shRNA Technology Research Articles

A Novel Human TBCK- Neuronal Cell Model Results in Severe Neurodegeneration and Partial Rescue with Mitochondrial Fission Inhibition.

TBCK syndrome is a rare fatal pediatric neurodegenerative disease caused by biallelic loss-of-function mutations in the TBCK gene. Previous studies by our lab and others have implicated mTOR, autophagy, lysosomes, and intracellular mRNA transport, however the exact primary pathologic mechanism is unknown. This gap has prevented the development of targeted therapies. We employed a human neural progenitor cell line (NPC), ReNcell VM, which can differentiate into neurons and astrocytes, to understand the role of TBCK in mTORC1 activity and neuronal autophagy and cellular mechanisms of pathology. We used shRNA technology to knockdown TBCK in ReNcells. These data showed that loss of TBCK did not inhibit mTORC1 activity in neither NPC nor neurons. Additionally, analysis of eight patient-derived cells and TBCK knock down HeLa cells showed that mTORC1 inhibition is inconsistent across different patients and cell types. We showed that TBCK knockdown in ReNcells affected NPC differentiation to neurons and astrocytes. Specifically, differentiation defects are coupled to cell cycle defects in NPC and increased cell death during differentiation. RNAseq analysis indicated the downregulation of several different neurodevelopmental and differentiation pathways. We observed a higher number of LC3-positive vesicles in the soma and neurites of TBCK knockdown cells. Further, TBCK knockdown altered mitochondrial dynamics and membrane potential in NPC, neurons and astrocytes. We found partial mitochondrial rescue with the mitochondrial fission inhibitor mdivi-1. This work outlines a new Human Cell Model for TBCK-related neurodegeneration and the essential role of mitochondrial health and partial rescue with mitochondrial fission inhibitor. This data, along with human neurons and astrocytes, illuminate mechanisms of neurodegeneration and provide a possible novel therapeutic avenue for affected patients.

Inhibition of ASAP1 Modulates the Tumor Immune Microenvironment and Suppresses Lung Cancer Metastasis via the p-STAT3 Signaling Pathway.

ADP ribosylation factor guanylate kinase 1 (ASAP1), a key protein regulating cell migration and invasion, has attracted extensive attention in oncological research in recent years. This study aims to explore the effects of ASAP1 inhibition on lung cancer metastasis and its potential mechanisms, particularly how it modulates the tumor immune microenvironment through the p-STAT3 signaling pathway. In this study, shRNA technology was employed to specifically inhibit ASAP1 expression in lung cancer cell lines A549, NCI-H1299, and PC-9. The effects of ASAP1 inhibition on lung cancer cell viability, apoptosis, migration, and invasion were evaluated using CCK-8, TUNEL apoptosis detection, and cell migration and invasion assays. Furthermore, animal experiments were conducted to assess the in vivo effects of ASAP1 inhibition on lung cancer metastasis, and immunohistochemical analysis was performed to investigate changes in immune cells in lung metastasis models, further exploring its impact on the tumor immune microenvironment. The experimental results demonstrated that ASAP1 inhibition significantly reduced lung cancer cell viability, induced apoptosis in A549, NCI-H1299, and PC-9 cells, and suppressed the migration and invasion abilities of these cells. In vivo experiments revealed that ASAP1 inhibition effectively suppressed lung cancer metastasis and altered the tumor immune microenvironment by regulating immune cells. Moreover, we found that ASAP1 inhibition could decrease tumor cell proliferation and induce tumor apoptosis in lung metastasis models by inhibiting the p-STAT3 signaling pathway. This study confirms that ASAP1 inhibition can suppress lung cancer metastasis by modulating the tumor immune microenvironment through the inhibition of the p-STAT3 signaling pathway. These findings provide new targets for lung cancer treatment and a theoretical basis for developing novel strategies against lung cancer metastasis. Future research will further explore the mechanisms of ASAP1 in lung cancer metastasis and how to optimize treatment strategies for lung cancer patients by targeting ASAP1.

Abstract LB192: AGR2 knockdown reduces cell proliferation and increases endoplasmic reticulum proteotoxic stress in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal solid malignancy with low survival rates due to a dense stroma, abnormal gene expression patterns, and limited treatment options. Anterior gradient 2 (AGR2) is often overexpressed in many cancers, including PDAC. AGR2 belongs to the protein disulfide isomerase family of proteins and is located in the endoplasmic reticulum, where it helps with protein-disulfide isomerase reactions to ensure proper protein folding. However, the molecular mechanisms underpinning AGR2 overexpression in PDAC pathophysiology are not well understood. Gene expression data obtained from the pancreatic adenocarcinoma (PAAD) dataset shows that AGR2 is overexpressed in PDAC tumors compared to normal tissues. We confirmed this gene over-expression in PDAC tumors and multiple pancreatic cancer cell lines using fluorescent microscopy, RT-qPCR, and western blotting. Using stable inducible short hairpin (shRNA) technology, we showed that AGR2 knockdown significantly decreases the viability and colony-forming ability of HPAF-II and PANC-1 cells. Gene Ontology (GO) enrichment analysis revealed that AGR2 knockdown suppresses a host of protein biosynthesis pathways, including protein targeting and localization to the endoplasmic reticulum (ER). Significantly, ER stress was induced in knockdown cells, shown by the activation of unfolded protein response, suggesting an accumulation of unfolded proteins. To determine the effects of unfolded protein accumulation on the ER, we performed transmission electron microscopy. Here, the results show an increase in the ER lumen size of knockdown cells compared to controls. Interestingly, we observed a decrease in mitochondria size and confirmed the increased expression of mitochondria fission proteins DRP1 and MFF. Finally, flow cytometry analysis revealed increased apoptosis in AGR2 knockdown cells compared to controls. Overall, our data provide insights into the role of AGR2 in PDAC tumorigenesis. However, more research is warranted to determine if AGR2 could be a potential therapeutic target. Citation Format: Philip Salu, Katie Reindl. AGR2 knockdown reduces cell proliferation and increases endoplasmic reticulum proteotoxic stress in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB192.

PitViper: a software for comparative meta-analysis and annotation of functional screening data.

Recent advancements in shRNA and Cas protein technologies have enabled functional screening methods targeting genes or non-coding regions using single or pooled shRNA and sgRNA. CRISPR-based systems have also been developed for modulating DNA accessibility, resulting in CRISPR-mediated interference (CRISPRi) or activation (CRISPRa) of targeted genes or genomic DNA elements. However, there is still a lack of software tools for integrating diverse array of functional genomics screening outputs that could offer a cohesive framework for comprehensive data integration. Here, we developed PitViper, a flexible and interactive open-source software designed to fill this gap, providing reliable results for the type of elements being screened. It is an end-to-end automated and reproducible bioinformatics pipeline integrating gold-standard methods for functional screening analysis. Our sensitivity analyses demonstrate that PitViper is a useful tool for identifying potential super-enhancer liabilities in a leukemia cell line through genome-wide CRISPRi-based screening. It offers a robust, flexible, and interactive solution for integrating data analysis and reanalysis from functional screening methods, making it a valuable resource for researchers in the field.

A knock down strategy for rapid, generic, and versatile modelling of muscular dystrophies in 3D-tissue-engineered-skeletal muscle

BackgroundHuman iPSC-derived 3D-tissue-engineered-skeletal muscles (3D-TESMs) offer advanced technology for disease modelling. However, due to the inherent genetic heterogeneity among human individuals, it is often difficult to distinguish disease-related readouts from random variability. The generation of genetically matched isogenic controls using gene editing can reduce variability, but the generation of isogenic hiPSC-derived 3D-TESMs can take up to 6 months, thereby reducing throughput.MethodsHere, by combining 3D-TESM and shRNA technologies, we developed a disease modelling strategy to induce distinct genetic deficiencies in a single hiPSC-derived myogenic progenitor cell line within 1 week.ResultsAs proof of principle, we recapitulated disease-associated pathology of Duchenne muscular dystrophy and limb-girdle muscular dystrophy type 2A caused by loss of function of DMD and CAPN3, respectively. shRNA-mediated knock down of DMD or CAPN3 induced a loss of contractile function, disruption of tissue architecture, and disease-specific proteomes. Pathology in DMD-deficient 3D-TESMs was partially rescued by a candidate gene therapy treatment using micro-dystrophin, with similar efficacy compared to animal models.ConclusionsThese results show that isogenic shRNA-based humanized 3D-TESM models provide a fast, cheap, and efficient tool to model muscular dystrophies and are useful for the preclinical evaluation of novel therapies.

CXCR2/Snail-1-Induced Epithelial-Mesenchymal Transition in the Formation and Progression of RCC with Inferior Vena Cava Tumour Thrombus.

Renal cell carcinoma (RCC), a common and highly invasive malignant tumour, presents clinical challenges due to its propensity for easy metastasis. Inferior vena cava tumour thrombus is a common RCC complication significantly impacting patient prognosis. This study investigates C-X-C chemokine receptor type 2 (CXCR2)/Snail-1-induced epithelial-mesenchymal transition (EMT) in RCC with inferior vena cava tumour thrombus. Tissues from 51 RCC patients were analysed for CXCR2 and Snail-1 Messenger Ribonucleic Acid (mRNA) levels using Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Elevated levels of both were observed in tumour and inferior vena cava tumour thrombus tissues. Using Short Hairpin RNA (shRNA) technology, we inhibited CXCR2 and Snail-1 expression to investigate their impact on EMT, invasiveness, and metastatic potential in RCC cells. Compared with that in the Short Hairpin RNA-Negative Control (ShNC) group, inhibition of CXCR2 and Snail-1 suppressed the degree of EMT, invasiveness, and metastatic ability of RCC cells (p < 0.01). Further mechanistic studies showed that CXCR2/Snail-1 participated in the formation and progression of RCC by regulating the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathways. Additionally, compared with that in the ShNC group, knockdown of CXCR2 and Snail-1 significantly inhibited the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9; p < 0.01), thereby regulating the metastasis of RCC. Our findings suggest that CXCR2/Snail-1-induced EMT plays an important role in the formation and progression of RCC with inferior vena cava tumour thrombus. CXCR2/Snail-1 participates in the invasion and metastasis of RCC by regulating the expression of multiple signalling pathways and related genes. These results provide new insights and directions for the treatment of RCC.

LRP11 promotes stem-like T cells via MAPK13-mediated TCF1 phosphorylation, enhancing anti-PD1 immunotherapy

BackgroundTumor-infiltrating T cells enter an exhausted or dysfunctional state, which limits antitumor immunity. Among exhausted T cells, a subset of cells with features of progenitor or stem-like cells has been...

TRIM28 recruits E2F1 to regulate CBX8-mediated cell proliferation and tumor metastasis of ovarian cancer.

Chromobox protein homolog 8 (CBX8) is a transcriptional suppressor participated in various cancers. However, the function and mechanism of CBX8 in the progression of ovarian cancer (OC) are unclear. In this study, we found that CBX8 was upregulated in OC tissues originating from GEPIA and TNM databases, OC patients' samples from hospital, and OC cell lines. Furthermore, CBX8 knockdown by short hairpin RNA (shRNA) technology markedly inhibited proliferation and invasion, induced migration, cell cycle arrest, and apoptosis in vitro. Mechanistically, CBX8 activated PI3K/AKT/mTOR signaling pathway to take effect. In addition, TRIM28 and E2F1 were enriched in OC tissues from the TNM database and OC patients' samples similar to the results of CBX8. Correlation analysis indicated positive correlations among TRIM28, E2F1, and CBX8. E2F1 was proved to bind to the promoter regions of CBX8 and TRIM28, while TRIM28 recruited E2F1 to increase the expression of CBX8 to further increase cell viability, proliferation, and invasion, and decrease migration, apoptosis, and cell cycle progression. Finally, CBX8 or TRIM28 knockdown repressed tumor growth and metastasis of OC in vivo. Therefore, our study showed that the promoting effect of CBX8 on tumor growth and metastasis of OC was participated in the PI3K/AKT/mTOR signaling, TRIM28 and E2F1. Our findings suggested that CBX8 could serve as a potential marker and therapeutic target for OC patients.

Deletion of osteopontin in non-small cell lung cancer cells affects bone metabolism by regulating miR-34c/Notch1 axis: a clue to bone metastasis.

Lung cancer is prone to bone metastasis, and osteopontin (OPN) has an important significance in maintaining bone homeostasis. The goal of this study was to explore the impact of OPN level on bone metabolism and the molecular mechanism of inhibiting bone metastasis in non-small cell lung cancer (NSCLC). The expression of OPN in NSCLC was ascertained by Western blot and immunohistochemistry, and the correlation between the expression level of OPN and survival of patients was analyzed. Then the shRNA technology was applied to reduce the expression of OPN in NSCLC cells, and CCK-8 assay was carried out to investigate the effect of low expression of OPN on the proliferation of NSCLC cells. In addition, the effects of low expression of OPN on osteoclast differentiation, osteoblast generation and mineralization were studied using osteoclast precursor RAW264.7 and human osteoblast SaOS-2 cells, and whether OPN could regulate miR-34c/ Notch pathway to affect bone metabolism was further explored. The findings showed that the high level of OPN in NSCLC was closely related to the poor prognosis of patients and the abnormal proliferation of NSCLC cell lines. The suppression of OPN was beneficial to inhibit the differentiation of osteoclasts and promote the mineralization of osteoblasts. Besides, this study confirmed that the deletion of OPN can regulate bone metabolism through the regulation of miR-34c/Notch1 pathway, which will contribute to inhibiting the occurrence of osteolytic bone metastasis in NSCLC.

Autophagy regulates sex steroid hormone synthesis through lysosomal degradation of lipid droplets in human ovary and testis

Autophagy is an evolutionarily conserved process that aims to maintain the energy homeostasis of the cell by recycling long-lived proteins and organelles. Previous studies documented the role of autophagy in sex steroid hormone biosynthesis in different animal models and human testis. Here we demonstrate in this study that sex steroid hormones estrogen and progesterone are produced through the same autophagy-mediated mechanism in the human ovary in addition to the human testis. In brief, pharmacological inhibition and genetic interruption of autophagy through silencing of autophagy genes (Beclin1 and ATG5) via siRNA and shRNA technologies significantly reduced basal and gonadotropin-stimulated estradiol (E2), progesterone (P4) and testosterone (T) production in the ex vivo explant tissue culture of ovary and testis and primary and immortalized granulosa cells. Consistent with the findings of the previous works, we observed that lipophagy, a special form of autophagy, mediates the association of the lipid droplets (LD)s with lysosome to deliver the lipid cargo within the LDs to lysosomes for degradation in order to release free cholesterol required for steroid synthesis. Gonadotropin hormones are likely to augment the production of sex steroid hormones by upregulating the expression of autophagy genes, accelerating autophagic flux and promoting the association of LDs with autophagosome and lysosome. Moreover, we detected some aberrations at different steps of lipophagy-mediated P4 production in the luteinized GCs of women with defective ovarian luteal function. The progression of autophagy and the fusion of the LDs with lysosome are markedly defective, along with reduced P4 production in these patients. Our data, together with the findings of the previous works, may have significant clinical implications by opening a new avenue in understanding and treatment of a wide range of diseases, from reproductive disorders to sex steroid-producing neoplasms, sex steroid-dependent malignancies (breast, endometrium, prostate) and benign disorders (endometriosis).

Abstract 3938: In vivo genome-wide CRISPR screen in pancreatic ductal adenocarcinoma defines HSPE1 as a potential oncogene by acting through G2/M cell cycle arrest

Abstract Superior knowledge of cancer biology has enabled unprecedented innovations in therapies targeting mutated driver genes. Despite the attempt of targeting cancer-inducing genes such as KRAS, the life expectancy of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) has poorly improved. We performed an unbiased genome wide in-vivo loss-of-function CRISPR screen to discover novel oncogenes and identified heat-shock protein HSPE1, whose function is still unknown in PDAC. By decreasing HSPE1 expression with shRNA and CRISPR technology, we detected a slowdown in cell growth of PDAC cancer cells. HSPE1 knock-out cells injected in mice displayed a drastic reduction in tumor volume. We exploited this vulnerability by disrupting HSPE1 function by using KHS101, a validated HSPD1-HSPE1 complex inhibitor. Several PDAC cell lines cultured in vitro were sensitive upon KHS101 treatment and in vivo administration of KHS101 reduced tumorigenesis. Compiling the negative fitness scores from DepMap database revealed a co-dependency between HSPE1 and PLK1, a protein involved in regulating G2/M checkpoint. Indeed, we detected a protein-level decrease of PLK1 in our experimental model of PDAC cells having either a drug-induced HSPE1 deficiency by KHS101 exposure or a HSPE1 gene-expression reduction by shRNA. We found that the cell cycle was disrupted through G2/M arrest in PDAC cells treated with KHS101. Our findings highlight a new role underlying PDAC tumorigenesis for HSPE1 and could unlock a new area of research towards precision medicine. Citation Format: Julien Boudreault, Ni Wang, Gang Yan, Meiou Dai, Sophie Poulet, Girija Daliah, Jean-Jacques Lebrun. In vivo genome-wide CRISPR screen in pancreatic ductal adenocarcinoma defines HSPE1 as a potential oncogene by acting through G2/M cell cycle arrest. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3938.

5613327 KNOCK-DOWN OF AUTOPHAGY-RELATED GENE 5 (ATG5) EXPRESSION IN THALASSEMIC CD34+ CELLS LEADS TO AMELIORATION OF ERYTHROPOIESIS

Background: β-thalassemia is characterized by ineffective erythropoiesis due to increased apoptosis of the thalassemic erythroid precursors. Recent studies documented enhanced initiation of autophagy in β-thalassemia erythroblasts, which may contribute to the elevated levels of apoptosis.1,2 Therefore, genetic manipulation of key genes of autophagy employing shRNA technology, could improve the pathophysiology of the disease. Aim: To this end, our study aimed to investigate the role of ATG5 in autophagy and/or apoptosis in β-thalassemia, by downregulating its expression via shRNA technology, and potentially exploit this effect for the amelioration of the disease pathophysiology. Materials and Methods: Lentiviral vectors (LVs), harboring three distinct shRNAs (sh11840, sh11841 and sh11842) targeting discrete regions of endogenous ATG5 mRNA, were used to transduce K562 cells at a multiplicity of infection (MOI) 25. A lentiviral vector carrying a shRNA against no human gene (shNT; No Target), was used as a control. Flow cytometry and confocal microscopy using an ATG5-specific antibody were employed to determine ATG5 expression at the protein level, while qPCR assessed ATG5 knock-down at the mRNA level. The sh11842 LV exhibited the highest ATG5 knock-down and was selected for in vitro assessment in thalassemic cells, following initial transduction of CD34+ cells at MOI 25, for a differentiation time period to include the early autophagy activation at basophilic stage. Autophagic flux in untransduced, shNT-transduced and shATG5-transduced CD34+ cells, was assessed using confocal microscopy to determine the level of the LC3/LAMP co-localization, markers of autophagosomes and lysosomes, while flow cytometry was employed to determine erythroid differentiation and apoptosis in the differentiated cells. Results: Transduction of K562 cells with the sh11842 LV led to the highest ATG5 knock-down, reaching 93% and 38% at mRNA and protein level, respectively (n=4). Transduction of thalassemic CD34+ cells with the sh11842 LV at day 6 of differentiation showed advanced erythropoiesis compared to shNT-transduced cells. Specifically, the percentage of basophilic erythroblasts was significantly reduced following sh11842-transduction as compared to shNT-transduction, (43.5 ± 4.5% vs 59.6 ± 8.1%, p=0.0127, n=4, respectively), leading to a transient increase of 28.9 ± 5.9% in polychromatic erythroblasts, compared to 16.3 ± 3.7% (p=0.0118, n=4) in shNT-transduced cells. As a result of this accelerated erythropoiesis, the percentage of orthochromatic erythroblasts increased to 5.5 ± 2.4%, compared to 2.8 ± 1.7% observed in shNT-transduced cells (p=0.199, n=4). This was followed by a significant reduction of LC3/LAMP co-localization in sh11842-transduced cells, reaching a Pearson’s correlation coefficient of 0.103 ± 0.29%, compared to 0.165 ± 0.4% seen in shNT-transduced and 0.372 in untransduced cells (p<0.0001). Furthermore, no differences in apoptosis were observed between sh11842- and shNT- transduced cells (59.0 ± 18.2% vs 50.3 ± 8.6%, p=0.6618 n=3). Pilot studies indicate that transduction with sh11842 LV may lead to increased mitochondrial membrane potential, as demonstrated by increased Mitotracker® staining, suggesting a potential reduction in mitophagy, as well. Summary - Conclusion: Knock-down of ATG5 expression leads to improvement of the thalassemic erythropoiesis in vitro, highlighting its potential synergistic role in the amelioration of thalassemic pathophysiology.

Abstract P3-05-24: Correlation between the expression of JCAD in plasma exosomes and breast cancer

Abstract Background: Breast cancer is the most common cancer in women. The five-year survival rate of patients with stage 0 and stage I breast cancer is more than 95%, that of patients with stage II breast cancer is 80%, that of patients with stage III breast cancer is about 50%, and that of stage IV breast cancer is less than 30%. The stage of breast cancer is closely related to the prognosis. Exosomes are membrane vesicles secreted by living cells, which carry donor cell specific genetic information. They can exist stably in the circulatory system, so they are ideal biomarkers. Method: We collected 128 breast cancer patients (17 benign cases, 26 stage I cases, 36 stage II cases, 15 stage III cases and 34 stage IV cases) treated in our center from 2018 to 2019. We collected patients’ plasma and extracted exosomes, then we sent them for sequencing. We analyzed the difference between benign cases’ and stage IV cases’ sequencing results, and then we screen them by comprehensively considering the change trend of gene expression from benign cases to stage IV cases, the survival curve of patients and the lymph node metastasis. The clinicopathological factors and progression free survival (PFS) of breast cancer patients were analyzed by Cox regression. Finally, JCAD was selected. Using virus to stably overexpress JCAD and shRNA technology to knock down JCAD, the function of JCAD for the proliferation and metastasis of breast cancer was discussed through CCK8 cell proliferation experiment, EdU cell proliferation experiment, plate cloning experiment, transwell cell migration experiment. T-test and chi square test were performed on the data. P &amp;lt; 0.05 showed that the difference was statistically significant. Kaplan-Meier survival was used to analyze the long-term efficacy. Result 1. The expression of JCAD mRNA in exosomes increased from benign to stage 4 (P &amp;lt; 0.001). In addition, patients with lymph node metastasis had significantly higher expression of exosome JCAD (p=0.009), and the prognosis of patients with high expression of exosome JCAD was poor (p=0.003). This conclusion has also been verified in TCGA database. Finally, Cox regression analysis showed that JCAD was an independent prognostic factor affecting PFS (p=0.005). 2. JCAD is transmitted between cells in the form of exosomes: In breast cancer cells, stable transgenic strains with overexpression of JCAD and shRNA knockdown strains of JCAD were constructed. The exosomes were extracted by ultracentrifugation for RNA and protein level verification. It shows that there was a difference in the expression of JCAD in the exosomes of overexpression and knockdown strains. After adding exosome inhibitor GW4869 to culture cells, it was found that the level of JCAD mRNA secreted by exosomes decreased significantly. In addition, through PKH26 exosome fluorescence labeling method, it was found that JCAD was transmitted between cells in the form of exosomes. 3. JCAD can promote the proliferation and migration of breast cancer cells: This experiment verified the ability of JCAD to promote the proliferation of breast cancer cells through CCK8 cell proliferation experiment, EdU cell proliferation experiment, plate cloning experiment. Then the ability of JCAD to promote the migration of breast cancer cells was verified by transwell cell migration experiment. The exosomes were added to wild type cells for co-culture. It was further found that the exosomes with high expression of JCAD could promote the proliferation and migration of breast cancer cells. Conclusion JCAD is significantly overexpressed in stage IV patients and patients with lymph node metastasis. Moreover, JCAD is an independent prognostic factor affecting PFS, and the exosome JCAD is expected to become a potential prognostic liquid biopsy marker. Further cell experiments showed that JCAD can be transmitted between breast cancer cells through exosomes and promote the proliferation and metastasis of breast cancer cells. Citation Format: Douwaner Liu, Yayun Chi, Bingqiu Xiu, Qi Zhang, Wei-Ru Chi, Liyi Zhang, Min Xiong, Jiong Wu. Correlation between the expression of JCAD in plasma exosomes and breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-24.

Schwann cells-derived exosomes promote functional recovery after spinal cord injury by promoting angiogenesis.

Exosomes are small vesicles that contain diverse miRNA, mRNA, and proteins that are secreted by multiple cells, and play a vital function in cell-cell communication. Numerous exosomes produced by cells have been demonstrated to be protective against spinal cord injury (SCI). This study aims to investigate the neuroprotective effect of Schwann cells-derived exosomes (SCs-Exos) on spinal cord injury. We found that SCs-Exos can be taken directly by brain-derived endothelial cells.3 (bEnd.3 cells) and promoted to proliferate, migrate, and form bEnd.3 tube. Additionally, our results showed that the pro-angiogenesis molecules, Integrin-β1, were highly expressed in SCs-Exos. Moreover, we used special shRNA technology to investigate the role of Integrin-β1 in mediating the effect of SCs-Exos-induced angiogenesis on bEnd.3 cells. We observed that the pro-angiogenic effect of SCs-Exos on bEnd.3 cells was suppressed by inhibiting the expression of integrin-β1 in SCs-Exos. In the SCI model, we found that SCs-Exos attenuated tissue damage and improved functional recovery after SCI. Using immunofluorescence staining, we observed that SCs-Exos treatment promoted angiogenesis in SCI, and integrin-β1 was required to promote angiogenesis. In conclusion, our results indicate that SCs-Exos promote angiogenesis by delivering integrin-β1 and may serve as a promising novel therapeutic agent for enhancing neurological functional recovery after SCI.

APEX1 promotes the oncogenicity of hepatocellular carcinoma via regulation of MAP2K6.

Objective: Apurinic/apyrimidinic endonuclease 1 (APEX1), a key enzyme responsible for DNA base excision repair, has been linked to development and progression of cancers. In this work, we aimed to explore the role of APEX1 in hepatocellular carcinoma (HCC) and elucidate its molecular mechanism.Methods: The expression of APEX1 in HCC tissues and matched adjacent normal tissues (n = 80 cases) was evaluated by immunohistochemistry. Web-based tools UALCAN and the Kaplan-Meier plotter were used to analyze the Cancer Genome Atlas database to compare expression of APEX1 mRNA to 5-year overall survival. APEX1 was stably silenced in two HCC cell lines, Hep 3B and Bel-7402, with shRNA technology. An in vivo tumorigenesis model was established by subcutaneously injecting sh-APEX1-transfected Bel-7402 cells into mice, and tumor growth was determined. We performed high-throughput transcriptome sequencing in sh-APEX1-treated HCC cells to identify the key KEGG signaling pathways induced by silencing of APEX1.Results: APEX1 was significantly upregulated and predicted poor clinical overall survival in HCC patients. Silencing APEX1 inhibited the proliferation of HCC cells in vivo and in vitro, and it repressed invasion and migration and increased apoptosis and the percentage of cells in G1. Differentially expressed genes upon APEX1 silencing included genes involved in TNF signaling. A positive correlation between the expression of APEX1 and MAP2K6 was noted, and overexpressing MAP2K6 overcame cancer-related phenotypes associated with APEX1 silencing.Conclusion: APEX1 enhances the malignant properties of HCC via MAP2K6. APEX1 may represent a valuable prognostic biomarker and therapeutic target in HCC.

Polycystin-2 mediates mechanical tension-induced osteogenic differentiation of human adipose-derived stem cells by activating transcriptional co-activator with PDZ-binding motif.

Human adipose-derived stem cells (hASCs) have multi-directional differentiation potential including osteogenic differentiation. Mechanical stimulation is thought to be a key regulator of bone remodeling and has been proved to promote osteogenic differentiation of mesenchymal stem cells. However, the mechanism how mechanical tension-induced osteogenesis of hASCs still remains poor understood. Polycystin-2 (PC2), a member of the transient receptor potential polycystic (TRPP) family, is involved in cilia-mediated mechanical transduction. To understand the role of PC2 in osteogenic differentiation under mechanical stimuli in hASCs, PKD2 gene was stably silenced by using lentivirus-mediated shRNA technology. The results showed that mechanical tension sufficiently enhanced osteogenic differentiation but hardly affected proliferation of hASCs. Silencing PKD2 gene caused hASCs to lose the ability of sensing mechanical stimuli and subsequently promoting osteogenesis. PC2 knock-out also reduced the cilia population frequency and cilia length in hASCs. TAZ (transcriptional coactivator with PDZ-binding motif, also known as Wwtr1) could mediate the genes regulation and biological functions of mechanotransduction signal pathway. Here, mechanical tension also enhanced TAZ nuclear translocation of hASCs. PC2 knock-out blocked tension-induced upregulation of nuclear TAZ and suppress tension-induced osteogenesis. TAZ could directly interact with Runx2, and inhibiting TAZ could suppress tension-induced upregulation of Runx2 expression. In summary, our findings demonstrated that PC2 mediate mechanical tension-induced osteogenic differentiation of hASCs by activating TAZ.

Effect of CDCA5 on Proliferation and Metastasis of Triple Negative Breast Cancer Cells under shRNA Interference Technology.

Objective It was to explore the effect of cell division cycle associated 5 (CDCA5) under shRNA interference on proliferation and metastasis of triple negative breast cancer (TNBC) cells. Methods MDA-ME-231 and BT549 cells were selected as the research objects. According to the different interference methods and CDCA5 interference sequences, they were divided into the interference group 1MDA-ME-231, the interference group 2MDA-ME-231, the interference group 1BT549, the interference group 2BT549 (using shRNA technology), the control group MDA-ME-231, and the control group BT549 (breast cancer cells under normal culture conditions). MCF10A cells were routinely cultured as the negative control group to analyze the effect of CDCA5 expression on the proliferation and migration of cancer cells. Results The expression of CDCA5 protein in MDA-ME-231 and BT549 cells in control group was significantly higher than that in negative control group (P < 0.05). Compared with the control group, the inhibition rates of CDCA5 expression in 1MDA-ME-231, 2MDA-ME-231, 1BT549, and 2BT549 cells in the interference group were 39.01%, 42.98%, 49.57%, and 60.98%, respectively (P < 0.05). From 12 h, the proliferation level of TNBC cells at different culture time was lower than that of the control group (P < 0.05). Compared with the number of staining cells in the control group, the positive staining cells in 1MDA-ME-231 (61.42%), 2MDA-ME-231 (72.06%), 1BT549 (52.53%), and 2BT549 (59.65%) in the interference group were significantly decreased (P < 0.05). Conclusion The results show that the expression of CDCA5 in TNBC is increased, which plays an important role in the proliferation and migration of cancer cells. shRNA interference technology can knock down the expression of CDCA5 and inhibit its “promoting cancer” effect.

ARHGAP29 Regulates Keratinocyte Migration through the RhoA/ROCK Pathway

Genetic variants in Rho GTPase Activating Protein 29 (ARHGAP29) have been associated with non‐syndromic cleft lip and/or palate, one of the most common human birth defects. Orofacial clefting results from the failure of facial processes to fuse during development which can be caused by defects in proliferation, adhesion, and/or migration. ARHGAP29 is widely expressed and preferentially interacts with the small GTPase RhoA, a critical modulator of the actin cytoskeleton required for cell morphology and migration. Therefore, we hypothesized that ARHGAP29 would play a critical role in keratinocyte migration. To test this hypothesis, we knocked down ARHGAP29 in a human keratinocyte cell line using both shRNA and CRISPR technologies. To assess migration, we used an in vitro scratch assay on a confluent keratinocyte monolayer and followed scratch closure over 24 hours. ARHGAP29 knockdown keratinocytes displayed significantly increased cell area and significant delays in scratch closure. By tracing single cells throughout the duration of scratch closure, we found that ARHGAP29 knockdown cells were significantly slower and showed defects in directionality that were rescued with a ROCK inhibitor, Y‐27632, suggesting these migration defects are downstream of the RhoA pathway. Collectively, our results demonstrate that ARHGAP29 regulates keratinocyte morphology and promotes migration speed and directionality through the RhoA/ROCK pathway, providing potential insight into its importance in craniofacial development.

Mechanism of the inhibitory effects of thalidomide on expressions of VEGF and bFGF

Objective: To investigate the inhibitory effects of thalidomide on the expressions of VEGF and bFGF in human lung adenocarcinoma A549 cells and human hepatocellular carcinomas HepG2 cells mediated by cereblon (CRBN). Methods: shRNA technology was used to construct the A549 cell line (A549CRBN) and HepG2 cell line (HepG2CRBN) with stable knockdown of CRBN, which was verified by real-time PCR and Western blot. A549 cells were divided into negative control group (A549luciferase) and CRBN down-regulation group (A549CRBN); HepG2 cells were divided into negative control group (HepG2luciferase) and CRBN down-regulation group (HepG2CRBN). The above cells were seeded into 6-well plates at 3×105 cells/well, and cultured in a 37℃, 5% CO2 incubator for 24 h. Then, 1 ml medium containing 100 μmol/L thalidomide (thalidomide group) and 1 ml medium containing 1‰ DMSO (control group) were added respectively, and the culture was continued for 24 hours before subsequent experiments. Each group was designed with three replicate wells. The effect of thalidomide on the activity of A549 cell line was detected by MTS assay. Real-time PCR was performed to detect mRNA expression levels of VEGF, bFGF and c-jun. ELISA assay was performed to detect protein expressions of VEGF and bFGF. Results: Compared with the control group, thalidomide at the concentrations of 1, 10, 50 and 100 μmol/L had no significant effects on the proliferation of A549 and HepG2 cells (P＞0.05). VEGF and bFGF levels in the A549CRBN or HepG2luciferase groups were significantly lower than those in the A549CRBN or HepG2CRBN groups (P＜0.05). Compared with the control group of the A549luciferase or HepG2luciferase, thalidomide inhibited the expressions of VEGF and bFGF in A549luciferase and HepG2luciferase cells (P＜ 0.05), but did not inhibit the expressions of VEGF and bFGF in A549CRBN and HepG2CRBN cells. Compared with the control group of the HepG2luciferase, thalidomide inhibited c-Jun expression in HepG2luciferase cells (P＜0.01), but did not significantly inhibit c-Jun expression in HepG2CRBN cells. Conclusion: The inhibitory effects of thalidomide on VEGF and bFGF expressions may be mediated by CRBN in A549 and HepG2 cells, and c-Jun may be one of the key transcription factors responsible for this inhibition.

CircRNA_0008194 functions as a ceRNA to promote invasion of hepatocellular carcinoma via inhibiting miR-190a/AHNAK signaling pathway.

BackgroundHepatitis B virus infection was identified as the main risk factor of hepatocellular carcinoma (HCC) in China, which induced a high morbidity and mortality. In recent years, circRNAs were reported involving in the oncogenesis and development of multiple malignant tumors.MethodBioinformatical analysis has been employed to predict the relevant circRNA with AHNAK. The loss of function and gain of function have been used by knocking‐down circRNA through the shRNA technology while overexpressing through lentivirus infection. Dual‐luciferase reporter assay was used to detect circRNA binding to miRNA and target genes. We further used immunoprecipitation technique to detect the binding ability between non‐coding RNAs.ResultsIn this study, according to the previous report, we mainly focused on AHNAK, which has been confirmed as an oncogene involving in the metastasis of HCC. Bioinformatics analysis showed that circ_0008194 could be spliced by AHNAK. In this study, the abnormal upregulated circ_0008194 in tumor tissues was detected. The positive correlation between circ_0008194 and AHNAK was also confirmed. Through knockdown and overexpression of circ_0008194, we conducted in vitro functional studies. We found circ_0008194 could induce the invasion of cells in vitro. Mechanically, circ_0008194 presented the binding ability with miR‐190a causing the suppression of miR‐190a expression, causing the competitive inhibition of AHNAK, resulting in the promotion of EMT.ConclusionOur results suggested that circ_0008194 may act as a sponge to adsorb miR‐190a, thereby promoting the expression of AHNAK and promoting the metastasis of liver cancer tumors.