TRIM29 prevents hepatocellular carcinoma progression by inhibiting Wnt/β-catenin signaling pathway

TRIM29 plays an important role in many neoplasms.In this study,we aimed to elucidate its role in hepatocellular carcinoma (HCC) and explore the corresponding potential mechanism.The expression level of TRIM29 in HCC samples and hepatoma cell lines was detected.We found that TRIM29 was down-regulated in clinical HCC samples and cultured hepatoma cell lines by western blot analysis and quantitative polymerase chain reaction.In addition,we demonstrated that higher TRIM29 expression was associated with higher differentiation grade of HCC.To explore the effect of TRIM29 on hepatoma cells and its possible mechanisms,TRIM29-knockdown and overexpression cell models were constructed.The results showed that the depletion of TRIM29 promoted liver cancer cell proliferation,clone formation,migration and invasion in vitro probably through the Wnt/β-catenin signaling pathway.This study revealed the inhibitory roles of TRIM29 in HCC and the possible mechanisms.     

MiR-181c modulates the proliferation,migration, and invasion of neuroblastoma cells by targeting Smad7

MicroRNAs （miRNAs） function as key regulators of gene expression in various cancers. In this study, the aim is to explore the roles and regulation mechanism of miR-181c in neuroblastoma （NB） cells. We found that miR-181c was downregulated in metastatic NB tissues, compared with primary NB tissues. Then functional studies indicated that miR-181 c overexpression inhibited NB cell proliferation, migration, and invasion, while miR-181c inhibition increased cell proliferation, migration, and invasion. EGFP reporter assay, real-time polymerase chain reaction and western blot analysis validated that Smad7 was a direct target of miR- 181c. MiR-181c reduced Smad7 expression at both mRNA and protein levels. Finally, functional assays showed that the effect of Smad7 knockdown on cells was similar to that of miR-181c overexpression. Importantly, Smad7 overexpression could restore the antitumor effects that were induced by miR-181 c. In conclusion, our results demonstrated that miR- 181c inhibits NB cell growth and metastasis-related traits through the suppression of SmadT, functioning as a tumor suppressor. Moreover, our results suggested that miR-181c may serve as an important therapeutic target for NB patients.     

PZR promotes metastasis of colorectal cancer through increasing FAK and Src phosphorylation

Metastasis is the main cause of death in patients with colorectal cancer (CRC), but the molecular mechanism is not yet fully understood. Previous studies have shown that P zero-related protein (PZR), a member of the imm uno globulin family, can promote fibr onectin-depe ndent migratio n of mouse embryonic fibroblasts as well as invasion and metastasis of hepatic carcinoma cells. However, the role of PZR in CRC remains unclear. In this study, we determined the ectopic expression of PZR in CRC tissues, and results showed that PZR expression was in creased not only in tumors with higher pathological stage, but also in tumors with distant metastasis. Through PZRknockdown and overexpression in CRC cell lines, we found that the expression of PZR had significant effect on the invasion and migration of CRC cells as well as the phosphorylation of prometastasis proteins including focal adhesion kinase (FAK) and Src. Taken together, this study indicates that PZR may promote the invasion and migration of CRC cells through increasing the phosphorylation of FAK and Src, which provides a new theoretical basis and a possible marker for the diag nosis or prog nosis of CRC metastasis.     

Piezo1 regulates migration and invasion of breast cancer cells via modulating cell mechanobiological properties

Cell migration and invasion are two essential processes during cancer metastasis. Increasing evidence has shown that the Piezo1 channel is involved in mediating cell migration and invasion in some types of cancers. However, the role of Piezo1 in the breast cancer and its underlying mechanisms have not been clarified yet. Here, we show that Piezo1 is high-expressed in breast cancer cell (BCC) lines, despite its complex expression in clinical patient database. Piezo1 knockdown (Piezo1-KD) promotes unconfined BCC migration, but impedes confined cell migration. Piezo1 may mediate BCC migration through the balances of cell adhesion, cell stiffness, and contractility. Furthermore, Piezo1-KD inhibits BCC invasion by impairing the invadopodium formation and suppressing the expression of metalloproteinases (MMPs) as well. However, the proliferation and cell cycle of BCCs are not significantly affected by Piezo1. Our study highlights a crucial role of Piezo1 in regulating migration and invasion of BCCs, indicating Piezo1 channel might be a new prognostic and therapeutic target in BCCs.     

miR-195-5p exerts tumor-suppressive functions in human lung cancer cells through targeting TrxR2

miR-195-5p has been widely explored in various cancers and is considered as a tumor-suppressive microRNA.However,its roles in human lung cancer pathogenesis are not fully elucidated.In this study,we aimed to explore how miR-195-5p is involved in malignant behaviors of lung adenocarcinoma(LUAD)cells.miR-195-5p expression was examined in the tumor tissues of patients with LUAD and human LUAD cell lines including A549 and PC-9.Thioredoxin reductase 2(TrxR2)was predicted to be an mRNA target of miR-195-5p using online tools and validated by the Dual-Luciferase Reporter Assay.Lentivirus infection was used for gene overexpression,while gene knockdown was achieved by RNA interference.Cell proliferation was determined by Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine methods,and cell migration and invasion were assayed with transwell experiments.Cell apoptosis was determined by annexin V staining-based flow cytometry.The antitumor effects of miR-195-5p were also evaluated in nude mice xenografted with A549 cells.We found that miR-195-5p was lowly expressed in human LUAD cells,and its overexpression markedly suppressed cell proliferation,migration,and invasion and increased the apoptosis of LUAD cells in vitro.TrxR2 knockdown phenocopied the tumor-suppressive effects of miR-195-5p overexpression,while simultaneous TrxR2 overexpression remarkably reversed the effects of miR-195-5p overexpression on malignant behaviors of A549 and PC-9 cells.Additionally,miR-195-5p overexpression inhibited the growth of xenografted A549 tumor in nude mice.Our work verified that miR-195-5p exerts tumor-suppressive functions in LUAD cells through targeting TrxR2 and suggested that the miR-195-5p/TrxR2 axis is a potential biomarker for LUAD therapy.     

RNA-binding protein QKI regulates contact inhibition via Yes-associate protein in ccRCC

Contact inhibition adjusts organ size to the proper size and ensures the cultured cells growing to a monolayer.By regulating the downstream coordinator YAP,the evolutionarily conserved Hippo transduction pathway attunes cell growth and death in response to cell contact inhibition,polarity,self-renewal,and differentiation.Dysregulation of this pathway is involved in various diseases such as cancer.RNA-binding protein QKI regulates cell proliferation,metabolism,division,and immunity in various cancer models,but its role in cancer cell contact inhibition remains unclear.In this study,we aimed to clarify the relationship between QKI and YAP,and the role of their interaction in cell contact inhibition.We found a lower QKI expression level in sparse condition,whereas a higher expression level in confluent condition by western blot analysis and immunofluorescence assay.QKI knockdown elevated cell proliferation and invasion both in vitro and in vivo.Strikingly,the results of CCK-8 assay,colony formation assay,and transwell assay showed that the phenomenon was in accord with the expression level of pYAP and reverse with YAP.Higher levels of Wnt3a and β-catenin were also found in xenografts of QKI-knockdown clear cell renal cell carcinoma (ccRCC) CAKI-1 cells by western blot analysis and immumohistochemical staining.Finally,a positive correlation between QKI and pYAP was found in clinical specimens by immunohistochemistry.Thus,as a negative regulator of YAP,QKI attuned the cell contact inhibition,leading to inhibition of cancer cell proliferation and invasion through Wnt and GPCR pathway.     

MiRNA-218, a new regulator of HMGB1, suppresses cell migration and invasion in non-small cell lung cancer

MieroRNAs （miRNAs） function as negative regulators of gene expression involved in cancer metastasis. The aim of this study is to investigate the potential roles of miR-218 in non-small cell lung cancer and validate its regulation mech- anism. Functional studies showed that miR-218 overexpres- sion inhibited cell migration and invasion, but had no effect on cell viability. Enhanced green fluorescent protein reporter assay, real-time polymerase chain reaction and western blot analysis confirmed that miR-218 suppressed the expression of high mobility group box-1 （HMGB1） by directly targeting its 31-untranslated region. Accordingly, silencing of HMGBI accorded with the effects of miR-218 on cell migration and invasion, and overexpression of HMGB1 can restore cell migration and invasion which were reduced by miR-218. In conclusion, these findings demon- strate that miR-218 functions as a tumor suppressor in lung cancer. Furthermore, miR-218 may act as a potential thera- peutic biomarker for metastatic lung cancer patients.     

Evidences of Hfq associates with tryptophanase and affects extracellular indole levels

In this study, we observed a novel property of Escherichia coli Hfq protein： it possibly influenced extracellular indole levels. The extracellular indole concentrations were increased in Hfq mutant cells and decreased in Hfq overexpression cells in a cell densitydependent manner. The decreased extracellular indole levels in Hfq overexpression cells caused the postponement of entering into stationary phase. Indole was produced by tryptophanase, the gene product of tnaA, which catalyzed tryptophan into indole, ammonia and pyruvate. Further studies showed that at cell density of 0.8 but not at 0.4, tryptophanase activities of total cell extracts were affected by Hfq mutation or overexpression. Protein puff-down assay and co-immunoprecipitation experiments revealed that Hfq associated with tryptophanase under relatively higher extracellular indole levels, suggesting this was a feedback control of indole production. The association of Hfq and tryptophanase might be indirect because purified Hfq could not affect the values of Km and Vmax of purified tryptophanase.     

mad—overexpression down regulates the malignant growth and p53 mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Mad protein has been shown as an antagonist of cMyc protein in some cell lines.The effect of Mad protein to the malignant phenotype of human hepatoma BEL-7404 cell line was investigated experimentally.An eukarryotic vector pCDNA Ⅲ containing full ORF fragment of mad cDNA was transfected into targeted cells.Under G418 selection,stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells.DNA synthesis,cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cells were partially inhibited in comparison to control cells.Flos cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase,resulting in the retardation of cell proliferation.RT-PCR detected a marked inhibition of the expression of cdc25A,an important regulator gene of G0/G1 to S phase in cell cycle.It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-74040M1 cells in the absence of serume.Thus,Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL-7404 cells.     

Antisense Tiam1 down-regulates the invasiveness of 95D cells in vitro

As a specific guanine nucleotide exchange factor of Rac 1, Tiam 1 (T-lymphoma invasion and metastasis inducing protein 1) is involved in a number of cellular events, such as cytoskeleton reorganization, cell adhesion, and cell migration. Since Tiaml was implicated in the invasion and metastasis of T-lymphoma cells and breast tumor cells, we compared the expression level of Tiaml in two human giant-cell lung carcinoma cell strains with high or low metastasis potential, and found that Tiaml expression level in high-metastatic 95D cells was higher than that in low-metastatic 95C cells. To further confirm the role of Tiam I in invasion and metastasis, we constructed the antisense Tiaml expression plasmid (pcDNA3-anti-Tiaml), which was transfected into 95D cells. A stable transfected clone with decreased Tiaml expression was screened and selected for further research. Transwell assay showed that down-regulation of endogenous Tiam1 by anti-Tiam1 can reduce the in vitro invasiveness of 95D cells. Our results suggested that Tiam1 signaling contributed to the invasion and metastasis of the human giant-cell lung carcinoma cells.     

The 5‘—flanking cis—acting elements of the human ε—globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins

The nuclear matrix attachment regions(MARs) and the binding nuclear matrix proteins in the 5‘-flanking cisacting elements of the human ε-globin gene have been examined.Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII,-446bp- -419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells,indicating that ε-PREII may be an erythroidspecific facultative MAR.In gel mobility shift assay and Southwestern blotting assay,an erythroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (ε-PREII).Furthermore,we demonstrated that the silencer (-392bp- -177bp) upstream of the human ε-globin gene could associate with the nuclear matrices from K562,HEL and Raji cells.In addition,the nuclear matrix proteins prepared from these three cell lines could also bind to this silencer,suggesting that this silencer element might be a constitutive nuclear matrix attachment region(constitutive MAR).Our results demonstrated that the nuclear matrix and nuclear matrix proteins might play an important role in the regulation of the human ε-globin gene expression.     

The antibody preparation and expression of human Pescadillo

To explore the biological roles of human Pescadillo and investigate its potential effect on tumorigene- sis, the cDNA of Pescadillo was fused with that of GST. After purification and elution, the purified GST-Pescadillo fusion protein was obtained, and the antibody against the fusion protein was generated. Endogenous Pescadillo protein was observed to be remarkably induced by estrogen. It was mainly distributed in the tissues such as breast, ovary and intestine, all of which contain proliferating cells, and was also detected in many cell lines of human cancer: renal carcinoma, hepatoma, ovarian cancer, colon carcinoma, and breast cancer. The expression level of Pescadillo was increased significantly in breast cancer tissues compared with their paired margin tissues. Taken together, these data suggest that Pescadillo may play important roles in the initiation and development of cancer and may be a po- tential target in cancer diagnosis and therapy.     

Proscillaridin A inhibits hepatocellular carcinoma progression through inducing mitochondrial damage and autophagy

Hepatocellular carcinoma(HCC)is one of the leading causes of cancer-related deaths globally.At present,drug options for systemic treatment of HCC are very limited.There is an urgent need to develop additional effective drugs for HCC treatment.In the present study,we found that proscillaridin A(ProA),a cardiac glycoside,exerted a strong anticancer effect on multiple HCC cell lines.ProA significantly inhibited the cell proliferation,migration,and invasion of HCC cells.ProA also had a marked inhibitory effect on the progression of HCC in the MHCC97H xenograft nude mouse model.ProA-mediated suppression of HCC was closely related to cell apoptosis.ProA-treated HCC cells displayed significant mitochondrial damage and elevated reactive oxygen species production,resulting in profound cell apoptosis.Meanwhile,ProA also played a role in autophagy induction in HCC cells.Defects in autophagy partially relieved ProA’s anticancer effect in HCC cells.Our findings demonstrate that ProA can effectively inhibit HCC progression and may serve as a potential therapeutic agent for HCC treatment.     

Involvement of regulatory volume decrease in the migration of nasopharyngeal carcinoma cells

The transwell chamber migration assay and CCD digital camera imaging techniques were used to investigate the relationship between regulatory volume decrease (RVD) and cell migration in nasopharyngeal carcinoma cells (CNE-2Z cells). Both migrated and non-migrated CNE-2Z cells, when swollen by 47% hypotonic solution, exhibited RVD which was inhibited by extracellular application of chloride channel blockers adenosine 5‘-triphosphate (ATP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen. However, RVD rate in migrated CNE-2Z cells was bigger than that of non-migrated cells and the sensitivity of migrated cells to NPPB and tamoxifen was higher than that of nonmigrated cells. ATP, NPPB and tamoxifen also inhibited migration of CNE-2Z cells. The inhibition of migration was positively correlated to the blockage of RVD, with a correlation coefficient (r) = 0.99, suggesting a functional relationship between RVD and cell migration. We conclude that RVD is involved in cell migration and RVD may play an important role in migratory process in CNE-2Z cells.     

Autophagy prevents autophagic cell death in Tetrahymena in response to oxidative stress

Autophagy is a major cellular pathway used to degrade long-lived proteins or organelles that may be damaged due to increased reactive oxygen species(ROS) generated by cellular stress. Autophagy typically enhances cell survival, but it may also act to promote cell death under certain conditions. The mechanism underlying this paradox, however, remains unclear. We showed that Tetrahymena cells exerted increased membranebound vacuoles characteristic of autophagy followed by autophagic cell death(referred to as cell death with autophagy) after exposure to hydrogen peroxide. Inhibition of autophagy by chloroquine or 3-methyladenine significantly augmented autophagic cell death induced by hydrogen peroxide. Blockage of the mitochondrial electron transport chain or starvation triggered activation of autophagy followed by cell death by inducing the production of ROS due to the loss of mitochondrial membrane potential. This indicated a regulatory role of mitochondrial ROS in programming autophagy and autophagic cell death in Tetrahymena. Importantly, suppression of autophagy enhanced autophagic cell death in Tetrahymena in response to elevated ROS production from starvation, and this was reversed by antioxidants. Therefore, our results suggest that autophagy was activated upon oxidative stress to prevent the initiation of autophagic cell death in Tetrahymena until the accumulation of ROS passed the point of no return, leading to delayed cell death in Tetrahymena.