Gallbladder Carcinoma Cell Proliferation Research Articles

EMP3 as a key downstream target of miR-663a regulation interferes with MAPK/ERK signaling pathway to inhibit gallbladder cancer progression

Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract, and its molecular pathogenesis remains unclear. Here we explore the functional roles of epithelial membrane protein 3 (EMP3) in GBC progression, which is aberrantly expressed in various types of cancers. The results showed that the expression level of EMP3 was reduced in human GBC tissues compared with non-malignant tissues. Further, the low expression of EMP3 was associated with the poor prognosis of GBC patients by Kaplan-Meier analysis. The ectopic expression of EMP3 inhibited GBC cell proliferation, migration and invasion in vitro and in vivo. Conversely, the depletion of EMP3 promoted GBC cell growth and metastasis. In addition, we found that EMP3 was a target gene of miR-663a, and the downregulation of EMP3 in GBC was attributed to the overexpression of miR-663a. MiR-663a was also shown to be a tumor-promoting factor mediating GBC development. In this study, we demonstrate that downregulation of EMP3 activates MAPK/ERK signaling, which regulates GBC progression. These data reveal the mechanism by which EMP3 inhibits the progression of GBC, suggesting that the miR-663a/EMP3/MAPK/ERK axis may be a new therapeutic target for GBC treatment.

MiR-195-5p suppresses the proliferation, migration, and invasion of gallbladder cancer cells by targeting FOSL1 and regulating the Wnt/β-catenin pathway.

BackgroundMicroRNA-messenger RNA (miRNA-mRNA) regulatory networks are essential factors that regulate tumor development and metastasis in various cancers including gallbladder carcinoma (GBC). Here, we identified the miR-195-5p/Fos-like antigen-1 (FOSL1) axis in GBC by bioinformatics analysis and aimed to investigate its role and regulatory mechanism in the development and progression of GBC.MethodsBioinformatics analysis was used to construct a miRNA-mRNA regulatory network. Real-time quantitative polymerase chain reaction (qRT-PCR), western blot, and dual luciferase reporter assays confirmed that miR-195-5p targets FOSL1 in GBC. Cell Counting Kit-8 (CCK-8), wound healing, transwell, flow cytometry assays, western blotting, and immunofluorescence were used to detect the biological effects of the miR-195-5p/FOSL1 regulatory axis and the Wnt/β-catenin signaling pathway on the proliferation, migration, invasion, and cell cycle of GBC cells. A nude mouse tumorigenesis model was constructed to verify the role of miR-195-5p in vivo.ResultsBioinformatics analysis and qRT-PCR confirmed that the miR-195-5p/FOSL1 regulatory axis was closely related to GBC cells. Overexpression of miR-195-5p inhibited the proliferation, migration, and invasion of GBC cells, and the cells were blocked in the G0/G1 phase. Dual luciferase reporter gene assays and western blot analysis showed that FOSL1 is targeted by miR-195-5p. The recovery experiment showed that miR-195-5p can inhibit cell proliferation, migration, invasion, and increase of cells in the G0/G1 phase, and the overexpression of FOSL1 could restore this effect by regulating the Wnt/β-catenin signaling pathway. Finally, we confirmed that miR-195-5p inhibited the growth of transplanted tumors in vivo.ConclusionsThe overexpression of miR-195-5p inhibits the proliferation and metastasis of GBC cells by directly targeting FOSL1 and regulating the Wnt/β-catenin signaling pathway.

MiR-4733-5p promotes gallbladder carcinoma progression via directly targeting kruppel like factor 7

ABSTRACT Gallbladder carcinoma (GBC) is highly aggressive with poor prognosis. Accumulating reports show that miRNAs play critical roles in tumor progression. Previous studies have identified several miRNAs that promoted or inhibited GBC cell proliferation and/or metastasis. Here, we used the Gene Expression Omnibus (GEO) dataset to identify dysregulated miRNAs in GBC, followed by validating the upregulation of the miR-4733-5p and downregulation of kruppel-like factor 7 (KLF7) in GBC biopsies by quantitative real-time PCR (RT-qPCR), in situ hybridization (ISH) staining, and immunohistochemistry (IHC) assays. GBC cell proliferation and invasion capacities mediated by miR-4733-5p were evaluated by a series of function assays in vitro, including CCK-8, colony formation assay, wound healing assay and transwell assay. Xenograft tumor model found that miR-4733-5p promoted GBC tumor growth in vivo. This study clarified that miR-4733-5p was upregulated in GBC and promoted GBC cell proliferation via directly binding to 3’ untranslated region (UTR) of KLF, which was downregulated and prohibited the proliferation and migration of GBC cells.

AFAP1-AS1/Hsa-miR-15a-5p/Bcl-2 Axis is a Potential Regulator of Cancer Cell Proliferation and Apoptosis in Gallbladder Carcinoma

Objective Gallbladder carcinoma (GBC) is a common malignancy of the biliary tract that, with late diagnosis, is often fatal. A better understanding of the underlying molecular mechanisms may facilitate targeted therapy for GBC. Therefore, this study aimed to investigate whether long non-coding RNA AFAP1-AS1 regulates GBC cell proliferation and apoptosis through hsa-miR-15a-5p. Methods SGC-996 and NOZ cell lines were transfected with an hsa-mR-15a-5p inhibitor or si-AFAP1-AS1, and GBC cell proliferation and apoptosis were measured. The expression levels of AFAP1-AS1, hsa-miR-15a-5p, apoptosis-related proteins, and bcl-2 were assessed. The dual-luciferase reporter assay was used to determine the binding between AFAP1-AS1 and hsa-miR-15a-5p or between hsa-miR-15a-5p and bcl-1. Results In SGC-996 and NOZ cells, AFAP1-AS1 was significantly expressed and hsa-miR-15a-5p was modestly expressed. Transfection of the hsa-miR-15a-5p inhibitor elevated proliferation of SGC-996 and NOZ cells and decreased apoptosis, whereas transfection of si-AFAP1-AS1 reduced the proliferation rate and increased apoptosis. In addition, AFAP1-AS1 bound hsa-miR-15a-5p and hsa-miR-15a-5p targeted bcl-2. Increased bcl-2 expression was observed in the GBC cells. AFAP1-AS1 may regulate GBC cell proliferation and apoptosis via has-miR-15a-5p to mediate bcl-2 expression. Conclusion The AFAP1-AS1/hsa-miR-15a-5p/bcl-2 axis regulates GBC cell proliferation and apoptosis, providing potential guidance for the clinical treatment of GBC.

MiR-4461 inhibits the progression of Gallbladder carcinoma via regulating EGFR/AKT signaling

ABSTRACT Increasing evidence has demonstrated that microRNAs (miRNAs) participated in the tumorigenesis, progression and recurrence of various malignancies including Gallbladder carcinoma (GBC). miR-4461 was reported to work as a tumor suppressor gene in renal cell carcinoma. However, the role of miR-4461 in GBC remains unknown. Herein, we show that miR-4461 is downregulated in gallbladder cancer stem cells (CSCs). Forced miR-4461 expression attenuates the self-renewal, tumorigenicity of gallbladder CSCs, and inhibits proliferation and metastasis of GBC cells. Conversely, miR-4461 knockdown promotes the self-renewal of gallbladder CSCs, and facilities proliferation and metastasis of GBC cells. Mechanistically, miR-4461 inhibits GBC progression via downregulating EGFR/AKT pathway. Special EGFR siRNA or AKT overexpression virus abolishes the discrepancy of self-renewal, tumorigenesis, growth, and metastasis between miR-4461 overexpression GBC cells and their control cells. In conclusion, miR-4461 suppresses GBC cells self-renewal, tumorigenicity, proliferation, and metastasis by inactivating EGFR/AKT signaling, and may therefore prove to be a potential therapeutic target for GBC patients.

Long Non-Coding RNA Myosin Light Chain Kinase Antisense 1 Plays an Oncogenic Role in Gallbladder Carcinoma by Promoting Chemoresistance and Proliferation.

BackgroundLong non-coding RNAs (lncRNAs) have been reported to play critical roles in human tumours, including gallbladder carcinoma (GBC). However, their biological functions and molecular mechanisms in tumorigenesis and progression remain largely unknown.MethodsQuantitative polymerase chain reaction (qPCR) was used to verify the expression of lncRNA myosin light chain kinase antisense RNA 1 (MYLK-AS1) in 120 pairs of GBC tissues and paired adjacent non-tumour tissues, as well as in six different GBC cell lines (NOZ, EH-GB1, OCUG-1, GBC-SD, SGC-996 and QBC-939). Cell counting kit 8 was applied to explore cell proliferation and drug sensitivity assays. The target miRNAs (miR) of MYLK-AS1 and downstream target genes were predicted using Starbase 3.0 software and confirmed by double luciferase reporting test. The expression of proteins was assessed using Western blot assay.ResultsHere, we demonstrated that MYLK-AS1 was significantly upregulated and correlated with a poor prognosis and poor clinical characteristics in GBC. Furthermore, the forced expression of MYLK-AS1 significantly promoted GBC cell proliferation and resistance to gemcitabine in vitro. Mechanistically, MYLK-AS1 functioned as an efficient miR-217 sponge, thereby releasing the inhibition of enhancer of zeste 2 polycomb repressive complex 2 (EZH2) subunit expression. MYLK-AS1 promoted GBC cell proliferation and resistance to gemcitabine by upregulating EZH2 expression, and EZH2 was confirmed as a direct target of miR-217.DiscussionOur results confirmed that the chemoresistant driver MYLK-AS1 might be a promising candidate as a therapeutic target for the treatment of advanced GBC.

Golgi phosphoprotein3 promotes the proliferation of gallbladder carcinoma cells via regulation of the NLRP3 inflammasome.

Golgi phosphoprotein 3 (GOLPH3) has been demonstrated to promote tumor progression in various gastrointestinal malignancies. However, its effects in gallbladder carcinoma (GBC) remain unknown. In the present study, the expression levels of GOLPH3 and nucleotide-binding domain leucine-rich repeat and pyrin domain containing receptor 3 (NLRP3) in human GBC tissues were detected by immunohistochemistry, and the clinical data and survival of these patients were analyzed. Next, whether GOLPH3 could affect tumor proliferation via regulation of the NLRP3 inflammasome was investigated in vitro. The results demonstrated that GOLPH3 could promote GBC cell proliferation, and that it regulated protein expression levels of NLRP3, as well as Caspase-1 P10. Conversely, knockdown of NLRP3 reversed the effects of GOLPH3 overexpression on GBC cell proliferation. GOLPH3 and NLRP3 expression levels were found to be upregulated in GBC tissues and their expression was positively correlated. The expression of GOLPH3 and NLRP3 was associated with the expression of the proliferative marker Ki-67 in tissues, and associated with poor survival, tumor stage, degree of differentiation, depth of invasion, carbohydrate antigen 19-9 and C-reactive protein levels in patients with GBC. In summary, these results indicate that GOLPH3 promotes GBC cell proliferation via a NLRP3/Caspase-1 pathway. GOLPH3 and NLRP3 participate in the process of human GBC growth and may serve as a potential therapeutic targets.

Expression and inhibitory effects of p53-upregulated modulator of apoptosis in gallbladder carcinoma.

The p53-upregulated modulator of apoptosis (PUMA) has been reported to be involved in various types of cancer. However, its potential biological role in gallbladder carcinoma (GBC) has not been fully elucidated. The present study aimed to determine the expression levels of PUMA and its biological effects on GBC. The mRNA and protein expression levels of PUMA in GBC tissues and cell lines were measured using reverse transcription-quantitative PCR and western blotting, respectively. The effects of PUMA overexpression on cell viability, proliferation and invasive ability were determined in vitro using the MTT, colony formation and Transwell invasion assays, respectively. The apoptotic rates were detected using the Annexin V-FITC apoptosis detection kit. Furthermore, follow-up of patients with GBC was performed to identify the association between PUMA expression levels and GBC prognosis. The results of the present study demonstrated that the expression levels of PUMA were significantly lower in the GBC tissues and cell lines compared with those in adjacent normal gallbladder tissues and normal gallbladder cells, respectively. Further experiments indicated that overexpression of PUMA inhibited the viability, proliferation and invasive ability of GBC cells compared with those in the control-transfected GBC cells. In addition, overexpression of PUMA significantly promoted apoptosis in GBC cells. Furthermore, overexpression of PUMA inhibited epithelial-mesenchymal transition, and promoted Bax upregulation and Bcl-2 downregulation compared with those in the control group. Low PUMA expression levels were associated with a short overall survival time in patients with GBC. In conclusions, PUMA may act as a tumor suppressor in GBC and may serve as a potential novel treatment target for human GBC.

ARRB1 Drives Gallbladder Cancer Progression by Facilitating TAK1/MAPK Signaling Activation.

Gallbladder carcinoma (GBC) is the most common malignancy of the biliary tract, with a dismal 5-year survival of 5%. Recently, ARRB1, as a molecular scaffold, has been proposed to participate in the progression of multiple malignancies. However, the effect and regulatory mechanisms of ARRB1 in GBC have not been investigated. Our study aimed to explore the biological functional status and the possible molecular mechanisms of ARRB1 with respect to GBC progression. The survey showed that human GBC tissues exhibited increased levels of ARRB1 compared with normal tissues, and the high expression of ARRB1 was associated with poor prognosis of GBC patients. A series of in vitro and in vivo functional experiments based on knockdown of ARRB1 uncovered that ARRB1 enhanced GBC cell proliferation, migration, and invasion. Furthermore, we reported that TAK1, a component of the TNF /MAPK pathway, is a vital downstream effector of ARRB1. In addition, siTAK1 could abolish the functional changes between ARRB1 overexpression GBC cells and control ones. Our data revealed that ARRB1 facilitated the carcinogenesis and development of GBC through TNF/TAK1/MAPK axis, suggesting that ARRB1 may be a promising biomarker and treatment target for GBC patients.

MiR-188-5p inhibits proliferation, migration, and invasion in gallbladder carcinoma by targeting Wnt2b and Smad2.

Gallbladder carcinoma (GBC) commonly occurs in gastrointestinal malignancy and has the fifth highest mortality rate among gastrointestinal malignancy. Recently, miR-188-5p, a small noncoding RNA, has been implicated in various types of cancer such as nasopharyngeal carcinoma, oral squamous cell carcinoma, liver cancer, and prostate cancer. However, the effect of miR-188-5p on GBC remains unclear. Here, we demonstrated that miR-188-5p was downregulated in GBC tissues, and downregulation of miR-188-5p correlated with larger tumor size, lymph node metastasis, and extensive metastasis. In addition, the overall survival time of patients with higher miR-188-5p expression was significantly longer than that of patients with low-miR-188-5p expression. Moreover, downregulation of miR-188-5p promoted the proliferation, migration, and invasion of GBC cells, while its overexpression inhibited cell invasion and induced cell apoptosis, and arrested GBC growth in vivo. Importantly, miR-188-5p-dependent tumorigenesis was correlated with Wnt/β-catenin signaling and p-38/JNK signaling. In conclusion, miR-188-5p plays a direct role in GBC tumorigenesis. Our study suggests that miR-188-5p could serve as a novel diagnosis marker and therapeutic target in GBC.

Oroxylin A Exerts Its Antitumor Effects in Human Gallbladder Cancer via Inhibition of the PTEN/PI3K/AKT Signaling Pathway.

Gallbladder carcinoma (GBC) is one of the most common carcinomas of the biliary tract and is associated with aggressive malignancy and poor prognosis. Current therapeutic strategies, including surgery, radiotherapy, and chemotherapy, are not sufficient for the treatment of GBC, and new therapeutic strategies are urgently needed. The antitumor effects of oroxylin A (OrA), a natural flavonoid extracted from the dried roots of medicinal plants such as Scutellariae species (Radix Scutellariae), have been widely reported in various cancers. In this study, we first evaluated the antitumor activity and the underlying mechanism of action of OrA on GBC cells in vitro. Our results revealed that OrA significantly attenuated the proliferation, migration, and invasion of GBC cells, simultaneously promoting their apoptosis. Suppression of the phosphate on and tension homology deleted chromosome ten (PTEN)/phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT) signaling pathway was found to be the underlying mechanism involved in the antitumor activity of OrA. In addition, experiments using a tumor xenograft mouse model confirmed the antitumor effects of OrA in vivo. Taken together, our findings indicate that OrA could be a potential antitumor agent for the prospective treatment of GBC.

MUC16 C-terminal binding with ALDOC disrupts the ability of ALDOC to sense glucose and promotes gallbladder carcinoma growth

The MUC16 C-terminal (MUC16c) level is associated with tumor serum CA-125 levels, however, the roles remain unclear in gallbladder carcinoma (GBC). In this study, we found that MUC16c promoted glucose uptake and glycolysis for GBC cell proliferation. Mass spectrometry analysis suggested that MUC16c could combine with aldolase. The ALDOC mRNA and protein are overexpressed in GBC tumors. The IHC results also showed the consistent up-regulation of.ALDOC and MUC16c level in GBC tumor tissues than in peritumor tissues. We determined that MUC16c combining with ALDOC promoted ALDOC protein stability and disrupted the ability of ALDOC sensing glucose deficiency, which activated AMPK pathway and increased GBC cell proliferation. ALDOC knockdown significantly inhibited the glucose uptake and glycolysis induced by MUC16c. Our study established important roles of MUC16c promoting GBC cell glycolysis and proliferation and revealed the underlying mechanism of CA-125-related heavy tumor metabolic burden in GBC.

RNA Sequencing Revealed Signals of Evolution From Gallbladder Stone to Gallbladder Carcinoma.

Gallbladder stone is a major risk factor for gallbladder carcinoma (GBC), while there is still a controversy whether period of follow-up since newly diagnoses of asymptomatic gallstones increases the risk of GBC. In this study, 10 GBC patients and 30 patients with gallstones were admitted to our hospital. Patients with gallstones were divided into 3 groups according to the follow-up time, involving 10 patients with follow-up period of 1–3 years (GS3 group), 10 patients with follow-up period of 5–10 years (GS5 group), and 10 patients with follow-up period of more than 10 years (GS10 group). Tumor and para-tumor tissues of GBC patients, and gallbladder tissues of gallstone patients were collected. RNA sequencing was performed on the 50 samples. Besides, 1,704 differentially expressed genes (DEGs) were identified in tumors compared with para-tumor tissues of 10 GBC patients, which were enriched into some well-known cancer-related pathways, such as PI3K-Akt, mitogen-activated protein kinase (MAPK), Ras, and Wnt signaling pathways, and the most significant pathway was neuroactive ligand-receptor interaction. Patients with gallstones with periods of follow-up equal to 1–3 and > 10 years showed to have higher cancer risk than those with 5–10 years. ALPP and GPR87 are potential biomarkers for predicting cancer risk in patients with gallstones. The in vitro results revealed that GPR-87 can promote the proliferation, migration, and invasion of GBC cells. Herein, we explored the relationship between GBC patients and patients with gallstones with different periods of follow-up in transcriptome level.

TFAP2A is a novel regulator that modulates ferroptosis in gallbladder carcinoma cells via the Nrf2 signalling axis.

Ferroptosis is a recently identified form of controlled cell death generally associated with the accumulation of lipid-associated reactive oxygen species (ROS). However, the molecular mechanisms underlying ferroptosis have not been established. Microarray expression data for three human gallbladder carcinoma (GBC) and matched non-tumour specimens were downloaded from the Gene Expression Omnibus (GEO) repository. Candidate genes were filtered using bioinformatic analysis. After cell transfection, candidate gene impacts on cell proliferation, migration, invasion and ferroptosis (ferrous iron (Fe2+) and malondialdehyde (MDA) levels) were assessed. We screened 626 differentially expressed genes (DEGs) including 465 that were downregulated and 161 that were upregulated in the three tissue pairs. These DEGs were used to construct a protein-protein interaction (PPI) network. Functional enrichment analysis revealed the top three modules in the network and four hub genes. Transcription factor AP-2 alpha (TFAP2A) was screened and showed overexpression in The Cancer Genome Atlas (TCGA) digestive system tumour data and a relationship with clinical survival. In vitro, GBC exhibited upregulated expression of TFAP2A, whose inhibition reduced GBC cell proliferation, migration, and invasion. Fe2+ and MDA levels were elevated. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed TFAP2A enrichment in oxidative stress. Subsequent experiments demonstrated that TFAP2A silencing attenuated the expression of key genes associated with oxidative stress such as heme oxygenase 1 (HO-1), nuclear factor erythroid 2 like 2 (Nrf2), ferritin heavy chain 1 (FTH1) and NAD(P)H quinone dehydrogenase 1 (NQO1). Bioinformatic and experimental analyses reveal that TFAP2A plays a vital role in ferroptosis and hence is a potential therapeutic target for GBC treatment.

Increased expression of RBMS3 predicts a favorable prognosis in human gallbladder carcinoma.

Multiple regions in the short arm of chromosome 3 are frequently deleted in a variety of solid tumors including gallbladder carcinoma (GBC). RNA binding motif, single-stranded interacting protein 3 (RBMS3), a tumor suppressor gene (TSG), is located in this region. However, the role of RBMS3 in GBC remains unclear. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were performed to evaluate the mRNA and protein expression levels of RBMS3 in 41 fresh frozen GBC tissues and paired normal tissues. An immunohistochemical assay was performed on a tissue microarray (TMA, consisting of 125 cases GBC and 47 normal controls). Microvessel density (MVD) counts were determined using CD34 immunohistochemical staining. Moreover, univariate and multivariate analyses were performed to determine the correlations between RBMS3 expression, MVD and patient prognosis. Cellular functions including proliferation, clonogenicity and apoptosis, were assessed to further identify in vitro roles of RBMS3. It was revealed that both mRNA and protein expression levels of RBMS3 were significantly lower in GBC tissues than in normal controls. Multivariate Cox regression analyses demonstrated cytoplasmic RBMS3 expression as an independent prognostic factor correlated with GBC angiogenesis, histopathological differentiation and TNM stage. Kaplan-Meier curves revealed that patients with lower cytoplasmic RBMS3 levels had a significantly worse OS than patients with higher cytoplasmic RBMS3 expression. Additionally, ectopic expression of RBMS3 markedly suppressed GBC cell proliferation and clonogenicity and promoted apoptosis in vitro. These findings indicated the potential of cytoplasmic RBMS3 as a tumor prognostic biomarker and a promising therapeutic target for GBC.

TASP1 Promotes Gallbladder Cancer Cell Proliferation and Metastasis by Up-regulating FAM49B via PI3K/AKT Pathway.

The highly conserved protease TASP1 not only takes part in critical site-specific proteolysis, but also plays an important role in numerous liquid and solid malignancies. However, the TASP1 expression and its biological regulation function in malignant gallbladder carcinoma (GBC) remain fully unknown. Here we observed that TASP1 levels were substantially overexpressed in GBC samples compared with non-tumor tissues. High TASP1 level was closely associated with T stage and metastasis, and was also correlated with poor prognosis in GBC patients. The depletion of TASP1 inhibited GBC cell proliferation and metastasis in vitro and in vivo. Furthermore, we first revealed that FAM49B had biological function and was positively regulated by TASP1 activating PI3K/AKT signaling pathway in GBC. At the same time, FAM49B also promoted GBC cell proliferation and migration. Inhibition of PI3K/AKT with LY294002 or FAM49B expression abrogated Myc-TASP1/Lv-shTASP1-induced GBC cell proliferation and motility. In conclusion, these findings demonstrate that TASP1 is critical for GBC progression via TASP1-PI3K/AKT-FAM49B axis and it may be a novel prognostic factor. The therapeutic targeting TASP1 may be a potential treatment approach for GBC patients.

NOP2/Sun RNA methyltransferase 2 promotes tumor progression via its interacting partner RPL6 in gallbladder carcinoma.

NOP2/Sun domain family, member 2 (NSUN2) is a nuclear RNA methyl‐transferase catalyzing 5‐methylcytosine formation. Evidence shows that NSUN2 is correlated with cell unlimited proliferation. However, its functional role in gallbladder carcinoma (GBC), which is the most common biliary tract malignancy and has a poor prognosis, remains to be determined. Here we found that NSUN2 was highly expressed in GBC tissues as well as cell lines. NSUN2 silencing repressed GBC cell proliferation and tumorigenesis both in vitro and in vivo. Conversely, upregulation of NSUN2 enhanced GBC cell growth and colony formation. We further discovered that RPL6 was a closely interacting partner with NSUN2. Silencing RPL6 resulted in insufficient NSUN2 translational level and accumulative NSUN2 transcriptional level. Exogenous expression of NSUN2 partially rescued the effect of RPL6 in gallbladder cancer progression. Taken together, our data provided novel mechanic insights into the function of NSUN2 in GBC, thus pointing to NSUN2 as a potential and effective therapeutic approach to GBC treatment.

Effect of silencing phosphatidylinositol 3-kinase regulatory subunit gamma gene by small interfering RNA on proliferation and migration capability of gallbladder carcinoma cells

Objective To observe the effect of small interfering RNA (siRNA) silencing phosphatidylinositol 3-kinase regulatory subunit gamma (PIK3R3) gene on the proliferation and migration of gallbladder cancer cells NOZ and GBC-SD. Methods Small interfering RNA (si-PIK3R3) against PIK3R3 was constructed and real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting were used to detect PIK3R3 mRNA and protein in transfected gallbladder cancer cells NOZ and GBC-SD. The expression of cell counting kit-8 (CCK-8) proliferation assay was used to detect the proliferation of transfected 0-5 d cells. Transwell chamber assay was used to detect the migration of gallbladder cancer cells 48h after transfection. Results The si-PIK3R3 transfection group and the si-NC negative control group were successfully transfected into the NOZ and GBC-SD of gallbladder cancer cells. The expression of PIK3R3 mRNA (0.119±0.447) in the NOZ cell si-PIK3R3 group was significantly lower than that of NC group (1.000±0.398) (t=165.100, P<0.05), the PIK3R3 mRNA (0.085±0.593) in the si-PIK3R3 group of GBC-SD cells was significantly lower than that in the si-NC group (1.000±0.669) (t=500.200, P<0.05); the expression of PIK3R3 protein in the transfected group was significantly lower than that in the negative control group. The results of CCK-8 in NOZ cells showed that the absorbance values of 0-5 d si-PIK3R3 transfection group were 0.185±0.007, 0.163±0.013, 0.229±0.019, 0.240±0.017, 0.273±0.012, 0.350±0.069 (t=11.960, P<0.05); The results of 48 h Transwell showed that the number of cells migrated in the si-PIK3R3 transfection group [(69.0±4.5) cells] was significantly lower than that in the si-NC group (t=25.120, P<0.05). The results of CCK-8 in GBC-SD cells showed that the absorbance values of the 0-5 d si-PIK3R3 transfection group were 0.153±0.003, 0.158±0.002, 0.201±0.010, 0.240±0.012, 0.297±0.018, 0.483±0.024 (t=14.160, P<0.05); 48 h Transwell results showed that the number of cells migrated in the si-PIK3R3 transfection group [(91.0±3.5) cells] was significantly lower than that in the si-NC group (t=25.390, P<0.05). Conclusion si-PIK3R3 can inhibit the proliferation and migration of human gallbladder cancer cells, suggesting that PIK3R3 plays an important role in the development of gallbladder carcinoma. Key words: Gallbladder cancer; Small interfering RNA; Phosphatidylinositol 3-kinase regulatory subunit gamma

Modulation on gallbladder carcinoma by TGF-β1 via IGFBP-2.

Gallbladder carcinoma (GC) occupies more than 90% of all cancers in biliary tract with an increasing incidence. Most patients with GC are already at terminal stage at the time of primary diagnosis, causing unfavorable prognosis and high mortality. Transformation growth factor-beta (TGF-β) is up-regulated in GC. However, the mechanism by how TGF-β is involved in GC remains unclear. The aim of this study was to investigate the effect and mechanism of TGF-β in GC using GC cell line NOZ cells.In vitro cultured NOZ cell was randomly assigned into control, si-NC and TGF-β1 siRNA groups and were transfected with siRNA negative control (NC) or TGF-β1 siRNA followed by analysis of TGF-β1 expression by Real-time PCR, cell proliferation by MTT assay, cell apoptosis and cell invasion, as well as expression of proteins in epithelial-mesenchymal transition (EMT), p38, Smad2/3 and Smad4 phosphorylation by Western blot, Insulin-like growth factor-binding protein-2 (IGFBP-2) level by ELISA. After transfecting TGF-β1 siRNA into NOZ cells, TGF-β1 expression was suppressed and cell proliferation and invasion were inhibited, together with enhanced Caspase-3 activity. Meanwhile, E-cadherin expression was increased, with decreased Vimentin, IGFBP-2, p38, Smad2/3 and Smad4 phosphorylation (P< 0.05 comparing to control group). In conclusion, inhibition of TGF-β1 expression facilitates GC cell apoptosis, inhibits GC cell proliferation, invasion and EMT occurrence.

Hesperidin Induces ROS-Mediated Apoptosis along with Cell Cycle Arrest at G2/M Phase in Human Gall Bladder Carcinoma

A natural predominant flavonoid hesperidin rich in citrus fruits exhibits multifunctional medicinal properties. The anticancerous potential of hesperidin has been widely explored; however, the gall bladder carcinoma (GBC) still remains untouched due to the unavailability of efficient experimental model. The aim of our study was to identify the apoptotic and antiproliferative potential of hesperidin in GBC. The promising efficacy of hesperidin was assessed through the generation of reactive oxygen species (ROS), cellular apoptosis, and loss of mitochondrial membrane potential (MMP) in the primary cells generated from surgically removed cancerous gall bladder tissues. Moreover, cell cycle analysis and caspases-3 activity were performed to confirm the apoptosis inducing potential of hesperidin. Results revealed that hesperidin exposure for 24 h at a dose of 200 µM reduced the cell proliferation of GBC cells significantly. In addition, hesperidin treatment further resulted in an increased ROS generation and nuclear condensation at the same dose. Caspase-3 activation and cell cycle arrest at G2/M phase were also accelerated in a dose-dependent manner. Together, these results suggest that hesperidin can be considered as a potential anticancerous compound for the treatment of GBC. Furthermore, evaluation of the pharmacological aspects of hesperidin is desirable for drug development.