MicroRNA-190b confers radio-sensitivity through negative regulation of Bcl-2 in gastric cancer cells

Objectives

To determine the role of miR-190b in radio-sensitivity of gastric cancer (GC).

Results

In radio-resistant GC cells, down-regulation of miR-190b and up-regulation of Bcl-2 were observed. The protein expression of Bcl-2 was negatively regulated by miR-190b. Overexpression of miR-190b significantly decreased cell viability and enhanced radio-sensitivity of GC cells. Of note, these effects of miR-190b on GC cells radio-sensitivity were abolished by Bcl-2.

Conclusion

miR-190b confers radio-sensitivity of GC cells, possibly via negative regulation of Bcl-2.

     

Inhibition of integrin β3, a binding partner of kallistatin,leads to reduced viability,invasion and proliferation in NCI-H446 cells

Background

Kallistatin is a serine proteinase inhibitor and heparin-binding protein. It is considered an endogenous angiogenic inhibitor. In addition, multiple studies demonstrated that kallistatin directly inhibits cancer cell growth. However, the molecular mechanisms underlying these effects remain unclear.

Methods

Pull-down, immunoprecipitation, and immunoblotting were used for binding experiments. To elucidate the mechanisms, integrin β3 knockdown (siRNA) or blockage (antibody treatment) on the cell surface of small the cell lung cancer NCI-H446 cell line was used.

Results

Interestingly, kallistatin was capable of binding integrin β3 on the cell surface of NCI-H446 cells. Meanwhile, integrin β3 knockdown or blockage resulted in loss of antitumor activities induced by kallistatin. Furthermore, kallistatin suppressed tyrosine phosphorylation of integrin β3 and its downstream signaling pathways, including FAK/-Src, AKT and Erk/MAPK. Viability, proliferation and migration of NCI-H446 cells were inhibited by kallistatin, with Bcl-2 and Grb2 downregulation, and Bax, cleaved caspase-9 and caspase 3 upregulation.

Conclusions

These findings reveal a novel role for kallistatin in preventing small cell lung cancer growth and mobility, by direct interaction with integrin β3, leading to blockade of the related signaling pathway.

     

Three-dimensional co-culture microfluidic model and its application for research on cancer stem-like cells inducing migration of endothelial cells

Objectives

To build a three-dimensional co-culture model in a microfluidic device for cancer research and evaluate its feasibility by investigating cancer stem-like cells (SCs) induced migration of human umbilical vein endothelial cells (ECs).

Results

The microfluidic device provided two-dimensional and three-dimensional (2D/3D) culture and co-culture environments without affecting cell viability. The device also provided an effective concentration for the chemiotaxis of cells, and to support real-time monitoring of cell behavior. In this model, SCs significantly increased the migration area of ECs with a hepatocarcinoma cell line (MHCC97H; MCs). The presence of ECs also induced both MCs and SCs invasion into Matrigel. The migration area of MCs and SCs significantly increased when co-cultured with ECs.

Conclusions

This 3D co-culture microfluidic model is a suitable model in cancer research. Compared with MCs, SCs had greater potential in inducing EC migration and interacting with ECs.

     

Adipocyte-derived IL-6 and leptin promote breast Cancer metastasis via upregulation of Lysyl Hydroxylase-2 expression

Background

Adipocytes make up the major component of breast tissue, accounting for 90% of stromal tissue. Thus, the crosstalk between adipocytes and breast cancer cells may play a critical role in cancer progression. Adipocyte-breast cancer interactions have been considered important for the promotion of breast cancer metastasis. However, the specific mechanisms underlying these interactions are unclear. In this study, we investigated the mechanisms of adipocyte-mediated breast cancer metastasis.

Methods

Breast cancer cells were cocultured with mature adipocytes for migration and 3D matrix invasion assays. Next, lentivirus-mediated loss-of-function experiments were used to explore the function of lysyl hydroxylase (PLOD2) in breast cancer migration and adipocyte-dependent migration of breast cancer cells. The role of PLOD2 in breast cancer metastasis was further confirmed using orthotopic mammary fat pad xenografts in vivo. Clinical samples were used to confirm that PLOD2 expression is increased in tumor tissue and is associated with poor prognosis of breast cancer patients. Cells were treated with cytokines and pharmacological inhibitors in order to verify which adipokines were responsible for activation of PLOD2 expression and which signaling pathways were activated in vitro.

Results

Gene expression profiling and Western blotting analyses revealed that PLOD2 was upregulated in breast cancer cells following coculture with adipocytes; this process was accompanied by enhanced breast cancer cell migration and invasion. Loss-of-function studies indicated that PLOD2 knockdown suppressed cell migration and disrupted the formation of actin stress fibers in breast cancer cells and abrogated the migration induced by following coculture with adipocytes. Moreover, experiments performed in orthotopic mammary fat pad xenografts showed that PLOD2 knockdown could reduce metastasis to the lung and liver. Further, high PLOD2 expression correlated with poor prognosis of breast cancer patients. Mechanistically, adipocyte-derived interleukin-6 (IL-6) and leptin may facilitate PLOD2 upregulation in breast cancer cells and promote breast cancer metastasis in tail vein metastasis assays. Further investigation revealed that adipocyte-derived IL-6 and leptin promoted PLOD2 expression through activation of the JAK/STAT3 and PI3K/AKT signaling pathways.

Conclusions

Our study reveals that adipocyte-derived IL-6 and leptin promote PLOD2 expression by activating the JAK/STAT3 and PI3K/AKT signaling pathways, thus promoting breast cancer metastasis.

     

MiRNA-145 suppresses lung adenocarcinoma cell invasion and migration by targeting N-cadherin

Objective

To investigate the roles of miR-145 in lung adenocarcinoma (LAC) and to clarify the regulation of N-cadherin by miR-145.

Results

In 57 paired clinical LAC tissues, diminished miR-145 was significantly correlated with the lymph node metastasis and was negatively correlated with N-cadherin mRNA level expression. Wound healing and transwell assays revealed a reduced capability of tumor metastasis induced by miR-145 in LAC. miR-145 negatively regulated the invasion of cell lines through targeting N-cadherin by directly binding to its 3′-untranslated region. Silencing of N-cadherin inhibited invasion and migration of LAC cell lines similar to miR-145 overexpression.

Conclusions

MiR-145 could inhibit invasion and migration of lung adenocarcinoma cell lines by directly targeting N-cadherin.

     

An increase in integrin-linked kinase non-canonically confers NF-κB-mediated growth advantages to gastric cancer cells by activating ERK1/2

Background

Increased activity or expression of integrin-linked kinase (ILK), which regulates cell adhesion, migration, and proliferation, leads to oncogenesis. We identified the molecular basis for the regulation of ILK and its alternative role in conferring ERK1/2/NF-κB-mediated growth advantages to gastric cancer cells.

Results

Inhibiting ILK with short hairpin RNA or T315, a putative ILK inhibitor, abolished NF-κB-mediated the growth in the human gastric cancer cells AGS, SNU-1, MKN45, and GES-1. ILK stimulated Ras activity to activate the c-Raf/MEK1/2/ERK1/2/ribosomal S6 kinase/inhibitor of κBα/NF-κB signaling by facilitating the formation of the IQ motif-containing GTPase-activating protein 1 (IQGAP1)-Ras complex. Forced enzymatic ILK expression promoted cell growth by facilitating ERK1/2/NF-κB signaling. PI3K activation or decreased PTEN expression prolonged ERK1/2 activation by protecting ILK from proteasome-mediated degradation. C-terminus of heat shock cognate 70 interacting protein, an HSP90-associated E3 ubiquitin ligase, mediated ILK ubiquitination to control PI3K- and HSP90-regulated ILK stabilization and signaling. In addition to cell growth, the identified pathway promoted cell migration and reduced the sensitivity of gastric cancer cells to the anticancer agents 5-fluorouracil and cisplatin. Additionally, exogenous administration of EGF as well as overexpression of EGFR triggered ILK- and IQGAP1-regulated ERK1/2/NF-κB activation, cell growth, and migration.

Conclusion

An increase in ILK non-canonically promotes ERK1/2/NF-κB activation and leads to the growth of gastric cancer cells.

     

Circ_0027599/PHDLA1 suppresses gastric cancer progression by sponging miR-101-3p.1

Background

Pleckstrin homology-like domain family A member 1 (PHLDA1) is a tumor suppressor gene in gastric cancer, but its role regulated by circular RNAs (circRNAs) is not known. CircRNAs are important regulators in cancer growth and progression, however, the molecular roles of circRNAs in gastric cancer are rarely known. The study was aimed to investigate the role of circRNAs in regulating PHLDA1 expression in gastric cancer.

Results

The circRNA expression profile in the gastric cancer tissues by circRNA microarray showed that hsa_circ_0027599 (circ_0027599) was significantly down-regulated in gastric cancer patients and cells when comparing with the controls. Circ_0027599 overexpression suppressed gastric cancer cell proliferation and metastasis. By using bioinformatics tools and luciferase reporter assays, circ_0027599 was verified as a sponge of miR-101-3p.1 (miR-101) and suppressed cancer cell survival and metastasis. It was also verified that PHLDA1 was regulated by circ_0027599 in gastric cancer cells.

Conclusions

The study uncovered that PHLDA1 was regulated by circ_0027599/miR-101, which suppressed gastric cancer survival and metastasis in gastric cancer.

     

RNA binding protein Lin28A promotes osteocarcinoma cells progression by associating with the long noncoding RNA MALAT1

Objectives

To explore the effects of Lin28A on progression of osteocarcinoma (OS) cells.

Results

Lin28A mRNA and protein expressions were significantly increased in OS tissues compared with that in normal adjacent tissues. Expressions of Lin28A and long noncoding RNA MALAT1 were positively correlated. Patients with higher Lin28A expression had shorter overall survival. Moreover, Lin28A knockdown inhibited OS cells proliferation, migration, invasion and promoted cell apoptosis; Lin28A was found to harbor binding sites on MALAT1 sequences and associated with MALAT1, and increased MALAT1 stability and expression. Notably, the inhibition of Lin28A knockdown was attenuated or even reversed by MALAT1 overexpression.

Conclusions

RNA binding protein Lin28A could facilitate OS cells progression by associating with the long noncoding RNA MALAT1.

     

Long non-coding RNA H19 regulates E2F1 expression by competitively sponging endogenous miR-29a-3p in clear cell renal cell carcinoma

Background

Numerous recent studies indicate that the long non-coding RNAs (lncRNAs) are frequently abnormal expressed and take critical roles in many cancers. Renal cell carcinoma is the secondary malignant tumors in the urinary system and has high mortality and morbidity. Around 80% of RCCs is clear cell renal cell carcinoma (ccRCC) and is characterized by high metastasis and relapse rate. However, the clinical significances of lncRNAs in ccRCC are still unknown.

Methods

The human cancer lncRNA PCR array (Yingbio) was performed to detect the differentially expressed lncRNAs in human ccRCC samples. Real-time PCR (RT-PCR), dual-luciferase assay, RNA binding protein immunoprecipitation (RIP) assay, transwell assay, CCK-8 assay, and western blot were performed to explore the molecular mechanism of lncRNAs in ccRCC cell migration and invasion.

Results

In this study, lncRNA-H19 was high expressed and negatively correlated with miR-29a-3p in ccRCC. By bioinformatics software, dual-luciferase reporter and RIP assays, we verified that miR-29a-3p was identified as a direct target of lncRNA-H19. RT-PCR and western blot demonstrated that down-regulated lncRNA-H19 could affect the expression of miR-29a-3p targeting E2F1 with competitively binding miR-29a-3p. Furthermore, transwell assays indicated that lncRNA-H19 knockdown inhibited cells migration and invasion, but this effect was attenuated by co-transfection of lncRNA-H19 siRNA and miR-29a-3p inhibitor. Over expression of E2F1 could rescue lncRNA-H19 siRNA induced suppression on cell migration and invasion in ccRCC cells.

Conclusions

These results show a possible competing endogenous RNAs regulatory network involving lncRNA-H19 regulates E2F1 expression by competitively sponging endogenous miR-29a-3p in ccRCC. This mechanism may contribute to a better understanding of ccRCC pathogenesis, and lncRNA-H19 may be further considered as a potential therapeutic target for ccRCC intervention.

     

A novel bispecific antibody fusion protein co-targeting EGFR and CD47 with enhanced therapeutic index

Objective

To promote targeting specificity of anti-CD47 agents, we have constructed a novel bispecific antibody fusion protein against EGFR and CD47, which may minimize the “off-target” effects caused by CD47 expression on red blood cells.

Results

The novel bispecific antibody fusion protein, denoted as Bi-SP could simultaneously bind to EGFR and CD47 and exhibited potent phagocytosis-stimulation effects in vitro. Bi-SP treatment with a low dose more effectively inhibited tumor growth than either EGFR-targeting antibody, Pan or the SIRPα variant-Fc (SIRPαV-Fc) in the A431 xenograft tumor model. In addition, the treatment with Bi-SP produced less red blood cell (RBC) losses than the SIRPαV-Fc treatment, suggesting its potential use for minimizing RBC toxicity in therapy.

Conclusions

Bi-SP with improved therapeutic index has the potential to treat CD47+ and EGFR+ cancers in clinics.

     

PKM2 knockdown influences SREBP activation and lipid synthesis in bovine mammary-gland epithelial MAC-T cells

Objective

The purpose of the article is to evaluate the changes in lipid metabolism in bovine mammary-gland epithelial MAC-T cells after PKM2 knockdown.

Results

MAC-T cells stably expressing low levels of PKM2 were established with lentivirus-mediated small hairpin RNA. Although the knockdown of PKM2 had no effect on MAC-T cell growth, the reduced expression of PKM2 attenuated the mRNA and protein expression of key enzymes involved in sterol synthesis through the SREBP pathway.

Conclusions

The downregulation of PKM2 significantly influenced lipid synthesis in bovine mammary-gland epithelial MAC-T cells. These findings extend our understanding of the crosstalk between glycolysis and lipid metabolism in bovine mammary-gland epithelial cells.

     

Identification of trunk mutations in gastric carcinoma: a case study

Background

Intratumor heterogeneity (ITH) poses an urgent challenge for cancer precision medicine because it can cause drug resistance against cancer target therapy and immunotherapy. The search for trunk mutations that are present in all cancer cells is therefore critical for each patient.

Case presentation

In this study, we aimed to evaluate the efficiency of multiregional sequencing for the identification of trunk mutations present in all regions of a tumor as a case study. We applied multiregional whole-exome sequencing (WES) to investigate the genetic heterogeneity and homogeneity of a case of gastric carcinoma. Approximately 83% of common missense mutations present in two samples and approximately 89% of common missense mutations present in three samples were trunk mutations. Notably, trunk mutations appeared to have higher variant allele frequencies (VAFs) than non-trunk mutations.

Conclusions

Our results indicate that small-scale multiregional sampling and subsequent screening of low VAF somatic mutations might be a cost-effective strategy for identifying the majority of trunk mutations in gastric carcinoma.

     

Parthenolide induces apoptosis and autophagy through the suppression of PI3K/Akt signaling pathway in cervical cancer

Objective

To investigate the effect of parthenolide on apoptosis and autophagy and to study the role of the PI3K/Akt signaling pathway in cervical cancer.

Results

Parthenolide inhibits HeLa cell viability in a dose dependent-manner and was confirmed by MTT assay. Parthenolide (6 µM) induces mitochondrial-mediated apoptosis and autophagy by activation of caspase-3, upregulation of Bax, Beclin-1, ATG5, ATG3 and down-regulation of Bcl-2 and mTOR. Parthenolide also inhibits PI3K and Akt expression through activation of PTEN expression. Moreover, parthenolide induces generation of reactive oxygen species that leads to the loss of mitochondrial membrane potential.

Conclusion

Parthenolide induces apoptosis and autophagy-mediated growth inhibition in HeLa cells by suppressing the PI3K/Akt signaling pathway and mitochondrial membrane depolarization and ROS generation. Parthenolide may be a potential therapeutic agent for the treatment of cervical cancer.

     

miR-1260b promotes cell migration and invasion of hepatocellular carcinoma by targeting the regulator of G-protein signaling 22

Objectives

To investigate whether miR-1260b can regulate migration and invasion of hepatocellular carcinoma (HCC) by targeting RGS22.

Results

miR-1260b was up-regulated in HCC tissues compared with their corresponding non-cancerous tissues. Over-expression of miR-1260b increased migration and invasion of HepG2 and SMMC-7721 cells associated with HCC. Regulator of G-protein signaling 22 (RGS22) was identified as a directly target of miR-1260b and was inhibited by miR-1260b. Knockdown of RGS22 increased proliferation of HCC cells.

Conclusions

The new identified miR-1260b/RGS22 axis provides useful therapeutic methods for treatment of HCC deepening on our understanding of underlying mechanisms of HCC tumorigenesis.