlying potential mechanisms. First, we determined that ICA at appropriate concentrations has the ability to promote cell migration and angiogenesis using wound healing assays and tube formation assays. Then, at the molecular level, we observed the upregulation of VEGFA, VEGFR2, ANGI, ANGII, and Tie2 mRNA and detected the upregulation of TGFβ1 protein by Western blotting. We also demonstrated that angiogenic concentrations of ICA can effectively activate autophagy. The autophagy inhibitor 3-MA significantly suppressed TGFβ1 expression and tube formation in EA.hy926 cells. Overall, we hope that our studies might help to further understand the effect of ICA on vascular endothelial cells and provide a theoretical basis for future angiogenic applications of ICA

LncRNA SNHG7 shows a strong relationship with malignant behavior of cancer cells and poor clinical outcome in cancer. The resistance of ovarian cancer for Paclitaxel seriously limits the clinical efficacy in chemotherapy for ovarian cancer patients. In this study, we investigated whether lncRNA SNHG7 was involved in Paclitaxel sensitivity of ovarian cancer as well as the underlying mechanism regulating the behavior of ovarian cancer cells with Paclitaxel resistance. The experiment results of wound healing and transwell showed that in paclitaxel-resistant ovarian cancer cells, transfection with siRNA-SNHG7 in ovarian cancer cells reduced cell migration and invasion. And cell cycle was observed by means of Flow cytometry. RNA immunoprecipitation assay was performed to analyze the interaction of lncRNA SNHG7 and EIF4G2. Overexpression of EIF4G2 by transfection with Ov- EIF4G2 plasmids efficiently blocked the changes of migration and invasion, as well as G0/1 arrest caused by lncRNA SNHG7 silencing. Taken together, these results demonstrated that lncRNA SNHG7 could affect the degradation of EIF4G2 to regulate the sensitivity of ovarian cancer to Paclitaxel, inhibit cell viability, migration, and invasion. The interaction of lncRNA SNHG7 and EIF4G2 plays an important role in the migrative and invasive activity and Paclitaxel resistance of ovarian cancer cells.

Colorectal cancer (CRC) is a global public health issue with increasing prevalence. MicroRNA-934 (miR-934) is a kind of non-coding RNA involved in the regulation of diverse cancers. Though previous researches have revealed part of association between miR-934 and CRC, the role of miR-934 in CRC pathogenesis has not been completely explored yet. In this study, we aim to investigate the effect of miR-934 on cell proliferation, migration, invasion and angiogenesis in CRC. Accordingly, miR-934 was found to be over-expressed in SW480 and HCT116 cells, two typical CRC cell lines. Meanwhile, miR-934 knockdown significantly inhibited cell proliferation and induced cell cycle arrest in SW480 and HCT116 cells. It was further validated that miR-934 knockdown displayed an inhibitory effect on cell migration and invasion in SW480 and HCT116 cells. Additionally, miR-934 deficiency markedly decreased VEGF expression in SW480 and HCT116 cells and suppressed capability of CRC cells to promote tube formation in vascular endothelial cells, which suggests the pro-angiogenesis role of miR-934 in vitro. Dual luciferase reporter assay further showed that miR-934 directly bound to B-cell translocation gene 2 (BTG2). BTG2 knockdown reversed the inhibitory effect of miR-934 silencing on cell proliferation, migration, invasion, and angiogenesis in SW480 and HCT116 cells. In summary, this study suggests that miR-934 facilitates CRC progression by targeting BTG2, and further highlights the role of miR-934 in pathogenesis of CRC.

Wound healing is related to proliferation, migration, and angiogenesis of keratinocytes. Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) is an important N6-methyladenosine (m6A) reader, which is involved in multiple processes, including wound healing. However, the function and mechanism of IGF2BP2 in keratinocyte processes are largely uncertain. In the present study, expression levels of IGF2BP2 and heparanase (HPSE) were detected by quantitative reverse transcription polymerase chain reaction and western blotting assays. Cell proliferation was investigated by cell counting kit-8 (CCK-8) analysis. Cell migration was determined through wound healing assay. Angiogenesis was measured by tube formation assay and vascular endothelial growth factor (VEGF) level using enzyme linked immunosorbent assay (ELISA). The interaction between IGF2BP2 and HPSE was analyzed by RNA immunoprecipitation, pull-down and luciferase reporter analyses. The results showed that IGF2BP2 expression was enhanced in wound healing. IGF2BP2 downregulation constrained HaCaT cell proliferation, migration, and angiogenesis. IGF2BP2 knockdown decreased HPSE expression. IGF2BP2 could regulate HPSE stability by binding with 3ʹ untranslated region (UTR) of HPSE. HPSE upregulation attenuated silencing IGF2BP2-mediated suppression of proliferation, migration, and angiogenesis. As a conclusion, IGF2BP2 knockdown repressed proliferation, migration, and angiogenesis of HaCaT cells by decreasing HPSE stability.

We aim to evaluate the efficacies of combination therapy with low-frequency ultrasound-stimulated microbubbles (USMB) and radiofrequency ablation (RFA) on suppressing the proliferation of pancreatic cancer cell and treating Panc02 subcutaneous xenograft mice. The proliferation of HPDE6-C7 and Panc02 cells after the treatment of USMB and RFA alone or combination were evaluated by CCK-8 assay. Scratch test was performed to assess the cell migration capability. Panc02-bearing mice were received 14-day treatment of USMB and RFA alone or combination. Tumor size and survival rate were recorded once two days. The serum levels of immune-related factors and changes of apoptosis- and autophagy-related factors were detected by ELISA and western blotting methods. As a result, CKK-8 assays revealed significant inhibition on Panc02 cell proliferation in combination therapy with USMB and RFA relative to other groups (all p

Long non-coding RNA (lncRNA) five prime to Xist (FTX) exerts important functions in human cancer, while its role in retinoblastoma (RB) remains unclear. This study aimed to investigate the role of FTX in RB. The expression levels of FTX were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was evaluated by cell counting kit-8 (CCK-8), 5‐ethynyl‐2′‐deoxyuridine (EdU) staining and colony formation assays. Cell migration and invasion were detected by Transwell assay. The relationship among FTX, microRNA-320a (miR-320a) and with-no-lysine kinase 1 (WNK1) was also investigated. In the present study, we found that the expression levels of FTX were notably elevated in RB tissues and cancer cell lines. Overexpression of FTX exacerbated the aggressive phenotypes (cell proliferation, migration and invasion) of RB cells. Downregulation of miR-320a obviously attenuated the inhibitory effects of knockdown of FTX in RB malignant phenotypes, and knockdown of WNK1 also reversed the impacts of miR-320a inhibitor on malignant phenotypes. In vivo experiments further confirmed that knockdown of FTX efficiently prevents tumor growth in vivo. Our results revealed that FTX promoted RB progression by targeting the miR-320a/WNK1 axis (graphical abstract), suggesting that FTX might be a novel therapeutic target for RB.

Objective: Angiogenesis is involved in multiple biological processes, including atherosclerosis (AS) and cancer. Dickkopf1 (DKK1) plays many roles in both tumors and AS and has emerged as a potential biomarker of cancer progression and prognosis. Targeting DKK1 is a good choice for oncological treatments. Many anticancer therapies are associated with specific cardiovascular toxicity. However, the effects of DKK1 neutralizing therapy on AS are unclear. We focused on how DKK1 affected angiogenesis in AS and ox-LDL-induced human umbilical vein endothelial cells (HUVECs). Methods: ApoE-/- mice were fed a high-fat diet and then injected with DKK1i or DKK1 lentivirus to study the effects of DKK1. In vitro, promoter assays, protein analysis, database mining, dual-luciferase reporter assay (DLR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and coimmunoprecipitation (co-IP) were used to study the mechanism of DKK1 biogenesis. Cell migration and angiogenesis assays were performed to investigate the function and regulatory mechanisms of DKK1. Results: DKK1 participated in angiogenesis both in the plaques of ApoE-/- mice by knockdown or overexpression of DKK1 and ox-LDL-induced HUVECs. DKK1 induced angiogenesis (increasing migration and capillary formation, inducing expression of VEGFR-2/VEGF-A/MMP) via the CKAP4/PI3K pathway, independent of Wnt/β-catenin. ox-LDL increased the expression and nuclear transfer of Ets-1 and c-jun, and induced the transcriptional activity of DKK1 in HUVECs. Ets-1, along with c-jun and CBP, could bind to the promoter of DKK1 and enhance DKK1 transcription. MiR33a-5p was downregulated in ox-LDL induced HUVECs and aortic artery of high-fat diet ApoE-/- mice. Ets-1 was a direct target of miR33a-5p. MiR33a-5p/Ets-1/ DKK1 axis contributed to angiogenesis. Conclusions: MiR33a-5p/Ets-1/DKK1 signaling participated in ox-LDL-induced angiogenesis of HUVECs via the CKAP4/PI3K pathway. These new findings provide a rationale and notable method for tumor therapy and cardiovascular protection.

BET bromodomain BRD4 and RAC1 oncogenes are considered important therapeutic targets for cancer and play key roles in tumorigenesis, survival and metastasis. However, combined inhibition of BRD4-RAC1 signaling pathways in different molecular subtypes of breast cancer including luminal-A, HER-2 positive and triple-negative breast (TNBC) largely remains unknown. Here, we demonstrated a new co-targeting strategy by combined inhibition of BRD4-RAC1 oncogenic signaling in different molecular subtypes of breast cancer in a context-dependent manner. We show that combined treatment of JQ1 (inhibitor of BRD4) and NSC23766 (inhibitor of RAC1) suppresses cell growth, clonogenic potential, cell migration and mammary stem cells expansion and induces autophagy and cellular senescence in molecular subtypes of breast cancer cells. Mechanistically, JQ1/NSC23766 combined treatment disrupts MYC/G9a axis and subsequently enhances FTH1 to exert antitumor effects. Furthermore, combined treatment targets HDAC1/Ac-H3K9 axis, thus suggesting a role of this combination in histone modification and chromatin modeling. C-MYC depletion and co-treatment with vitamin-C sensitizes different molecular subtypes of breast cancer cells to JQ1/NSC23766 combination and further reduces cell growth, cell migration and mammosphere formation. Importantly, co-targeting RAC1-BRD4 suppresses breast tumor growth in vivo using xenograft mouse model. Clinically, RAC1 and BRD4 expression positively correlates in breast cancer patient's samples and show high expression patterns across different molecular subtypes of breast cancer. Both RAC1 and BRD4 proteins predict poor survival in breast cancer patients. Taken together, our results suggest that combined inhibition of BRD4-RAC1 pathways represents a novel and potential therapeutic approach in different molecular subtypes of breast cancer and highlights the importance of co-targeting RAC1-BRD4 signaling in breast tumorigenesis via disruption of C-MYC/G9a/FTH1 axis and down regulation of HDAC1.

The present study was aimed to evaluate the expression profile of Zinc finger C3H1 domain-containing protein (ZFC3H1) using bioinformatic analysis of public datasets from The Cancer Genome Atlas database (TCGA). The results showed that the expression levels of ZFC3H1 were notably lower than the corresponding non-cancerous tissues in prostate adenocarcinoma (PRAD), and patients in the high ZFC3H1-expression group showed poor survival. We hypothesized that the low expression of ZFC3H1 in tumor tissue might have be an inhibitory effect on the autoimmune system. We predicted the regulatory target and protein interaction partner network of ZFC3H1, and identified a PPI network composed of 26 node genes in PRAD. Furthermore, we found that the expression levels of MPHOSPH6 (encoding M-phase phosphoprotein 6) and MRPS31 (encoding mitochondrial ribosomal protein S31) were lower in PRAD tissues than in non-cancerous tissues, and the survival time of patients with high MPHOSPH6 and MRPS31 expression was poor. To further demonstrate the role of ZC3H1 in PRAD, we knocked-down the ZFC3H1 expression and found that the inhibition of ZFC3H1 significantly inhibited PRAD cell migration and invasion. Furthermore, ZFC3H1 siRNA treatment could reduce cell viability and increase the number of apoptotic cells in PRAD cells. Taken together, ZFC3H1 could represent a new marker for PRAD prognosis and provide a reference for the development of new therapies to treat PRAD.

Aberrant expression of P68 RNA helicase (p68), a prototypical member of the DEAD box family of RNA helicases, contributes to tumor development and progression. P68 tyrosine phosphorylation induced by PDGF signaling facilitates cancer metastasis by promoting EMT. In this report, we show that p68 promotes breast cancer cell EMT and cell migration by upregulation of PDGF receptor β (PDGFR-β). Knockdown of p68 in MDA-MB-231 and BT549 cells significantly decreases PDGFR-β both in mRNA and protein levels. P68 promotes EMT and cell migration in response to PDGF-BB stimulation via upregulation of PDGFR-β, suggesting that p68 enhances PDGF signaling by a positive feedback loop in cancer cells. Furthermore, our study reveals that p68 mediates the effects of PDGFR-β in regulation of androgen receptor (AR) in breast cancer cells. We demonstrate that p68 and PDGFR-β co-regulate AR expression and promote androgen-mediated proliferation in breast cancer cells. Our studies uncover an important pathway of p68-PDGFR-β axis in promoting breast cancer progression.

Ophiopogonin B (OP-B), a kind of saponin compound that exists in Radix Ophiopogonis is frequently adopted for the treatment of lung disease as traditional Chinese medicine. The present work aimed to explore the anti-tumor activity of OP-B on non-small cell lung carcinoma (NSCLC) and its possible mechanism. We found that OP-B-treated cells suppressed the viability and proliferation of cells depending on its concentration, as assayed by MTT and Alamar Blue (IC50 were 14.22 ± 1.94, 12.14 ± 2.01, and 16.11 ± 1.83 μM in A549, NCI-H1299, and NCI-H460 cells, respectively). Then, the suppressive effect of OP-B on the invasion and migration of NSCLC was observed through wound healing and Transwell assays, and the epithelial-mesenchymal transition (EMT) markers was detected by immunofluorescence and western blotting. In addition, a dose-dependent reduction of β-catenin both within cytoplasm and nucleus was observed, and the downstream proteins cyclin D1 and c-Myc of Wnt/β-catenin pathway were also reduced. We further constructed β-catenin-overexpression cell models to reveal the underlying mechanism. The results showed that 10 μM of OP-B notably reduced β-catenin protein levels, as well as cell migration and invasion. In spite of the increasement of β-catenin, activation of Wnt pathway and EMT progression, knockdown of Axin leaded to de-function of OP-B on cell metastasis. Taken together, OP-B reduced NSCLC migration and invasion by strengthening the Axin/β-catenin interaction and reducing β-catenin protein translocation.

Epidermal growth factor receptor (EGFR) signalling and the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) are aberrantly activated in ovarian cancer. However, inhibition of EGFR signalling in ovarian cancer patients resulted in a disappointing clinical benefit. In this study, we found that EGFR could activate IL-6-STAT3 pathway in ovarian cancer cells. However, we also demonstrated that EGFR knockdown could increase STAT3 phosphorylation in HO8910 and OVCAR-3 ovarian cancer cells. Interestingly, we further demonstrated that the non-coding RNA miR-146b could simultaneously block both the EGFR and IL-6-STAT3 pathways. Finally, our data demonstrated that miR-146b overexpression resulted in a greater suppression of cell migration than STAT3 pathway inhibition alone.These results suggest a complex and heterogeneous role of EGFR in ovarian cancer. Combined blockade of EGFR and IL-6-STAT3 pathways by miR-146b might be a strategy for improving the clinical benefit of targeting the EGFR pathway in ovarian cancer patients in the future.

Background: Although we previously revealed that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and important for gemcitabine resistance, the role of DNA-PKcs in the progression and metastasis of PDAC remain unclear. To date, the upstream signaling events stimulating DNA-PKcs overexpression in PDAC are still not well characterized. Methods: Expression of DNA-PKcs was measured by western blot. The levels of miRNA-101 and lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) were detected by real-time PCR. Cell viability was determined by CCK-8. Cell migration and cell invasion were measured by transwell assay. The regulatory relationship between NEAT1 and miR-101 was determined by a luciferase assay. Results: DNA-PKcs expression was significantly elevated in human PDAC tissues and cells. DNA-PKcs overexpression was correlated with TNM stage and lymph node metastasis. Higher expression of DNA-PKcs was closely correlated with patients of worse overall survival (OS). DNA-PKcs knockdown suppresses malignant behaviors of PDAC cells. Further study showed that miRNA-101 level was decreased in PDAC tissues and cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in PDAC cells. Moreover, NEAT1 functions as an oncogene influencing cell proliferation, migration and invasion in part by serving as a competing endogenous RNA (ceRNAs) modulating miR-101 expression, leading to up-regulation of DNA-PKcs. Conclusion: These findings suggest that NEAT1/miR-101-dependent up-regulation of DNA-PKcs promotes the malignant behaviors of PDAC cells. The NEAT1/miR-101/DNA-PKcs axis may serve as a viable prognostic marker and therapeutic target for PDAC.

To study the regulation of miR-148a-3p on CDK6 and its mechanism in the progress of acute myeloid leukemia (AML), differential miRNAs were analyzed by bioinformatics, and the miR-148a-3p levels in AML cell lines were detected. Results showed that miR-148a-3p played a crucial role in AML, and the level was lower in AML cells, especially in J111 and KG-1a cells. In J111 and KG-1a cells, the up-regulation of miR-148a-3p mimics blocked the cell growth by arresting cell cycle at G2/M and enhancing cell apoptosis. Transwell and EMT markers detection indicated that miR-148a-3p reduced the cell migration and invasion. Afterward, through bioinformatics analysis, it showed that the CDK6 is one of the direct target genes of miR-148a-3p. DLR assay confirmed the target regulation. CDK6 overexpression reversed the effects of miR-148a-3p on AML cells. Collectively, miR-148a-3p inhibited the process of AML cells through disturbing the CDK-6 expression, implying that the trageting miR-148a-3p might be regarded as effective therapy of AML., Graphical abstract

miR-17-5p has been proved that play important roles in many kinds of tumors progression. This study aimed at explore the function and mechanism of miR-17-5p in thyroid cancer (TC). RT-qPCR was used to detect miR-17-5p and Early growth response 2 (EGR2) expression in TC tissues and cells. CCK8 and colony formation assay were used to analyze cell proliferation. Cell migration and cell invasion was detected by Wound-healing assay and Transwell assay. Detection of protein expression using Western blot analysis. Dual-Luciferase assay was used to analyze the relationship between miR-17-5p and EGR2. In vivo experiment was performed by establishing Xenograft animal model to observe the function of miR-17-5p. We found that miR-17-5p is significantly increased in thyroid cancer tissues and cells. miR-17-5p inhibition repressed cell proliferation, clonal formation, cell migration, and cell invasion in thyroid carcinoma. Moreover, miR-17-5p inhibition suppressed tumorigenesis in vivo. Dual-Luciferase assay and Western blotting assay further proved that miR-17-5p has a negative regulation to EGR2. EGR2 was decreased in TC tissues and cells. Overexpressed EGR2 inhibited the development of thyroid carcinoma both vivo and in vivo. EGR2 knockdown remarkably decreased the anti-cancer effect of miR-17-5p inhibition. miR-17-5p is a thyroid cancer oncomir by modulation of the EGR2., Graphical abstract

Circular RNA (circRNA) features prominently in the progression of hepatocellular carcinoma (HCC), of which the biological function and potential mechanism of circ_0008274 in HCC are obscure. The present study aims to explore circ_ 0008274’s biological functions and underlying mechanisms in HCC. The expressions of circ_0008274, miR-140-3p and Granulin (GRN) mRNA in HCC tissues and cells were investigated by quantitative real-time polymerase chain reaction. Besides, GRN protein expression was measured by Western blot. Furthermore, chi-square test was used to probe the interrelation between circ_0008274 expression and clinicopathological parameters. In addition, cell counting kit-8 (CCK-8) and EdU assays were applied to detect cell proliferation. Moreover, transwell assay was used to detect cell migration and invasion. What’s more, bioinformatics prediction, dual-luciferase reporter gene assay and RNA Immunoprecipitation experiments were used to corroborate the targeting interrelations among circ_0008274, miR-140-3p and GRN. Herein we reported that circ_0008274 was highly expressed in HCC, and its high expression enhanced the proliferation, migration, and invasion of HCC cells, while depleting circ_0008274 inhibited the malignant biological behaviors of HCC cells. Mechanistically, circ_0008274 upregulates GRN expressions via adsorbing miR-140-3p to expedite the progression of HCC., GRAPHICAL ABSTRACT

Nasopharyngeal carcinoma (NPC) is known for its potential to progress to the lymph nodes and distant metastases at an early stage. As an important regulator in tumorigenesis biological processes, the functions of lncRNA in NPC tumor development remain largely unclear. In this research, the expression of EPB41L4A-AS2 in NPC tissues and cells was analyzed via real-time quantitative polymerase chain reaction (qRT-PCR). CCK8, colony formation, and EDU experiments were used to determine the viability of NPC cells. Transwell and wound healing assays were performed to test NPC cell migration and invasion. RNA pull-down and mass spectrometry analysis were used to identify potential binding proteins. Then, a popliteal lymph node metastasis model was established to test NPC metastasis. EPB41L4A-AS2 is repressed by transforming growth factor-beta, which is downregulated in NPC cells and tissue. It is associated with the presence of distant metastasis and adverse outcomes. The univariate and multivariate survival assays confirmed that EPB41L4A-AS2 expression was an independent predictor of progression-free survival (PFS) in patients with NPC. Biological analyses showed that overexpression of EPB41L4A-AS2 reduced the metastasis and invasion of NPC in vitro and in vivo, but had no significant effect on cell proliferation. Mechanistically, in the nucleus we identified that EPB41L4A-AS2 relies on binding to YBX1 to reduce the stability of Snail mRNA to enhance the expression of E-cadherin and reverse the progression of epithelial-to-mesenchymal transition (EMT). In the cytoplasm, we found that EPB41L4A-AS2 blocked the invasion and migration of NPC cells by promoting LATS2 expression via sponging miR-107. In a whole, the findings of this study help to further understand the metastasis mechanism of NPC and could help in the prevention and treatment of NPC metastasis.

Background: Hepatocellular carcinoma (HCC) is a major leading cause of cancer mortality worldwide. Thyroid hormone responsive (THRSP) gene is primarily known for regulating responses to thyroid hormones, but its expression has been correlated with differential outcomes in some cancers. To date, however, its role in the progression of HCC remains unknown. Methods: The mRNA and protein expression of THRSP was measured in HCC tissues and cell lines via qPCR and western blot assays. Lentiviral transfection was used to establish stable cell lines overexpressing THRSP and shRNA was used to silence THRSP. The effects of THRSP on cell growth were then determined in vivo and in vitro. Cell migration and invasion of HCC cells were investigated using transwell and wound healing assays. Results: In tissue samples from patients, HCC tissues had decreased THRSP expression relative to adjacent healthy tissues. Further, patients with decreased THRSP protein and mRNA expression had worse outcomes. Knockdown of THRSP led to increased cell growth, migration, and invasion of HCC cells, and THRSP overexpression exerted an anti-tumor effect in vivo and in vitro. We found that increased expression of THRSP inhibited hepatocellular carcinogenesis by inhibiting the process of epithelial-to-mesenchymal transition through acting on the ERK/ZEB1 signaling pathway. Conclusion: THRSP may act as a functional tumor suppressor and was frequently reduced in HCC tissue samples. We identified a novel pathway for the THRSP/ERK/ZEB1-regulated suppression of HCC tumorigenesis and invasion. Restoring THRSP expression may represent a promising approach for HCC therapies.

Metabolic reprogramming, especially Warburg effect, is a key event in tumor initiation and progression. ZEB1 plays a vital role in metastasis of various cancers. We previously found that ZEB1 was excessively expressed in hepatocellular carcinoma (HCC) and its high expression was closely correlated with metastasis and recurrence of HCC. We want to know whether glycolytic enzymes are regulated by ZEB1 and contribute to carcinogenesis and metastasis of HCC. Methods: To explore whether ZEB1 could enhance glycolysis in HCC, we knocked down ZEB1 by short hairpin RNA (shRNA) in MHCC-97H and HCC-LM3 cells and performed glucose uptake, lactate production, ECAR and OCR assays. To investigate how ZEB1 enhances glycolysis, the protein levels of glycolytic enzymes were detected in the same cell lines using Western blot. The regulatory effect of ZEB1 on PFKM mRNA level was confirmed by RT-qPCR, luciferase report assay and ChIP assay. In order to assess the role of ZEB1-PFKM axis in cell proliferation, cell counting and CCK-8 assays were performed in MHCC-97H and HCC-LM3 cell lines knocked down for ZEB1 and further re-expressed for either ZEB1 or PFKM or not. To explored whether the ZEB1-PFKM axis also functions in HCC cell migration, invasion and metastasis, the same MHCC-97H and HCC-LM3 cell lines were performed for wound healing assays, transwell assays and colony formation assays, meanwhile, MHCC-97H cell lines were performed for orthotopic liver transplantation assays. Finally, the expression of ZEB1 and PFKM were examined in human liver cancer specimens and non-tumorous liver tissues using immunohistochemical and Western blot. Results: We found that ZEB1 transcriptionally upregulates the expression of the muscle isoform of phosphofructokinase-1 (PFKM), a rate-limiting enzyme in glycolysis. Intriguingly, a non-classic ZEB1-binding sequence in the promoter region of PFKM was identified through which ZEB1 directly activates the transcription of PFKM. Silencing of ZEB1 in MHCC-97H and HCC-LM3 cell leads to impaired PFKM expression, glycolysis, proliferation and invasion, and such impairments are rescued by exogenous expression of PFKM. Importantly, in-situ HCC xenograft assays and studies from TCGA database demonstrate that ZEB1-PFKM axis is crucial for carcinogenesis and metastasis of HCC. Conclusions: Our study reveals a novel mechanism of ZEB1 in promoting HCC by activating the transcription of PFKM, establishing the direct link of ZEB1 to the promotion of glycolysis and Warburg effect and suggesting that inhibition of ZEB1 transcriptional activity toward PFKM may be a potential therapeutic strategy for HCC.

Long noncoding RNAs (lncRNAs) are involved in the progression of various cancers, including hepatocellular carcinoma (HCC). However, the biological functions of lncRNA small ubiquitin-like modifier 1 pseudogene 3 (SUMO1P3) and the underlying mechanisms remain unclear. In this study, we revealed that SUMO1P3 expression was enhanced in HCC tissues and cell lines, positively associating with tumor size and number, poor differentiation, lymphatic and distant metastasis, TNM stage, and poor prognosis in HCC patients. In vitro assays showed that SUMO1P3 depletion reduced HCC cell viability and proliferation by hindering cyclin D1 expression and Akt phosphorylation. SUMO1P3 knockdown induced HCC cell apoptosis, as indicated by increased Bax and cleaved caspase-3 expression and the decreased Bcl-2 level. SUMO1P3 silencing suppressed HCC cell migration and invasion by increasing epithelial marker E-cadherin expression and decreasing mesenchymal marker vimentin expression, as well as reducing matrix metalloproteinase (MMP)-2 and MMP-9 levels. Consistently, SUMO1P3 depletion in HCC cells retarded tumor growth and lung metastasis in vivo. Overall, these results supported the applicability of SUMO1P3 as a useful predictor of HCC prognosis and a potential therapeutic target for HCC patients.

Background: Lung cancer is a malignant tumor in mammary gland epithelium with high morbidity and mortality among women worldwide. Long noncoding RNA GAS5 (GAS5) has been proved to be closely related with tumor progression. However, the influence of GAS5 on lung cancer and the specific mechanism remain unclear. Methods: Cell invasion, cell migration, cell apoptosis and cell cycle were investigated after transfection with pcDNA-GAS5 and sh-GAS5. Sizes of tumors were measured by establishing transplanted tumor model in vivo. E-cadherin and N-cadherin expressions were investigated. Results: Cell invasion and migration were inhibited markedly in GAS5 overexpressed cell line. Cell cycle results indicated that the percentage of S-phase cells was increased, and G2-phase was reduced in the GAS5 overexpression cell line. Tumor size was suppressed obviously after GAS5 overexpression treatment. GAS5 markedly inhibited the expression of E-cadherin and induced the expression of N-cadherin. GAS5 overexpression significantly inhibited lung cancer cell proliferation by increasing the E-cadherin and decreasing N-cadherin. Conclusions: These findings provide novel evidence that GAS5 can be viewed as an anti-lung cancer agent through affecting EMT pathway.

Vegfc/Vegfr3 signaling is critical for lymphangiogenesis, the sprouting of lymphatic vessels. In zebrafish, cells sprouting from the posterior cardinal vein can either form lymphatic precursor cells or contribute to intersegmental vein formation. Both, the Vegfc-dependent differential induction of Prox1a in sprouting cells as well as a Notch-mediated pre-pattern within intersegmental vessels have been associated with the regulation of secondary sprout behavior. However, how exactly a differential lymphatic versus venous sprout cell behavior is achieved is not fully understood. Here, we characterize a zebrafish mutant in the adaptor protein Grb2b, and demonstrate through genetic interaction studies that Grb2b acts within the Vegfr3 pathway. Mutant embryos exhibit phenotypes that are consistent with reduced Vegfr3 signaling outputs prior to the sprouting of endothelial cells from the vein. During secondary sprouting stages, loss of grb2b leads to defective cell behaviors resulting in a loss of parachordal lymphangioblasts, while only partially affecting the number of intersegmental veins. A second GRB2 zebrafish ortholog, grb2a, contributes to the development of lymphatic structures in the meninges and in the head, but not in the trunk. Our results illustrate an essential role of Grb2b in vivo for cell migration to the horizontal myoseptum and for the correct formation of the lymphatic vasculature, while being less critically required in intersegmental vein formation. Thus, there appear to be higher requirements for Grb2b and therefore Vegfr3 downstream signaling levels in lymphatic versus vein precursor-generating sprouts. Supplementary Information The online version contains supplementary material available at 10.1007/s10456-021-09774-w.

Cell migration and invasion are modulated by epithelial-to-mesenchymal transition (EMT) and the reverse MET process. Despite the detection of microRNA-362 (miR-362, both the miR-362-5p and -3p species) in cancers, none of the identified miR-362 targets is a mesenchymal or epithelial factor to link miR-362 with EMT/MET and metastasis. Focusing on the TGF-β/SMAD signaling pathway in this work, luciferase assays and western blot data showed that miR-362 targeted and negatively regulated expression of SMAD4 and E-cadherin, but not SNAI1, which is regulated by SMAD4. However, miR-362 knockdown also down-regulated SMAD4 and SNAI1, but up-regulated E-cadherin expression. Wound-healing and transwell assays further showed that miR-362 knockdown suppressed cell migration and invasion, effects which were reversed by over-expressing SMAD4 or SNAI1, or by knocking down E-cadherin in the miR-362 knockdown cells. In orthotopic mice, miR-362 knockdown inhibited metastasis, and displayed the same SMAD4 and E-cadherin expression profiles in the tumors as in the in vitro studies. A scheme is proposed to integrate miR-362 negative regulation via SMAD4, and to explain miR-362 positive regulation of SMAD4 via miR-362 targeting of known SMAD4 suppressors, BRK and DACH1, which would have resulted in SMAD4 depletion and annulment of subsequent involvement in TGF-β signaling actions. Hence, miR-362 both negatively and positively regulates SMAD4 expression in TGF-β/SMAD signaling pathway to suppress cell motility and invasiveness and metastasis, and may explain the reported clinical association of anti-miR-362 with suppressed metastasis in various cancers. MiR-362 knockdown in miR-362-positive cancer cells may be used as a therapeutic strategy to suppress metastasis.

Reportedly, long non-coding RNAs (lncRNAs) are implicated in hepatocellular carcinoma (HCC) progression, yet little is known concerning the biological functions of TTN antisense RNA 1 (TTN-AS1) in HCC. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) was performed for detecting TTN-AS1, SPOCK1 mRNA, and miR-139-5p expressions in HCC cells and tissues. After TTN-AS1 was overexpressed or knocked down in HCC cells, CCK-8 and 5-Ethynyl-2ʹ-deoxyuridine (EdU) assays were carried out for examining cell multiplication. Transwell assays were conducted for evaluating HCC cell migration and invasion. Dual-luciferase reporter assay was employed for verifying the binding relationships between miR-139-5p and TTN-AS1, and between SPOCK1 3ʹUTR and miR-139-5p. Western blot was employed to measure SPOCK1, E-cadherin, N-cadherin, and Vimentin protein expressions. We demonstrated that, TTN-AS1 and SPOCK1 expression levels were remarkably enhanced in HCC cells and tissues, whereas miR-139-5p expression was observably reduced. Functional experiments suggested that TTN-AS1 knockdown markedly repressed HCC cell multiplication, migration, epithelial-mesenchymal transition (EMT), and invasion. In addition, TTN-AS1 interacted with miR-139-5p and decreased its expression. Moreover, SPOCK1 was a miR-139-5p target, and miR-139-5p inhibitors were able to reverse TTN-AS1 knockdown-induced inhibitory effect on SPOCK1 expression. SPOCK1 overexpression plasmid could counteract TTN-AS1 knockdown-induced inhibiting impact on HCC cell multiplication, migration, invasion, and EMT. In conclusion, TTN-AS1 expression level is remarkably enhanced in HCC, and TTN-AS1 can promote the multiplication, migration, invasion, and EMT of HCC cells via regulating miR-139-5p/SPOCK1 axis.

Liraglutide, a glucagon-like peptide 1 (GLP1) receptor agonist, is known to inhibit the atherosclerosis of apoE mice and suppress the cellular behaviors of VSMCs induced by AngII. This study aimed to explore whether liraglutide can reduce the proliferation, invasion and phenotypic transformation of VSMCs induced by Hcy and the underlying mechanism. Hcy was used to induce the proliferation of VSMCs, and liraglutide was then used to expose the cells for assessing cell proliferation. Afterward, the cell migration and phenotypic switch were evaluated to observe the effects of liraglutide. Meanwhile, the expression of PCSK9 and LDLR was detected. After overexpressing PCSK9, the changes in proliferation, cell migration and phenotypic switch were estimated again. Hcy promoted cell proliferation of VSMCs, whereas liraglutide blocked the proliferation, migration and phenotypic switch of Hcy-induced VSMCs. Furthermore, the expression of PCSK9 was downregulated and LDLR expression was upregulated after liraglutide administration in Hcy-induced VSMCs. After overexpressing PCSK9, the proliferation, migration and phenotypic switch of Hcy-induced VSMCs were enhanced. Liraglutide blocked the proliferation, migration and phenotypic switching of Hcy-induced VSMCs by suppressing PCSK9/LDLR. This finding provided the basis for the future application of liraglutide as an effective drug for therapeutic strategy in targeting AS.

Based on many studies, trichosanthin (TCS) has an antiviral effect that regulates immune response, and targets cancer cells to exert broad-spectrum anti-tumor pharmacological activities. It is speculated that TCS may be a potential natural active drug for preventing as well as treating cervical cancer. But the clearer impact along with underlying TCS mechanism on cervical cancer are still unclear. The purpose of this study is to investigate the function and potential mechanism of TCS in cervical cancer. We measured the viability of cervical cancer cell lines (HeLa & caski cells) using CCK-8 analysis, detected cell proliferation efficiency through Ki-67 staining, analyzed cell apoptosis rate via flow cytometry as well as annexin V-FITC/PI double staining, performed apoptosis-related protein expression through western blotting, evaluated cell migration along with invasion by wound as well as transwell assays, carried out MMP via JC-1 and Rh123 fluorescent probes, as well as detected intracellular ATP and ROS levels by flow cytometry, respectively, to evaluate the effects of TCS. We found that TCS inhibited viability along with proliferation, induced apoptosis, as well as inhibited HeLa & caski cell migration along with invasion in a time- and dose-dependent manner. Additionally, TCS also reduced MMP, and the production of adenosine triphosphate, as well as induced the increase of intracellular reactive oxygen species in cancer cell lines. In accordance with the present studies, TCS inhibits HeLa & caski cell proliferation along with migration but promotes their apoptosis, which may be mediated by regulating oxidative stress., Graphical Abstract

Previous studies have demonstrated that protein tyrosine phosphatase 1B (PTP1B) can promote tumor progression in breast cancer, colon cancer and prostate cancer. Additionally, PTP1B also acts as a tumor suppressor in esophageal cancer and lymphoma. These findings suggest that PTP1B functions as a double-faceted molecule in tumors. However, the role of PTP1B in malignant melanoma (MM) is still unknown. PTP1B expression in normal and melanoma tissues was evaluated by GEO analysis and immunohistochemistry. The effects of PTP1B on cell migration and invasion were evaluated in melanoma cells with up – and downregulated PTP1B expression. In this study, we initially demonstrated that the expression of PTP1B in malignant melanoma tissue is significantly higher than its expression in benign nevus tissue and indicated poor survival of malignant melanoma patients. In vitro studies have demonstrated that inhibition of PTP1B suppresses and overexpression of PTP1B promotes migration and invasion of melanoma cells. Moreover, we found that PTP1B could interact with Src via coimmunoprecipitation and dephosphorylation of the Src at Tyr530 site. Collectively, our study revealed that PTP1B can promote melanoma cell metastasis by interacting with Src and provides a theoretical basis for future applications of PTP1B inhibitors in the treatment of malignant melanoma.

Ginsenoside Rg3, a natural compound, has been reported to function as an anticancer agent for hepatoma carcinoma, while the mechanisms underlying the anticancer effects are not clear. Therefore, the objective of our study was to explore the impact of RG3 on cell migration and invasion by regulating the lncRNA HOX antisense intergenic (HOTAIR) expression involving PI3K/AKT signaling pathway. qRT-PCR was utilized to measure the mRNA expression of HOTAIR. Furthermore, HOTAIR overexpression plasmids were transfected to SMMC-7721 and SK-Hep-1 cells. Additionally, MTT assay was used to evaluate the proliferation of transfected cells. The scratch and transwell assays were used to determine the migration and invasion ability of the cell. The protein levels were determined with Western blot. lncRNA HOTAIR was overexpressed in SMMC-7721 and SK-Hep-1 cells. Ginsenoside-Rg3 reduced the level of lncRNA HOTAIR. Overexpressed lncRNA HOTAIR offset ginsenoside-Rg3 inhibited proliferation, migration and invasion of HCC cells. Furthermore, ginsenoside-Rg3 decreased the expression of p-AKT, p-PI3K, matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9), which was reversed after the treatment of HOTAIR. LncRNA HOTAIR was overexpressed in SMMC-7721 cells. Ginsenoside-Rg3 could reduce the expression of lncRNA HOTAIR, resulting in the inhibited cell proliferation, migration and invasion. Furthermore, ginsenoside-Rg3 inhibited cell proliferation and invasion ability through the PI3k/AKT pathway. Thus, ginsenoside-Rg3 might be a potential and effective treatment for HCC., Graphical Abstract

Gastric cancer is a prevalent yet heterogeneous disease which ranks as the fifth most common cancer in the world. Dietary habit, genetic background, Helicobacter Pylori infections were the risk factors of gastric cancer. MicroRNA miR-425 is highly expressed in gastric cancer, but little attention has been devoted to the mechanism of miR-425 in tumorigenesis. This study aim to investigate the role of miR-425 in gastric cancer. The expression of miR-425 and Dickkopf-related protein-3(DKK-3) were analyzed by qRT-PCR. Gastric cell line BGC-823 and SGC-7901 were transfected miR-425 inhibitors or NC. Then, cell viability was determined by CCK-8, cell apoptosis and cell cycle were assessed by flow cytometer. Cell migration and cell invasion were analyzed by wound healing and trans-well assays. Luciferase reporter assay was conducted to assess the correlation between miR-425 and DKK-3. Downstream regulators, such as p-ASK1 and p-JNK, were analysis by western blot. Compared with normal gastric epithelium cell line, miR-425 was obviously upregulated in gastric cancer cell lines. MiR-425 inhibitor suppressed the cell viability, cell migration and cell invasion. The Luciferase assay data identified that DKK-3 is a target of miR-425. While miR-425 could lower the expression of DKK-3 which mediate tumorigenesis in a certain way., Graphical abstact

Preeclampsia (PE) is a potentially fatal pregnancy complication; however, its pathogenesis remains unclear. Long non-coding RNAs (lncRNAs) are associated with occurrence and progression of PE. Therefore, this study aimed to explore the function of the lncRNA prospero homeobox 1-antisense RNA 1 (PROX1-AS1) and elucidate its underlying molecular mechanism of action. We found that the expression levels of PROX1-AS1 were elevated in both the placental tissues and blood samples of the patients with PE. Moreover, the results of the flow cytometry and transwell assay showed that the knockdown of PROX1-AS1 inhibited the apoptosis and promoted the migration and invasion of HTR-8/SVneo cells. We also assessed the interactions between PROX1-AS1, caspase-9, and microRNA (miR)-211-5p via dual-luciferase reporter and RNA pull-down analyses. The data indicated that PROX1-AS1 acted as a sponge for miR-211-5p to regulate the expression of caspase-9. Moreover, the expression of miR-211-5p was reduced in PE and negatively related to PROX1-AS1, while that of caspase-9 was increased in PE and negatively regulated by miR-211-5p. Furthermore, inhibition of miR-211-5p rescued the facilitation of cell apoptosis, migration and invasion induced by the knockdown of PROX1-AS1. We also found that caspase-9 improved the apoptosis rate, and suppressed the cell migration and invasion induced by the overexpression of miR-211-5p. In conclusion, the knockdown of PROX1-AS1 promoted the cell morbidity of the trophoblast cells by modulating the miR-211-5p/caspase-9 axis, which may alleviate the progression of PE. This novel regulatory network may contribute to the pathogenesis and progression of PE., Graphical Abstract

Long noncoding RNAs (lncRNAs) are known to exert their effects to tumor progression. In this study, the role of the lncRNA GAS5 (growth arrest specific 5) was confirmed in reducing non-small cell lung cancer (NSCLC) cisplatin (DDP) resistance. In NSCLC tissue samples, GAS5 expression decreased significantly. Low GAS5 levels were positively correlated with NSCLC characteristics including TNM, tumor size and lymphatic metastasis. Functionally, GAS5 significantly reduced NSCLC/DDP cell migration, invasion and epithelial-mesenchymal transition (EMT) progression in vitro. In vivo, GAS5 upregulation inhibited remarkably NSCLC/DDP cell tumor growth. Mechanism analysis suggested that GAS5 was a molecular sponge of miR-217, inhibiting the expression of phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP). In conclusion, this study reveals that the GAS5/miR-217/LHPP pathway reduces NSCLC cisplatin resistance and that LHPP may serve as a potential therapeutic target for NSCLC cisplatin resistance.

Extensive desmoplasia in cholangiocarcinoma (CCA) is associated with tumor aggressiveness, indicating a need for further understanding of CCA cell–matrix interaction. This study demonstrated laminin as the most potent attractant for CCA cell migration and the vast elevation of its receptor integrin β4 (ITGB4) in CCA cell lines. Besides, their high expressions in CCA tissues were correlated with lymphatic invasion and the presence of ITGB4 was also associated with short survival time. ITGB4 silencing revealed it as the receptor for laminin-induced HuCCA-1 migration, but KKU-213 utilized 37/67-kDa laminin receptor (LAMR) instead. These findings highlight the role of ITGB4 and LAMR in transducing laminin induction of CCA cell migration and the potential of ITGB4 as diagnostic and prognostic biomarkers for CCA.

Oral squamous cell carcinoma (OSCC) is one of the most common digestive tumors, which has high mortality rate. Long non-coding RNAs (lncRNA) and MicroRNAs (miRNAs) are associated with the cell cycle and differentiation during the occurrence and development of malignant tumors. This research aimed to investigate the effects of lncRNA SNHG20 on the progress of oral squamous cell carcinoma (OSCC) cells. Ninety pairs of tumor tissues and paracancerous tissues were collected from patients with OSCC and the CAL27 and SCC25 OSCC cells were selected for the following experiments. RT-qPCR was used for detecting the expression of SNHG20, miR-19b-3p, and RAB14. Western blotting was used to detect the protein levels of RAB14. MTT assay was employed to assess cell proliferation. Transwell assay was used to determine the cell migration and invasion abilities. Furthermore, luciferase reporter and RNA pull-down assays were used to verify the binding of SNHG20/RAB14 to miR-19b-3p. Then, the function of the SNHG20/miR-19b-3p/RAB14 axis in OSCC was explored. The results indicated that lncRNA SNHG20 was upregulated in the tissues. Furthermore, bioinformatic analysis showed that both SNHG20 and RAB14 could bind to miR-19b-3p. RAB14 was upregulated, and miR-19b-3p was downregulated in the tissues. The knockdown of SNHG20 inhibited cell proliferation, migration, and invasion. Contrarily, the knockdown of miR-19b-3p reversed the effects of si-SNHG20 on cell proliferation, migration, and invasion, and the overexpression of RAB14 reversed the effects of miR-19b-3p mimic on the cell biological functions. LncRNA SNHG20 affects cell proliferation, migration, and invasion via the miR-19b-3p/RAB14 axis in OSCC., Graphical abstract

Background: Invasive growth is one of the most typical features of aggressive types of malignant cancer, including glioblastoma. Lysosomal cysteine protease-cathepsin S (CTSS), has been reported to be involved in invasive growth and distant metastasis of cancer cells. However, the underlying mechanisms remained elusive. Methods: U87 and U251 human glioblastoma cell lines were applied in this study. Cell migration and invasion ability were measured by wound healing assay and transwell assay. Western blot was employed to detect the expression levels of proteins. Immunofluorescence assays of cells and tissues were used to visualize the localization and expression of proteins. The SPSS software was used for statistical analysis. Results: Our results showed that the high expression of CTSS was link with the grades of glioma tissues. The CTSS inhibitor-Z-FL-COCHO (ZFL), could attenuate TGF-β-induced invasive growth as proven by wound healing and transwell assays. Furthermore, inhibition of CTSS could reverse TGF-β-induced epithelial-to-mesenchymal transition (EMT) and restore TGF-β-triggered tight junction proteins turnover, thus decreasing glioblastoma cell mobility. We also observed that TGF-β could change the morphology of glioblastoma cells, redistribute intermediate-filament, vimentin, which was highly relevant to mesenchymal type cells and enhanced mobility. However, inhibition of CTSS could significantly restore this transformation. Our results proved that PI3K/AKT/mTOR pathway was significantly suppressed in the TGF-β+ZFL (CTSS inhibitor) groups, and AKT activator-SC79, could reverse the anti-invasion effect of CTSS, indicating an important role of PI3K/AKT/mTOR pathway in this process. Conclusion: Z-FL-COCHO (ZFL), a CTSS inhibitor, could reverse TGF-β-induced EMT and change of tight junction proteins via PI3K/AKT/mTOR pathway.