A quadratic isothermal amplification fluorescent biosensor without intermediate purification for ultrasensitive detection of circulating tumor DNA.

Circulating tumor DNA (ctDNA) is an auspicious tumor biomarker released into the bloodstream by tumor cells, offering abundant information concerning cancer genes. It plays a crucial role in the early diagnosis of cancer. However, due to extremely low levels in body fluids, achieving a simple, sensitive, and highly specific detection of ctDNA remains challenging. Here, we constructed a purification-free fluorescence biosensor based on quadratic amplification of ctDNA by combining nicking enzyme mediated amplification (NEMA) and catalytic hairpin assembly (CHA) reactions. After double isothermal amplification, this biosensor achieved an impressive signal amplification of nearly 107-fold, enabling it to detect ctDNA with ultra-sensitivity. And the detection limit of this biosensor is as low as 2 aM. In addition, we explored the influence of human serum on the performance of the biosensor and found that it showed favorable sensitivity in the presence of serum. This biosensor eliminates the need for an intermediate purification step, resulting in enhanced sensitivity and convenience. Thus, our purification-free fluorescent biosensor exhibits ultra-high sensitivity when compared to other biosensors and has the potential to serve as an effective diagnostic tool for early detection of cancer.

Circular RNA circHECTD1 facilitates glioma progression by regulating the miR-296-3p/SLC10A7 axis.

Glioma is the most common type of primary brain tumor. Treatment options for recurrent gliomas include surgery, chemotherapy, and radiation therapy, but the clinical outcome is usually limited. In recent years, circular RNAs have been found to play a vital role in several human cancers. Gene Expression Omnibus database was utilized to verify the differentially expressed circRNAs. Then we detected that the expression of circular RNA circHECTD1 was significantly increased. The expression and function of circHECDT1 has not yet been reported in glioma. Then we confirmed that the level of circHECTD1 was significantly increased both in glioma tissues and cell lines, which is negatively correlated with the overall survival of patients. Knockdown of circHECTD1 inhibited proliferation and invasion in vitro, and also reduced the growth of tumor and prolonged the prognosis in vivo. Knockdown of circHECTD1 significantly elevated the miR-296-3p expression in LN229 and T98G cells. Luciferase reports and RNA immunoprecipitation data indicated that miR-296-3p was a direct target of circHECTD1 and that the miR-296-3p expression negatively regulated SLC10A7. Rescue experiments showed that the overexpression of SLC10A7 could impede the effects of circHECTD1 silencing on the proliferation and invasion of glioma cells. In this study, we identified that circHECTD1 regulates SLC10A7 by interacting with miR-296-3p in glioma cells. In conclusion, this study investigated a novel biomarker panel consisting of the circHECTD1/miR-296-3p/SLC10A7 axis, which is critical for glioma tumorigenesis and invasiveness and may represent a novel therapeutic target for intervening in glioma progression.

SENP7 inhibits glioblastoma metastasis and invasion by dissociating SUMO2/3 binding to specific target proteins.

Background: The poor surgical efficacy and recurrence of glioblastoma (GBM) are due to its lack of visible infiltrative features. Our bioinformatics study suggests that low expression of small ubiquitin-like modifier (SUMO)-specific protease 7 (SENP7) indicates poor prognosis in GBM. Objectives: This study investigated the effect of SENP7 expression on the invasion, migration, and proliferation of GBM cells and aims to identify the SUMO target proteins affected by SENP7. Methods: SENP7 expression was analyzed in eight GBM tumor samples and four GBM cell lines, comparing them to normal brain tissue. The effect of SENP7 overexpression on GBM LN229 cell migration, invasion, and proliferation was examined through in vitro assays. Furthermore, four SUMO target proteins involved in tumor invasion and proliferation (CDK6, matrix metalloproteinase-9 [MMP9], AKT, and HIF-1α) were studied to explore SENP7's molecular mechanism. Results: SENP7 expression was significantly lower in GBM tumors compared to normal tissue. SENP7 overexpression in LN229 cells inhibited migration and invasion without affecting proliferation. Overexpression reduced the levels of MMP9, AKT, and HIF-1α, but not CDK6. Immunohistochemical analysis showed decreased MMP9 and CD31 levels, suggesting reduced tumor invasion and angiogenesis. However, SENP7 overexpression did not affect tumor growth in vivo. Conclusions: SENP7 inhibits GBM invasion by dissociating proteins associated with tumor invasion from SUMO2/3, providing a potential target for future GBM therapies.

miR-596-3p suppresses brain metastasis of non-small cell lung cancer by modulating YAP1 and IL-8.

Brain metastasis (BM) frequently occurs in advanced non-small cell lung cancer (NSCLC) and is associated with poor clinical prognosis. Due to the location of metastatic lesions, the surgical resection is limited and the chemotherapy is ineffective because of the existence of the blood brain barrier (BBB). Therefore, it is essential to enhance our understanding about the underlying mechanisms associated with brain metastasis in NSCLC. In the present study, we explored the RNA-Seq data of brain metastasis cells from the GEO database, and extracted RNA collected from primary NSCLC tumors as well as paired brain metastatic lesions followed by microRNA PCR array. Meanwhile, we improved the in vivo model and constructed a cancer stem cell-derived transplantation model of brain metastasis in mice. Our data indicated that the level of miR-596-3p is high in primary NSCLC tumors, but significantly downregulated in the brain metastatic lesion. The prediction target of microRNA suggested that miR-596-3p was considered to modulate two genes essential in the brain invasion process, YAP1 and IL-8 that restrain the invasion of cancer cells and permeability of BBB, respectively. Moreover, in vivo experiments suggested that our model mimics the clinical aspect of NSCLC and improves the success ratio of brain metastasis model. The results demonstrated that miR-596-3p significantly inhibited the capacity of NSCLC cells to metastasize to the brain. Furthermore, these finding elucidated that miR-596-3p exerts a critical role in brain metastasis of NSCLC by modulating the YAP1-IL8 network, and this miRNA axis may provide a potential therapeutic strategy for brain metastasis.

MET inhibitor, capmatinib overcomes osimertinib resistance via suppression of MET/Akt/snail signaling in non-small cell lung cancer and decreased generation of cancer-associated fibroblasts.

BACKGROUND: Patients with non-small cell lung cancer (NSCLC) initially responding to tyrosine kinase inhibitors (TKIs) eventually develop resistance due to accumulating mutations in the EGFR and additional lesser investigated mechanisms such as the participation of the tumor microenvironment (TME). METHODS: Here, we examined the potential for MET inhibitor capmatinib for the treatment of osimertinib-resistant NSCLCs and normalizing the TME. RESULTS: We first established that HCC827 and H1975 cells showed increased resistance against osimertinib when co-cultured with CAFs isolated from osimertinib-resistant patients. Additionally, we showed that CAFs promoted epithelial-mesenchymal transition (EMT) and self-renewal ability in both HCC827 and H1975 cells. We subsequently found that both CAF-cultured HCC827 and H1975 showed a significantly higher expression of MET, Akt, Snail and IL-1ß, which were associated with survival and inflammatory responses. These cells in turn, promoted the generation of CAFs from normal lung fibroblasts. Subsequently, we observed that the treatment of capmatinib resulted in the re-sensitization of CAF-co-cultured H1975 and HCC827 to osimertinib, in association with reduced EMT and self-renewal ability. MET-silencing experiment using siRNA supported the observations made with capmatinib while with a greater magnitude. MET-silenced cell exhibited a severely hindered expression of inflammatory markers, IL-1ß and NF-κB; EMT markers, Snail and Vimentin, while increased E-cadherin. Finally, we demonstrated that the combination of capmatinib and osimertinib led to an increased tumor inhibition and significantly lower number of CAFs within the patient derived xenograft (PDX) model. CONCLUSION: Taken together, our findings suggested that an increased MET/Akt/Snail signaling was induced between the NSCLC cells and their TME (CAFs), resulting in osimertinib resistance. Suppression of this pathway by capmatinib may bypass the EGFR activating mutation and overcomes osimertinib resistance by targeting both tumor cells and CAFs.

HOXB9 enhances the ability of lung cancer cells to penetrate the blood-brain barrier.

Even after multimodal therapy, the prognosis is dismal for patients with brain metastases from non-small cell lung cancer (NSCLC). Although the blood-brain barrier (BBB) limits tumor cell penetration into the brain parenchyma, some nevertheless colonize brain tissue through mechanisms that are not fully clear. Here we show that homeobox B9 (HOXB9), which is commonly overexpressed in NSCLC, promotes epithelial-to-mesenchymal transition (EMT) and tumor migration and invasion. Animal experiments showed that HOXB9 expression correlates positively with the brain metastatic potential of human NSCLC cells, while brain metastatic cells derived through in vivo selection showed greater HOXB9 expression than their cells of origin. Comparable results were obtained after immunohistochemical analysis of clinical primary NSCLC and matched brain metastasis samples obtained after surgery. Using an in vitro BBB model, knockdown and overexpression experiments showed that HOXB9-dependent expression of MMP9 in NSCLC cells leads to reduced expression of junctional proteins in cultured human vascular endothelial cells and enhanced transmigration of tumor cells. These data indicate that HOXB9 enables NSCLC cells to break away from the primary tumor by inducing EMT, and promotes brain metastasis by driving MMP9 production and degradation of intercellular adhesion proteins in endothelial cells comprising the BBB.

Modulating lncRNA SNHG15/CDK6/miR-627 circuit by palbociclib, overcomes temozolomide resistance and reduces M2-polarization of glioma associated microglia in glioblastoma multiforme.

BACKGROUND: Accumulating evidence demonstrates the oncogenic roles of lncRNA (long non-coding RNA) molecules in a wide variety of cancer types including glioma. Equally important, However, tumorigenic functions of lncRNA in glioma remain largely unclear. A recent study suggested lncRNA SNHG15 played a role for regulating angiogenesis in glioma but its role in the tumor microenvironment (TME) was not investigated. METHODS: First, we showed that SNHG15 was upregulated in GBM cells and associated with a poor prognosis for the patients of GBM using public databases. Next, we collected temozolomide sensitive (TMZ-S) and resistant (TMZ-R) clinical samples and demonstrated that co-culturing TMZ-R cells with HMC3 (microglial) cells promoted M2-polarization of HMC3 and the secretion of pro-GBM cytokines TGF-ß and IL-6. RESULTS: Comparative qPCR analysis of TMZ-S and TMZ-R cells showed that a significantly higher level of SNHG15, coincidental with a higher level of Sox2, ß-catenin, EGFR, and CDK6 in TMZ-R cells. Subsequently, using bioinformatics tool, a potential mechanistic route for SNHG15 to promote GBM tumorigenesis was by inhibiting tumor suppressor, miR-627-5p which leads to activation of CDK6. Gene-silencing technique was employed to demonstrate that suppression of SNHG15 indeed led to the suppression of GBM tumorigenesis, accompanied by an increase miR-627-5p and decreased its two oncogenic targets, CDK6 and SOX-2. In addition, SNHG15-silenced TMZ-R cells became significantly sensitive towards TMZ treatment and less capable of promoting M2-phenotype in the HMC3 microglial cells. We then evaluated the potential anti-GBM activity of CDK6 inhibitor, palbociclib, using TMZ-R PDX mouse models. Palbociclib treatment significantly reduced tumorigenesis in TMZ-R/HMC3 bearing mice and SNHG15 and CDK6 expression was significantly reduced while miR-627-5p level was increased. Additionally, palbociclib treatment appeared to overcome TMZ resistance as well as reduced M2 markers in HMC3 cells. CONCLUSION: Together, we provided evidence supporting the usage of CDK6 inhibitor for TMZ-resistant GBM cases. Further investigation is warranted for the consideration of clinical trials.

Long non-coding RNA linc00645 promotes TGF-ß-induced epithelial-mesenchymal transition by regulating miR-205-3p-ZEB1 axis in glioma.

Accumulating evidence indicates long noncoding RNAs (lncRNA) play a vital role in tumor progression. However, the role of linc00645-induced accelerated malignant behavior in glioblastoma (GBM) remains unknown. In the present study, linc00645 expression was significantly upregulated in GBM tissues and cell lines. High level of linc00645 was associated with poor overall survival in GBM patients. Knockdown of linc00645 suppressed the proliferation, stemness, migration, invasion, and reversed transforming growth factor (TGF)-ß-induced motility of glioma cell lines. Furthermore, linc00645 directly interacted with miR-205-3p and upregulated of miR-205-3p impeded efficiently the increase of ZEB1 induced by linc00645 overexpression. Moreover, knockdown of linc00645 significantly suppressed the progression of glioma cells in vivo. miR-205-3p was a target of linc00645 and linc00645 modulates TGF-ß-induced glioma cell migration and invasion via miR-205-3p. Taken together, our findings identified the linc00645/miR-205-3p/ZEB1 signaling axis as a key player in EMT of glioma cells triggered by TGF-ß. These data elucidated that linc00645 plays an oncogenic role in glioma and it may serve as a prognostic biomarker and a potential therapeutic target for the treatment of glioma in humans.