shRNA Control Group Research Articles

TXNIP knockdown protects rats against bupivacaine-induced spinal neurotoxicity via the inhibition of oxidative stress and apoptosis

Bupivacaine (BUP) is an anesthetic commonly used in clinical practice that when used for spinal anesthesia, might exert neurotoxic effects. Thioredoxin-interacting protein (TXNIP) is a member of the α-arrestin protein superfamily that binds covalently to thioredoxin (TRX) to inhibit its function, leading to increased oxidative stress and activation of apoptosis. The role of TXNIP in BUP-induced oxidative stress and apoptosis remains to be elucidated. In this context, the present study aimed to explore the effects of TXNIP knockdown on BUP-induced oxidative stress and apoptosis in the spinal cord of rats and in PC12 cells through the transfection of adeno-associated virus-TXNIP short hairpin RNA (AAV-TXNIP shRNA) and siRNA-TXNIP, respectively. In vivo, a rat model of spinal neurotoxicity was established by intrathecally injecting rats with BUP. The BUP + TXNIP shRNA and the BUP + Control shRNA groups of rats were injected with an AAV carrying the TXNIP shRNA and the Control shRNA, respectively, into the subarachnoid space four weeks prior to BUP treatment. The Basso, Beattie & Bresnahan (BBB) locomotor rating score, % MPE of TFL, H&E staining, and Nissl staining analyses were conducted. In vitro, 0.8 mM BUP was determined by CCK-8 assay to establish a cytotoxicity model in PC12 cells. Transfection with siRNA-TXNIP was carried out to suppress TXNIP expression prior to exposing PC12 cells to BUP. The results revealed that BUP effectively induced neurological behavioral dysfunction and neuronal damage and death in the spinal cord of the rats. Similarly, BUP triggered cytotoxicity and apoptosis in PC12 cells. In addition, treated with BUP both in vitro and in vivo exhibited upregulated TXNIP expression and increased oxidative stress and apoptosis. Interestingly, TXNIP knockdown in the spinal cord of rats through transfection of AAV-TXNIP shRNA exerted a protective effect against BUP-induced spinal neurotoxicity by ameliorating behavioral and histological outcomes and promoting the survival of spinal cord neurons. Similarly, transfection with siRNA-TXNIP mitigated BUP-induced cytotoxicity in PC12 cells. In addition, TXNIP knockdown mitigated the upregulation of ROS, MDA, Bax, and cleaved caspase-3 and restored the downregulation of GSH, SOD, CAT, GPX4, and Bcl2 induced upon BUP exposure. These findings suggested that TXNIP knockdown protected against BUP-induced spinal neurotoxicity by suppressing oxidative stress and apoptosis. In summary, TXNIP could be a central signaling hub that positively regulates oxidative stress and apoptosis during neuronal damage, which renders TXNIP a promising target for treatment strategies against BUP-induced spinal neurotoxicity.

Shugoshin as a key mediator in the metastasis of triple negative breast cancer.

e13098 Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by the lack of estrogen, progesterone, or human epidermal growth factor 2 (HER2) receptor expression. TNBC has poor prognosis due to its high metastatic potential and limited treatment options. Shugoshin-1 (Sgo1), a centromeric protein that maintains stability in chromatids during mitosis, has been thought to play an important role in the progression of TNBC. Previous publications from our group have shown that the transient downregulation of Sgo1 in TNBC cells decrease the epithelial-to-mesenchymal transition (EMT), which is a precursor of metastasis. Our purpose with this study is to evaluate the role of long-term effects of Sgo1 downregulation in cell invasion, cell migration, tumor growth, and metastasis. Methods: To achieve our goal, MDA-MB-231 cells were stably transfected with a lentivirus containing a shRNA Sgo1 sequence or a scramble shRNA control. Cell invasion and migration were evaluated by Boyden chamber assays. Meanwhile, tumor growth and metastasis were evaluated using an immunocompromised orthotopic mice model. Female NOD-SCID Gamma (NSG) mice were injected either with 2.5 X 106 shRNA Control (n= 10) or shRNA Sgo1 (n= 10) MDA-MB-231 cells. Tumor growth was measured for eight weeks using a vernier caliper. Afterwards, primary, and metastatic tumors as well as primary organs were harvested. Lung and liver samples were embedded in paraffin and stained with hematoxylin and eosin. A Student t test was used to evaluate the differences between groups. A p-value < 0.05 was considered significant. Results: Our results show a significant reduction in cell invasion and migration in MDA-MB-231 cells treated with shRNA Sgo1. Moreover, we observed a significant reduction in tumor growth and metastasis to lymph nodes, liver, and lungs in the mice treated with shRNA Sgo1 when compared to shRNA Control group. Conclusions: These results suggest that Sgo1 is an important regulator in TNBC tumor growth and metastasis through the modulation of EMT genes. Thus, targeting Sgo1 early in the course of TNBC can be a potential therapeutic treatment of TNBC.

Prosaposin, tumor‐secreted protein, promotes pancreatic cancer progression by decreasing tumor‐infiltrating lymphocytes

Glycoproteins produced by tumor cells are involved in cancer progression, metastasis, and the immune response, and serve as possible therapeutic targets. Considering the dismal outcomes of pancreatic ductal adenocarcinoma (PDAC) due to its unique tumor microenvironment, which is characterized by low antitumor T‐cell infiltration, we hypothesized that tumor‐derived glycoproteins may serve as regulating the tumor microenvironment. We used glycoproteomics with tandem mass tag labeling to investigate the culture media of three human PDAC cell lines, and attempted to identify the key secreted proteins from PDAC cells. Among the identified glycoproteins, prosaposin (PSAP) was investigated for its functional contribution to PDAC progression. PSAP is highly expressed in various PDAC cell lines; however, knockdown of intrinsic PSAP expression did not affect the proliferation and migration capacities. Based on the immunohistochemistry of resected human PDAC tissues, high PSAP expression was associated with poor prognosis in patients with PDAC. Notably, tumors with high PSAP expression showed significantly lower CD8+ T‐cell infiltration than those with low PSAP expression. Furthermore, PSAP stimulation decreased the proportion of CD8+ T cells in peripheral blood monocytes. Finally, in an orthotopic transplantation model, the number of CD8+ T cells in the PSAP shRNA groups was significantly increased, resulting in a decreased tumor volume compared with that in the control shRNA group. PSAP suppresses CD8+ T‐cell infiltration, leading to the promotion of PDAC progression. However, further studies are warranted to determine whether this study contributes to the development of a novel immunomodulating therapy for PDAC.

Ceramide synthase 3 affects invasion and metastasis of hepatocellular carcinoma via the SMAD6 gene.

Patients with hepatocellular carcinoma (HCC) have poor prognosis due to lack of early diagnosis and effective treatment. Therefore, there is an urgent need to better understand the molecular mechanisms associated with HCC and to identify effective targets for early diagnosis and treatment. This study is to explore the expression and biological role of ceramide synthase 3 (CerS3) in HCC. A total of 159 pairs of HCC tissues and adjacent non-tumor tissues were obtained from the patients underwent radical resection in Shenzhen People's Hospital, and the total RNA and proteins from HCC tissues and adjacent non-tumor tissues were obtained. The expression of CerS3 protein and mRNA in HCC was detected by immunohistochemistry, Western blotting and real-time PCR. In vitro experiments, Hep3B cells were divided into a control vector group and a CerS3 vector group, and the cells were transfected with retroviral vector containing control cDNA or CerS3 cDNA, respectively. HCCLM3 cells were divided into a normal control shRNA group and a CerS3 shRNA group, and the cells were transfected with lentiviral vectors containing normal control shRNA or CerS3 shRNA, respectively. MTT, EdU, Transwell and scratch method were used to detect cell proliferation, migration and invasion. RNA sequencing was performed to determine the downstream signal of CerS3. Compared with the corresponding adjacent tissues,the mRNA and protein levels of CerS3 were elevated in the HCC tissues, with significant difference (both P<0.05). The Univariate and multivariate analysis showed that the overall survival rate was significantly correlated with the presence of venous invasion (95% CI 1.8-9.2, P<0.01), TNM stage (95% CI 2.3-5.2, P<0.05), poor histological grade (95% CI 1.4-6.8, P<0.05), and CerS3 (95% CI 1.5-3.9, P<0.05). Furthermore, the high CerS3 expression levels in tumor tissues were significantly associated with shorter overall survival rates compared with the low CerS3 expression (P<0.05). Compared with the vector control group, the Hep3B cell viability, EdU positive cells, and migration and invasion cell numbers in the CerS3 vector group were significantly increased (all P<0.05). Compared with the shRNA normal control group, the HCCLM3 cell viability, EdU positive cells, and numbers of migrating and invasive cells in the CerS3 shRNA group were significantly lower (all P<0.05). The RNA sequencing confirmed that the small mothers against decapentaplegic family member 6 (SMAD6) gene as an oncogenic gene could promote the HCC metastasis. Clinically, the overexpression of CerS3 is closely related to poor clinical features and poor prognosis. Functionally, CerS3 participates in the proliferation, invasion and metastasis of liver cancer cells via activating SMAD6 gene.

Shugoshin‐1 Depletion Reduces Tumor Growth in Triple‐Negative Breast Cancer

Breast cancer is the most common and deadliest cancer that affects females around the world. Triple‐negative breast cancer (TNBC) is a subtype of cancer characterized by the lack of progesterone, estrogen, and Her2 receptors expression. TNBC have the worst prognosis because of its elevated growth rate, limited options for treatment, rapid development of chemoresistance, and its high metastatic potential. Recent studies have correlated Shugoshin‐1 (SGO1) with epithelial‐to‐mesenchymal transition and tumor growth in cancer; however, none of the studies published so far were done in breast cancer. Therefore, we aimed to identify the role of SGO1 in tumor growth of TNBC. We hypothesize that tumor growth will be reduced in shSGO1, given its biological role in other cancers. To test our hypothesis, 2.5x106 MDA‐MB‐231 cell containing either shRNA Control or shRNA SGO1 were injected into female NSG mice that were 6‐8 weeks old. Tumors were allowed to growth for an 8‐weeks period. Following this timepoint, mice were sacrificed to harvest tumors (primary and secondary). Protein lysates were isolated from tumor samples and SGO1 depletion was characterized by Western blot. Tumors were measured, embedded in paraffin, sectioned, and stained with Hematoxylin and Eosin. A student t‐ test was used to evaluate statistical significance. Our results show that the average tumor size for shRNA Control group (n=10) was significantly higher than for shRNA SGO1 group (n=10) at any time point once the tumors were palpable. At the end‐point measurement the average tumor size for shRNA Control group was 848 mm3, while for shRNA SGO1 was 50 mm3. These results suggest that silencing SGO1 reduces tumor growth. Therefore, SGO1 may be a potential line of treatment for TNBC.

Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood\u2013brain barrier injury through the Wnt/\u03b2-catenin signalling pathway

BackgroundDisruption of the blood–brain barrier (BBB) after a stroke can lead to brain injury and neurological impairment. Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2 (LMP2) in the pathophysiology of ischemia stroke. However, the relationship between the immunoproteasome LMP2 and the BBB remains unclear.MethodsAdult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). Three days before MCAO, the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region. The rat brain microvascular endothelial cell (RBMVEC) line was exposed to oxygen–glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions in vitro. The RNA interference-mediated knockdown of LMP2 or β-catenin was analysed in vivo and in vitro. Analysis of the quantity of extravasated Evans blue (EB) and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB. Immunofluorescence and Western blotting were employed to detect the expression of target proteins. Cell migration was evaluated using a scratch migration assay. The results of immunofluorescence, Western blotting and cell migration were quantified using the software ImageJ (Version 1.53m). Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference (LSD) test.ResultsCerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins (occludin, claudin-1 and ZO-1) in the MCAO/R group compared with the sham group (P < 0.001). However, inhibition of the immunoproteasome LMP2 restored the expression of these proteins, resulting in higher levels of occludin, claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group (P < 0.001). In addition, inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability [e.g., the quantity of extravasated EB: LMP2-shRNA group (58.54 ± 7.37) µg/g vs. control-shRNA group (103.74 ± 4.32) µg/g, P < 0.001], and promoted the upregulation of Wnt-3a and β-catenin proteins in rats following MCAO/R. In vitro experiments, OGD/R induced marked upregulation of LMP2, proapoptotic protein Bax and cleaved caspase-3, and downregulation of occludin, claudin-1, ZO-1 and Bcl-2, as well as inhibition of the Wnt/β-catenin pathway Wnt-3a and β-catenin proteins in RBMVECs, compared with the control group under normal culture conditions (P < 0.001). However, silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R. Silencing of β-catenin by transfection of RBMVECs with β-catenin-siRNA aggravated the downregulation of tight junction proteins, and reduced the proliferation and migration of RBMVECs following OGD/R, compared with the control-siRNA group (P < 0.001). LMP2-siRNA and β-catenin-siRNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.ConclusionThis study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced BBB injury, and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.

NLRP3 Blockade Suppresses Pro-Inflammatory and Pro-Angiogenic Cytokine Secretion in Diabetic Retinopathy.

BackgroundInflammation and angiogenesis are the two dominant mechanisms of diabetic retinopathy (DR), which act more as mutual pathways rather than individual processes. However, the underlying mechanism of their interactions is still unclear. Here, we explored the potential crossing points between these pathways and the targeted therapeutic method in rats with DR.Materials and MethodsSprague–Dawley rats were randomly assigned to four groups: normal control group, streptozocin (STZ)-induced diabetes mellitus (DM) group, DM+shNC (non-specific negative control shRNA) group, and DM+shNLRP3 group. Silencing the NLR family pyrin domain containing 3 (NLRP3) protein was performed by intravitreal injections of NLRP3-targeted shRNA (shNLRP3) for rats in the DM+shNLRP3 group. All the rats’ retinas were collected for further morphological examination and pro-inflammatory and pro-angiogenic cytokine detection. Human retinal endothelial cells (HRECs) were also employed to explore the underlying mechanism.ResultsNLRP3-targeted shRNA given by intravitreal injection effectively alleviated the retinal histopathological changes in STZ-induced diabetic rats, which reduced the activation of the NLRP3 inflammasome and suppressed the expressions of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and inflammatory cytokines in diabetic rats’ retinas. In HRECs, NLRP3 over-expressing plasmid evoked an increase in pro-inflammatory cytokines and VEGF. In addition, YC-1, a HIF-1α inhibitor, could reverse the NLRP3 over-expression-induced VEGF production but not the pro-inflammatory cytokine expressions.ConclusionOur results suggest NLRP3 inflammasome as the potential cross-point between inflammation and pro-angiogenesis in DR and support the effectiveness of NLRP3-targeted shRNA administrated by intravitreal injection in animal models of DR. The protective effect of NLRP3-targeted shRNA may stem from the inhibition of both pro-inflammatory cytokines and HIF-1α/VEGF axis.

Long non-coding RNA CRNDE deteriorates intrauterine infection-induced neonatal brain injury

BackgroundThis study aimed to test the hypothesis that long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) could exacerbate brain injury caused by intrauterine infection in neonatal rats. MethodsIntrauterine infection was induced in pregnant rats by lipopolysaccharide (LPS). After delivery, newborn rats with brain injury caused by intrauterine infection were randomly divided into control, control shRNA, and CRNDE shRNA groups. CRNDE expression in serum and amniotic fluid of pregnant rats and neonatal brain tissues were determined by quantitative real-time PCR (qRT-PCR). Morris water maze (MWM) task was used to test the spatial learning and memory ability. Histological examination and apoptosis detection were performed by hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, respectively. Immunohistochemistry was conducted to evaluate the activation of astrocytes and microglia. ResultsLncRNA CRNDE was highly expressed in serum and amniotic fluid of maternal rats and in brain tissues of offspring rats. Furthermore, shRNA-mediated CRNDE downregulation could rescue the spatial learning and memory ability, improve brain histopathological changes and cell death, and inhibit the activation of astrocytes and microglia caused by LPS. ConclusionCRNDE silencing possessed a cerebral protective effect in neonatal rats with brain injury caused by interauterine infection.

Large tumor suppressor gene 2-mediated Hippo signaling pathway regulates the biological behavior of mesenchymal stem cells in vitro

To investigate the regulatory effect of Hippo signaling pathway mediated by large tumor suppressor gene 2 (LATS2) on biological behavior of mice bone marrow mesenchymal stem cells (BMSCs) in vitro. BMSCs of C57BL/6 mice were cultured in vitro and passed to the 6th to 7th generations for experiment. BMSCs with activated and inactivated LATS2 were constructed with lentiviral vectors transfections. The BMSCs were allocated to blank control group (MSC group), empty vector control group (MSC-eGFP group), LATS2-overexpressing group (MSC-LATS2 group), empty vector without LATS2 shRNA control group (MSC-shcontrol group) and LATS2-underexpressing group (MSC-shLATS2 group). The transduction efficiencies mediated by the lentiviral vectors were evaluated by flow cytometry. The mRNA and protein expressions of LATS2, phosphorylation of Yes associated protein (p-YAP) and 14-3-3 were quantified by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot respectively. Osteogenic and adipogenic differentiation of BMSCs were evaluated through alizarin red and oil red O staining. Proliferation of BMSCs was evaluated using the CCK-8 assay. The effect of Hippo pathway on horizontal and vertical migration ability of BMSCs was measured by the scratch test and Transwell chamber test. The transduction efficiencies mediated by the lentiviral vectors were 93.1%-97.1%. Compared with MSC-eGFP group, the expressions of LATS2, p-YAP and 14-3-3 in MSC-LATS2 group were significantly elevated [LATS2 mRNA (2-ΔΔCT): 2.55±0.13 vs. 1.08±0.05, LATS2/GAPDH: 2.63±0.11 vs. 1.06±0.08, p-YAP/total YAP: 1.67±0.11 vs. 1.00±0.04, 14-3-3/β-actin: 2.22±0.20 vs. 0.98±0.06, all P < 0.05], however, compared with MSC-shcontrol group, the expressions in MSC-shLATS2 group were significantly reduced [LATS2 mRNA (2-ΔΔCT): 0.10±0.01 vs. 1.01±0.05, LATS2/GAPDH: 0.09±0.01 vs. 1.05±0.06, p-YAP/total YAP: 0.10±0.02 vs. 1.10±0.09, 14-3-3/β-actin: 0.05±0.01 vs. 0.90±0.08, all P < 0.05]. It suggested that high and low expression of LATS2 could activate or inhibit Hippo pathway. The osteogenic and adipogenic differentiation and proliferation rate of BMSCs in MSC-LATS2 group were significantly lower than those in MSC-eGFP group, however, those in MSC-shLATS2 group were significantly higher than MSC-shontrol group (all P < 0.05). It suggested that high and low expression of LATS2 could inhibit or promote osteogenesis, adipogenesis and cell proliferation of BMSCs. Scratch test and Transwell chamber test showed that the degree of scratch healing in MSC-LATS2 group was significantly lower than that in MSC-eGFP group [(22.11±3.02)% vs. (45.99±6.58)%], while the number of cells migrating to the subventricular layer of Transwell was significantly reduced (cells/MP: 20.82±3.05 vs. 111.33±13.28, both P < 0.05); the degree of scratch healing in MSC-shLATS2 group was significantly higher than that in MSC-shcontrol group [(70.32±7.17)% vs. (39.28±2.98)%], the number of cells migrating to the subventricular layer of Transwell was increased significantly (cells/MP: 206.19±30.58 vs. 120.10±25.10, both P < 0.05). It suggested that high and low expression of LATS2 could inhibit or promote the horizontal and vertical migration of BMSCs. LATS2-mediated alteration of Hippo pathway could modulate differentiation, proliferation, and migration of mesenchymal stem cells in vitro.

PLK1 Expression in Mantle Cell Lymphoma and Its Clinical Significance

To explore the expression level of PLK1 in mantle cell lymphoma(MCL), and the effect of silencing PLK1 gene by RNA interference on the cell proliferation, apoptosis, and cell cycle. S-P immunohistochemistry technique was used to detect the expression of PLK1 in tissues of 42 patients with MCL and 30 patients with reactive proliferative lymphodenitis(RPL), their expression levels were compared and analyzed. The Jeko-1 cells were transfected with lentivirus contaiming PLK-1 shRNA, then the mRNA and protein expression of PLK-1 was detected by real-time guantitative PCR and Western blot nespectively, and the silencing efficacy of PLK-1 shRNA was identificd. The cell proliferation was detected by CCK method, the cell apoptosis was detected by Annexin V/PI double staining, the cell cycle was detected by PI single staining, the changes of apoptosis-related proteins BAX, BCL-2 and Caspase 3 were detected by Western blot. The positive expression rate of PLK-1 in tissue of MCL patients was 66.67%(28/42), which was significanfly higher than 20%(6/30) in tissue of RPL patients (P＜0.05). The PLK-1 positive expression correlated with B symptom, IPI score, Ann-Arbor stage(P＜0.05). After infection of Jeko-1 cells with lentivirus containing PLK-1 shRNA for 72 hours, the mRNA and protein expressions of PLK-1 were significantly down-regulated(P＜0.05), the proliferation rate of cells in group of PLK-1 shRNA was significanly lower than that in control and Neg shRNA groups(P＜0.05); the apoptosis rate of cells in PLK-1 shRNA group was (27.42±3.44)%, which was significantly higher than that in control group (1.23±0.42)% and Neg shRNA group (2.07±0.58) % (P＜0.05). The cell cycle analysis showed that the cell ratio in G2/M phase of PLK-1 shRNA group was (27.21±3.59) %, which was higher than that in control group (13.28±2.63)% and Neg shRNA group (14.34±2.37) %. The detection of apoptosis-related proteins showed that the expression of BAX was up-regulated, the expression of BCL-2 was down-regnlated and the expression of caspase 3 was up-regulated. The PLK-l overexpression appears in tissue of MCL patients. The silencing PLK-1 gene can inhibit the proliferation of Jeko-1 cells, induce the apopotosis of Jeko-1 cells and arrestes cell cycle in G2/M phase.

Smad4 gene silencing enhances the chemosensitivity of human lymphoma cells to adriamycin via inhibition of the activation of transforming growth factor β signaling pathway.

There are diverse investigations focused on the therapies of lymphoma. Our research was taken to identify the effects of lentiviral-mediated Smad4 gene silencing on chemosensitivity of human lymphoma cells to adriamycin (ADM) via transforming growth factor β (TGFβ) signaling pathway. Raji/ADM cells were cultured and infected with lentiviral particles Smad4-short hairpin (shRNA) and control-shRNA. Then, the messenger RNA (mRNA) and protein levels of TGFβ signaling pathway-related factors (Smad4, Smad3, cyclinE, cyclinD1, and p21) in Raji/ADM cells were determined. The effect of Smad4-shRNA on cell viability, invasion and migration, and apoptosis were also detected. Compared with the Raji group, increased mRNA and protein levels of Smad4, Smad3, cyclinE, cyclinD1, enhanced cell proliferation, migration and invasion as well as decreased mRNA, and protein levels of p21 and cell apoptosis rate were found in the Raji/ADM and control-shRNA groups. However, Smad4 gene silencing resulted in decreased mRNA and protein levels of Smad4, Smad3, cyclinE, and cyclinD1 along with inhibited cell proliferation, migration and invasion but increased expression of p21 together with cell apoptosis. Collectively, Smad4 gene silencing can inhibit the activation of TGFβ signaling pathway, thereby enhancing the chemosensitivity of human lymphoma cells to ADM.

Abstract P5-03-08: Eribulin-induced EpCAM expression provides drug resistance in triple-negative breast cancer cells

Abstract Breast cancer is the most common cancer in women in the world. Among breast cancer subtypes, triple-negative breast cancer is aggressive. Because there is no effective therapy for triple-negative breast cancer, development of a novel therapeutic strategy is required. Eribulin, a derivative of halicondrin B, is an anticancer chemical. Eribulin treatment has been approved in many countries for patients with metastatic or inoperable breast cancer, including triple-negative breast cancer. The known functions of Eribulin are inhibition of microtubule polymerization and induction of the expression of epithelial genes in breast cancer cells. However, the function of Eribulin-induced genes on the therapy for patients with triple-negative breast cancer remains unclear. EpCAM (Epithelial cell adhesion molecule) is one of epithelial markers and known as a cell-cell adhesion molecule. In addition, EpCAM expression is involved in drug resistance in cancer. We therefore focused on EpCAM expression in Eribulin-treated breast cancer cells. We used MDA-MB-231 cells that is a commonly used triple-negative breast cancer cell line. We treated cells with 10 nM Eribulin and observed inhibition of cell growth. In the same condition, we found up-regulation of EpCAM expression in both mRNA and protein levels. In mouse xenograft model with MDA-MB-231 cells, EpCAM expression was detected in whole tumor at 96 h after Eribulin administration. We analyzed the effect of other microtubule polymerization inhibitors, nocodazole and colchicine, on EpCAM induction. In the results, there was no up-regulation of EpCAM expression in cells treated with these chemicals, suggesting that EpCAM expression is not induced by inhibition of microtubule polymerization and growth inhibition. To analyze EpCAM function on Eribulin treatment, we constructed shRNA-mediated EpCAM knockdown system. We introduced EpCAM knockdown in MDA-MB-231 cells and treated with Eribulin. In the results of cell growth assays, the cell number of Eribulin-treated EpCAM-knockdown cells was significantly smaller than that of Eribulin-treated control-shRNA cells, whereas no change was observed between solvent-treated control-shRNA and EpCAM-knockdown groups. In mouse xenograft experiments, EpCAM-knockdown cells showed smaller tumor weights than control-shRNA cells after administration of Eribulin. These results indicate that EpCAM expression provides Eribulin resistance in breast cancer cells. In conclusion, we found Eribulin-induced EpCAM expression in triple-negative breast cancer cells. The EpCAM expression provides Eribulin resistance both in vitro and in vivo. These suggest that a combination of Eribulin treatment and EpCAM inhibition may improve therapy for breast cancer. Citation Format: Tanaka S, Itou J, Sato F, Toi M. Eribulin-induced EpCAM expression provides drug resistance in triple-negative breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-08.

Knock-Down of HOXB8 Prohibits Proliferation and Migration of Colorectal Cancer Cells via Wnt/β-Catenin Signaling Pathway.

BackgroundThere has been no research on the mechanism of HOXB8 action on colorectal cancer so far. This study was designed to investigate the mechanism of HOXB8 regulating colorectal cancer cell proliferation and invasion in vivo and in vitro.Material/MethodsHOXB8 shRNA, HOXB8 overexpression, and negative control vector were designed and stably transfected into HCT116 cells. MTT assays were performed to detect cell proliferation. Western blot was utilized to detect HOXB8 expression level in HCT116 stable cells. The invasive and migration abilities were detected by Transwell assay and wound-healing assay.ResultsHOXB8 knockdown inhibited cell proliferation. The invasiveness of HCT116 cells was significantly reduced following HOXB8 depletion compared with that in the shRNA control group, whereby the rates were reduced by 67% in HOXB8 knockdown group. The wound-healing rate of HOXB8 over-expression cells was significantly increased comparing with that of cells in the blank control group (P<0.05). HOXB8 knockdown promotes apoptosis of HCT116 cells. The expression of E-cadherin was restrained in the HOXB8 over-expression group and increased in the HOXB8 knockdown group.ConclusionsKnock-down of HOXB8 prohibits the proliferation and migration of colorectal cancer cells via the Wnt/β-catenin signaling pathway and the downregulation of various factors, such as MMP2, c-Myc, CyclinD1,and vimentin. Our data suggested that HOXB8 has great potential to be developed as a novel therapeutic agent for the treatment of human colorectal cancer.

Metformin reverses the resistance mechanism of lung adenocarcinoma cells that knocks down the Nrf2 gene.

The nuclear factor, erythroid 2 like 2 (Nrf2)/antioxidant response element (ARE) pathway has an important role in the drug resistance of adenocarcinoma, and may act via different mechanisms, including the mitogen-activated protein kinase (MAPK) pathway. However, it has remained elusive whether metformin affects Nrf2 and regulates Nrf2/ARE in adenocarcinoma. In the present study, reverse-transcription quantitative polymerase chain reaction, cell transfection, western blot analysis, a Cell Counting kit-8 assay and apoptosis detection were used to investigate the above in the A549 cell line and cisplatin-resistant A549 cells (A549/DDP). The results indicated that Nrf2, glutathione S-transferase α 1 (GSTA1) and ATP-binding cassette subfamily C member 1 (ABCC1) were dose-dependently reduced by metformin, and that the effect in A549 cells was greater than that in A549/DDP cells. Treatment with metformin decreased the proliferation and increased the apoptosis of A549 cells to a greater extent than that of A549/DDP cells, and the effect was dose-dependent. After transfection of A549/DDP cells with Nrf2 short hairpin RNA (shRNA), GSTA1 and ABCC1 were markedly decreased, compared with the shRNA-control group of A549/DDP, and low dose-metformin reduced the proliferation and increased apoptosis of A549/DDP cells. Metformin inhibited the Akt and extracellular signal-regulated kinase (ERK)1/2 pathways in A549 cells and activated the p38 MAPK and c-Jun N-terminal kinase (JNK) pathways. Furthermore, in the presence of metformin, inhibitors of the p38 MAPK and JNK signaling pathway at different concentrations did not affect the levels of Nrf2, but inhibitors of the Akt and ERK1/2 pathway at different doses reduced the expression of Nrf2. In addition, inhibitors of p38 MAPK and JNK did not affect the effect of metformin on Nrf2, while inhibitors of Akt and ERK1/2 dose-dependently enhanced the inhibitory effects of metformin in A549 cells. In conclusion, metformin inhibits the phosphoinositide-3 kinase/Akt and ERK1/2 signaling pathways in A549 cells to reduce the expression of Nrf2, GSTA1 and ABCC1. Metformin also reverses the resistance of A549/DDP cells to platinum drugs, inhibits the proliferation and promotes apoptosis of drug-resistant cells. These results may provide a theoretical basis and therapeutic targets for the clinical treatment of tumors.

Knockdown of metadherin inhibits cell proliferation and migration in colorectal cancer.

Metadherin (MTDH) is a multifunctional oncogene involved in tumor cell migration and metastasis through regulating a number of oncogenic signaling pathways in various human malignancies. Previous studies have demonstrated that MTDH is overexpressed in human colorectal cancer (CRC) and associated with cancer progression and a poor prognosis. However, the underlying mechanisms remain largely unknown. The present study investigated the expression and role of MTDH in CRC cells as well as the underlying mechanism of this. Western blot analysis and quantitative polymerase chain reaction were conducted to determine protein and mRNA expression of MTDH in three human CRC cell lines. A short hairpin RNA (shRNA) targeting MTDH was introduced into CRC HCT116 cells to stably inhibit MTDH expression. A Cell Counting Kit‑8 assay, colony formation assay, Transwell assay and flow cytometry were used to investigate the effect of MTDH‑knockdown on cell proliferation, migration, apoptosis and cell cycle arrest. Western blotting was performed to examine the protein expression levels of cell growth‑ and apoptosis‑associated genes. The results demonstrated that MTDH was commonly expressed in CRC cell lines. MTDH silencing significantly suppressed cell growth, colony forming ability and migration while inducing the apoptosis of HCT116 cells. In addition, MTDH depletion induced Sphase cell cycle arrest in HCT116 cells. Mechanistically, knockdown of MTDH markedly downregulated the expression of phosphorylated protein kinase B, c‑Myc, proliferating cell nuclear antigen and B‑cell lymphoma2 (Bcl‑2) protein in HCT116 cells, and the expression of p53 and Bcl‑2‑associated X protein was significantly increased compared with the negative control shRNA group (P<0.05), suggesting that MTDH may function through the expression of numerous types of apoptosis‑associated and signaling channel proteins in CRC cells. Taken together, these data indicated that MTDH may serve as a biomarker and candidate therapeutic target for CRC.

Effect of PAK1 gene silencing on proliferation and apoptosis in hepatocellular carcinoma cell lines MHCC97-H and HepG2 and cells in xenograft tumor.

This study intends to explore the effect of the PAK1 gene silencing on apoptosis and proliferation of hepatocellular carcinoma (HCC) MHCC97-H and HepG2 cells and cells in xenograft tumor. MHCC97-H and HepG2 cells and mice with xenograft tumor in vivo were randomly divided into control, empty vector and PAK1 shRNA groups. Morphology and the expression of green fluorescent protein of MHCC97-H and HepG2 cells and cells in xenograft tumor were observed. MTT assay and flow cytometry were used to detect proliferation, cell cycle and apoptosis of MHCC97-H and HepG2 cells and cells in xenograft tumor. The expressions of PAK1, PCNA, Ki67, Cyclin E, CDK2, p21, p53, Bax and Bcl-2 were measured using the quantitative reverse transcription polymerase chain reaction and western blotting. Compared with the control and empty vector groups, number of adherent cells of MHCC97-H and HepG2 cells and cells in xenograft tumor was reduced, and green fluorescent cells became round and reduced in the PAK1 shRNA group. Cell proliferation, the cells at S phase, the mRNA and protein expressions of PAK1, PCNA, Ki67, Cyclin E, CDK2 and Bcl-2 of MHCC97-H and HepG2 cells and cells in xenograft tumor were decreased, while the cells at G1 phase, apoptosis rate, the mRNA and protein expressions of p21, p53 and Bax of MHCC97-H and HepG2 cells and cells in xenograft tumor were increased in the PAK1 shRNA group. PAK1 gene silencing decreases proliferation of MHCC97-H cells, HepG2 cells and cells in xenograft tumor through the p53/p21 pathway.

Cyclin-dependent kinase 7 ( CDK7 ) expression in human hepatocellular carcinoma: association with HCC progression, prognosis and cell proliferative capacity

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world and contributes to a high cancer mortality globally. The multistep progression of HCC is highly related to the activation of oncogenes and the deactivation of cancer suppressor genes. The cyclin-dependent kinase 7 ( CDK7 ) gene is responsible for maintaining a normal cell cycle, acting as a gatekeeper, and plays an important part in RNA transcription. Although previous studies demonstrate that CDK7 is highly expressed in multiple cancers and is associated with the progression and prognosis of these cancers, the potential role of CDK7 in HCC still needs to be explored. Methods: Data consisting of CDK7 expression levels, patient phenotypes and their matched survival were acquired from The Cancer Genome Atlas (TCGA) database. The associations of the CDK7 expression level with clinicopathological factors and cell proliferation were analyzed. The HCC transcriptome data in the GEO database were inquired and analyzed using GEO2R. Two representative HCC cell models were built to observe the proliferation capacity of HCC cells when CDK7 expression was inhibited by either sh CDK7 or THZ1. Results: Based on the transcriptome and survival data in the TCGA database, the CDK7 expression level was inversely proportional to the overall survival of the HCC patients (pooled HR =1.51, 95% CI =1.06–2.15). For further investigation, the clinical features of HCC were integrated with the expression level of CDK7 . A high expression of CDK7 was proportional to the survival time of the HCC patients (P=2.38×10 −3 ), the neoplasm histologic grade (P −4 ) and cell proliferation (P −3 ). Moreover, CDK7 expression in the liver tumor was higher than that in non-tumor tissues (t-test =6.04, P=2.27×10 −8 ). For HCC patients, CDK7 expression was higher in the tumor tissues than in the para-carcinoma tissues, based on five GEO datasets (Z score =−6.20, P=5.64×10 −10 ). In the SMMC-7721 and Huh7 cell lines, CDK7 shRNA-transfected cells showed significantly lower capabilities of cell growth and proliferation than those of the shRNA control group after 48 hours of cell culture. Furthermore, the cell proliferation capacity was also inhibited when the SMMC-7721 cells (P=6.47×10 −2 ) or Huh7 cells (P=2.52×10 −2 ) were exposed to THZ1. Conclusions: The CDK7 expression level in HCC is associated with HCC progression, prognosis and the cell proliferation capacity. CDK7 may act as a potential target for HCC, and THZ1 might be a potent medicine for HCC treatment.

Stathmin inhibits proliferation and differentiation of dental pulp stem cells via sonic hedgehog/Gli.

The mineralization of dental pulp stem cells is an important factor in the tissue engineering of teeth, but the mechanism is not yet obvious. This study aimed to identify the effect of Stathmin on the proliferation and osteogenic/odontoblastic differentiation of human dental pulp stem cells (hDPSCs) and to explore whether the Shh signalling pathway was involved in this regulation. First, Stathmin was expressed in the cytoplasm and on the cell membranes of hDPSCs by cell immunofluorescence. Then, by constructing a lentiviral vector, the expression of Stathmin in hDPSCs was inhibited. Treatment with Stathmin shRNA (shRNA‐Stathmin group) inhibited the ability of hDPSCs to proliferate, as demonstrated by a CCK8 assay and flow cytometry analysis, and suppressed the osteogenic/odontoblastic differentiation ability, as demonstrated by alizarin red S staining and osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) activity, compared to that of hDPSCs from the control shRNA group. Molecular analyses showed that the Shh/GLI1 signalling pathway was inhibited when Stathmin was silenced, and purmorphamine, the Shh signalling pathway activator, was added to hDPSCs in the shRNA‐Stathmin group, real‐time PCR and Western blotting confirmed that expression of Shh and its downstream signalling molecules PTCH1, SMO and GLI1 increased significantly. After activating the Shh signalling pathway, the proliferation of hDPSCs increased markedly, as demonstrated by a CCK8 assay and flow cytometry analysis; osteogenic/odontoblastic differentiation‐related gene (ALP, BSP, OCN, DSPP) expression also increased significantly. Collectively, these findings firstly revealed that Stathmin‐Shh/GLI1 signalling pathway plays a positive role in hDPSC proliferation and osteogenic/odontoblastic differentiation.

Effect of OCT4A Gene on the Biological Characteristics of K562 Cells

To evaluate biological effects of OCT4A gene on K562 cells and explore the molecular mechanism of K562 cell apoptosis. Two recombinant lentiviral vectors were constructed, which could stablely up- regulate and down- regulate OCT4A protein. Recombinant lentivirus was generated by co-transfection of three-plasmids and transfec-ted into K562 cells. The experiments were divided into 5 groups: normal, pLVX-OCT4A-ZsGreen1, pLVX vector control, PLB-OCT4A shRNA and non-specific shRNA groups. Western blot was applied to detect the expression of OCT4A protein, the cell counting kit-8 was applied to evaluate the effect of OCT4A on proliferation of K562 cells. The apoptosis and differentiation of K562 cells were detected by flow cytometry with AnnexinV/7-AAD double staining. The mRNA expressions of caspase-3,BIM,BCL-xL,BAX in K562 cells were determined by real time PCR. The OCT4A fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR), the 2 lentiviral vectors were successfully constructed. In comparson with those in the control group, the expression of OCT4A protein of pLVX-OCT4A-ZsGreen1 group was significantly increased, but decreased in PLB-OCT4A shRNA group. CCK-8 assay showed that the higher the content of OCT4A protein, the faster the cell proliferation. The apoptosis rate was (3.48±0.52)% of pLVX-OCT4A-ZsGreen1 group, which was lower than that of control group, while the apoptosis rate PLB-OCT4A shRNA group was (7.25±0.57)%, which was higher than that of control group (P<0.05), however, the K562 cells differentiation was not influenced(P>0.05). Compared with control group, the gene expression of Caspase-3,BIM and BAX was down-regulated(P>0.05), but a significant up-regulation of BCL-xL gene expression was observed(P<0.05). Two lentiviral vectors have been successfully constructed, which can stably up- and down- regulate the expression of OCT4A in K562 cells respectively. OCT4A can promote the K562 cell proliferation and inhibit the apoptosis, the mechanism may be related with up-regulation of BCL-xl expression.

Construction of cochlear progenitor cells with recombinant short-hairpin RNA lentiviral vector inhibiting the expression of the β-catenin gene

Objective: To construct a recombinant short-hairpin RNA (shRNA) lentiviral vector targeting the β-catenin gene in cochlear precursor cells (CPCs) in mice, and to investigate its inhibitory effect. Methods: PCR was used for the multiplication of the β-catenin gene, and shRNA oligo was designed based on the β-catenin gene to construct an interference vector. Gateway Technology was used to construct shRNA lentiviral vector which carried the β-catenin gene, and then 293FT cells were transfected with the constructed lentiviral vector and helper plasmids pLV/helper-SL3, pLV/helper-SL4, and pLV/helper-SL5. The virus supernatant was collected to obtain viral particles, and then mouse CPCs were transiently infected with the recombinant lentivirus with four different concentrations (0, 5, 10, and 20 μl) . The shRNA control group was established. Quantitative real-time PCR and Western blot were used to investigate the inhibitory effect of shRNA β-catenin lentiviral vector on β-catenin. Results: The recombinant shRNA β-catenin lentiviral vector was successfully constructed, and the virus titers of shβ-catenin and shβ-catenin-control were 5.05×10(7) and 4.34×10(7), respectively. The results of in vitro experiments showed that in CPCs transfected with four different concentrations of recombinant lentivirus, the content of β-catenin protein gradually decreased with the increase in concentration, and there was a significant difference between groups (P<0.05) ; the CPCs transfected with shβ-catenin had significantly lower mRNA expression of β-catenin than those in the shβ-catenin-control group (P<0.05) . Conclusion: The constructed lentiviral vector targeting the β-catenin gene has a high infection efficiency, and the successful construction of lentiviral vectors provides a technical support for analyzing the role of β-catenin in the differentiation of CPCs into auditory hair cells.