MiR-135b-5p promotes cetuximab resistance in colorectal cancer by regulating FOXN3.

Despite advances in targeted therapies, primary and acquired resistance make the treatment of colorectal cancer (CRC) a pressing issue to be resolved. According to reports, the development of CRC is linked to miRNA dysregulation. Multiple studies have demonstrated that miR-135b-5p has an aberrant expression level between CRC tissues and adjacent tissues. However, it is unclear whether there is a correlation between miR-135b-5p and cetuximab (CTx) resistance in CRC. Use the GEO database to measure miR-135b-5p expression in CRC. Additionally, RT-qPCR was applied to ascertain the production level of miR-135b-5p in three human CRC cells and NCM460 cells. The capacity of cells to migrate and invade was examined utilizing the wound-healing and transwell assays, while the CCK-8 assay served for evaluating cell viability, as well as colony formation assays for proliferation. The expected target protein of miR-135b-5p in CRC cell cetuximab resistance has been investigated using western blot. Suppression of miR-135b-5p could increase the CTx sensitivity of CTx-resistant CRC cells, as manifested by the attenuation of proliferation, migration, and invasion ability. Mechanistic studies revealed miR-135b-5p regulates the epithelial-to-mesenchymal transition (EMT) process and Wnt/ß-catenin signaling pathway through downgulating FOXN3. In short, knockdowning miR-135b-5p could increase FOXN3 expression in CRC cells, promote the EMT process, and simultaneously activate the Wnt/ß-catenin signaling pathway to elevate CTx resistance in CRC cells.

Tumor microenvironment responsive nano-herb and CRISPR delivery system for synergistic chemotherapy and immunotherapy.

Chemoresistance remains a significant challenge for effective breast cancer treatment which leads to cancer recurrence. CRISPR-directed gene editing becomes a powerful tool to reduce chemoresistance by reprogramming the tumor microenvironment. Previous research has revealed that Chinese herbal extracts have significant potential to overcome tumor chemoresistance. However, the therapeutic efficacy is often limited due to their poor tumor targeting and in vivo durability. Here we have developed a tumor microenvironment responsive nanoplatform (H-MnO2(ISL + DOX)-PTPN2@HA, M(I + D)PH) for nano-herb and CRISPR codelivery to reduce chemoresistance. Synergistic tumor inhibitory effects were achieved by the treatment of isoliquiritigenin (ISL) with doxorubicin (DOX), which were enhanced by CRISPR-based gene editing to target protein tyrosine phosphatase non-receptor type 2 (PTPN2) to initiate long-term immunotherapy. Efficient PTPN2 depletion was observed after treatment with M(I + D)PH nanoparticles, which resulted in the recruitment of intratumoral infiltrating lymphocytes and an increase of proinflammatory cytokines in the tumor tissue. Overall, our nanoparticle platform provides a diverse technique for accomplishing synergistic chemotherapy and immunotherapy, which offers an effective treatment alternative for malignant neoplasms.

Identifying Factors Affecting the Survival of Patients with HIV-Associated B-Cell Lymphoma Using a Random Survival Forest Model.

Background: There have been no reports about the application of random survival forest (RSF) model to predict disease progression of HIV-associated B-cell lymphoma. Methods: A total of 44 patients with HIV-associated B-cell lymphoma who were referred to Nanjing Second Hospital from 2012 to 2019 were included. The RSF model was used to find predictors of survival, and the results of the RSF model were compared with those of the Cox model. The data were analyzed using R software (version 4.1.1). Results: One-, 2-, and 3-year survival rates were 74.5%, 57.7%, and 48.6%, respectively, and the median survival was 59.0 months. The first 3 most important predictors of survival included lactate dehydrogenase (LDH), absolute monocyte count (AMC), and white blood cells (WBCs) count. The median survival of high-risk patients was only 4.0 months. Areas under the curve (AUCs) of the RSF model remained at more than 0.90 at 1, 2, and 3 years. The RSF model displayed a lower prediction error rate (21.9%) than the Cox model (25.4%). Conclusions: Lactate dehydrogenase, AMC, and WBCs count are the most important prognostic predictors for patients with HIV-associated B-cell lymphoma. Much larger prospective and/or multicentre studies are required to validtae this RSF model.

Tumor-derived extracellular vesicles as a biomarker for breast cancer diagnosis and metastasis monitoring.

It is imperative to explore biomarkers that are both precise and readily accessible in the comprehensive management of breast cancer. A multicenter cohort, including 512 breast cancer patients and 198 nonneoplastic individuals, was recruited to detect the level of tumor-derived extracellular vesicles using our method based on dual DNA tetrahedral nanostructures. The level of tumor-derived extracellular vesicles was significantly higher in newly diagnosed breast cancer patients than in nonneoplastic individuals at a cutoff value of 3.58 U/µL. For postoperative metastasis monitoring, the level of tumor-derived extracellular vesicles was significantly higher in breast cancer patients with metastasis than in those without metastasis at a cutoff value of 3.91 U/µL. Its efficacy of diagnosis and metastasis monitoring was superior to traditional tumor markers. Elevated level of tumor-derived extracellular vesicles served as a predictive biomarker for diagnosis and metastasis monitoring in breast cancer patients.

Chimeric nanobody-decorated liposomes by self-assembly.

Liposomes as drug vehicles have advantages, such as payload protection, tunable carrying capacity and improved biodistribution. However, due to the dysfunction of targeting moieties and payload loss during preparation, immunoliposomes have yet to be favoured in commercial manufacturing. Here we report a chemical modification-free biophysical approach for producing immunoliposomes in one step through the self-assembly of a chimeric nanobody (cNB) into liposome bilayers. cNB consists of a nanobody against human epidermal growth factor receptor 2 (HER2), a flexible peptide linker and a hydrophobic single transmembrane domain. We determined that 64% of therapeutic compounds can be encapsulated into 100-nm liposomes, and up to 2,500 cNBs can be anchored on liposomal membranes without steric hindrance under facile conditions. Subsequently, we demonstrate that drug-loaded immunoliposomes increase cytotoxicity on HER2-overexpressing cancer cell lines by 10- to 20-fold, inhibit the growth of xenograft tumours by 3.4-fold and improve survival by more than twofold.

Bioinformatics analysis of the prognosis and biological significance of N6 methyladenine regulators in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a common type of cancer. We performed the present study to explore the function and specific regulatory mechanism of m6A in OSCC and to find a new diagnosis and treatment strategy for OSCC. METHODS: Using bioinformatics, we examined the associations between 20 genes associated with methylation and the epidemiological data about OSCC tumor samples. RESULTS: We created two subgroup curves based on the gene expression levels related to m6A methylation. In total, 14 genes were found to be differentially expressed. Significant differences in terms of survival rates, Grade and gender were found among subgroups with different m6A expression levels. Nine genes had areas under the curves greater than 0.7. Therefore, these genes may be utilized for the clinical diagnosis and prognosis of OSCC. Because of their high individual predictive value, HNRNPC and IGF2BP2 were chosen as the two potential predictors. The two regulatory elements were used to create the prognostic signals for OSCC. The developed prognostic signals made it possible to discern between the samples with good and poor prognoses without potential confounding factors. Four genes (HNRNPC, METTL14, YTHDF2 and ALKBH5) combined well with compounds, which had an anti-cancer effect. CONCLUSIONS: Our findings suggested that OSCC-related genes with m6A methylation could be beneficial treatment targets or prognostic indicators.

Expanding the applications of immune checkpoint inhibitors in advanced lung cancer beyond disease progression.

Background: Immunotherapy, particularly the utilization of immune checkpoint inhibitors (ICIs), assumes a pivotal role in the comprehensive management of advanced lung cancer. There has been substantial deliberation regarding the appropriateness of extending ICIs treatment beyond the point of disease progression. This study delves into the potential benefits of sustained utilization of ICIs subsequent to disease progression in patients. Methods: A retrospective analysis was conducted on a cohort of 248 patients diagnosed with advanced lung cancer who received treatment with ICIs. The study population comprised 99 patients in the treatment beyond progression (TBP) group and 42 patients in the non-treatment beyond progression (NTBP) group. Parameters including progression-free survival (PFS), overall survival (OS), objective response rate (ORR), and disease control rate (DCR) were assessed. The Cox proportional hazard regression model was employed to analyze prognostic factors related to immunotherapy. Results: Patients undergoing primary treatment with PD-1/PD-L1 inhibitors exhibited a median progression-free survival (mPFS) of 5.3 months. In the context of disease progression, a comparison between the TBP and NTBP groups was performed with respect to mPFS. The results demonstrated that the TBP group manifested an mPFS of 8.6 months, contrasting with the NTBP group's mPFS of 4.0 months (p=0.028). The mean overall survival (mOS) in the TBP group exhibited a statistically significant increase in comparison to the NTBP group (14.1 months vs. 6.0 months, p=0.028). Evaluation of the objective response rate (ORR) between the TBP and NTBP groups revealed a substantial distinction. The TBP group displayed an ORR of 12.1%, while the NTBP group exhibited a lower ORR of 2.4%. The statistical analysis yielded a p-value of 0.068, signifying a notable trend towards significance. The disease control rate (DCR) was also assessed and exhibited a noteworthy variance between the two groups, with a higher DCR of 92.9% in contrast to 71.4% in the control group (p = 0.001). Conclusion: Subsequent to ICIs treatment, a subset of patients may derive continued benefits from anticancer therapy, notwithstanding the progression of their advanced lung cancer.

Bioinspired engineering of fusogen and targeting moiety equipped nanovesicles.

Cell-derived small extracellular vesicles have been exploited as potent drug vehicles. However, significant challenges hamper their clinical translation, including inefficient cytosolic delivery, poor target-specificity, low yield, and inconsistency in production. Here, we report a bioinspired material, engineered fusogen and targeting moiety co-functionalized cell-derived nanovesicle (CNV) called eFT-CNV, as a drug vehicle. We show that universal eFT-CNVs can be produced by extrusion of genetically modified donor cells with high yield and consistency. We demonstrate that bioinspired eFT-CNVs can efficiently and selectively bind to targets and trigger membrane fusion, fulfilling endo-lysosomal escape and cytosolic drug delivery. We find that, compared to counterparts, eFT-CNVs significantly improve the treatment efficacy of drugs acting on cytosolic targets. We believe that our bioinspired eFT-CNVs will be promising and powerful tools for nanomedicine and precision medicine.

YTHDC2-mediated m6A mRNA modification of Id3 suppresses cisplatin resistance in non-small cell lung cancer.

Background: The methylation of adenosines at the N6 position (N6-methyladenosine; m6A) is one of the most conserved internal RNA modifications. m6A can modulate the expression of oncogenes or tumor suppressor genes, as well as m6A levels and the expression and activity of m6A enzymes, thus influencing tumor progression and therapeutic response. This study investigates the role of YTHDC2-mediated m6A messenger RNA (mRNA) modification of Id3 in controlling cisplatin resistance in non-small cell lung cancer (NSCLC). Methods: The expression of the m6A reader protein YTHDC2 was detected in an NSCLC cisplatin-resistant cell line (A549/DDP) using real-time fluorescence quantitative polymerase chain reaction (qPCR). YTHDC2 overexpression plasmids were constructed and transfected into A549/DDP and A549 cells respectively. We performed qPCR and western blot (WB) to detect changes in YTHDC2 and Id3 expression, and the effects of YTHDC2 overexpression on proliferation, apoptosis, invasion, and migration of drug-resistant cells were assessed by cell counting kit-8 (CCK-8), flow cytometry, and transwell and scratch assays. The m6A modification of Id3 by YTHDC2 was clarified by m6A-immunoprecipitation-PCR (m6A-IP-PCR) assay. Results: The CLIPdb online database predicted that YTHDC2 might bind to Id3. The results of qPCR showed that YTHDC2 was downregulated in the NSCLC cisplatin-resistant cell line A549/DDP compared to the cisplatin-sensitive cell line A549. Overexpression of YTHDC2 increased the expression of Id3, and the methylation inhibitor 3-deazaadenosine abrogated the regulatory effect of YTHDC2 on Id3. YTHDC2 overexpression significantly inhibited A549/DDP cell proliferation, migration, and invasion, and promoted apoptosis by synergistically promoting the effects of Id3. m6A-IP-PCR analysis revealed that YTHDC2 could inhibit the m6A level of Id3 mRNA. Conclusions: To regulate the activity of Id3, YTHDC2 requires modifications to m6A, which ultimately inhibit cisplatin resistance in NSCLC.

CPSF6-mediated XBP1 3'UTR shortening attenuates cisplatin-induced ER stress and elevates chemo-resistance in lung adenocarcinoma.

Alternative polyadenylation (APA) is a widespread mechanism generating RNA molecules with alternative 3' ends. Herein, we discovered that TargetScan includes a novel XBP1 transcript with a longer 3' untranslated region (UTR) (XBP1-UL) than that included in NCBI. XBP1-UL exhibited a lowered level in blood samples from lung adenocarcinoma (LUAD) patients and in those after DDP treatment. Consistently, XBP1-UL was reduced in A549 cells compared to normal BEAS-2B cells, as well as in DDP-treated/resistant A549 cells relative to controls. Moreover, due to decreased usage of the distal polyadenylation site (PAS) in 3'UTR, XBP1-UL level was lowered in A549 cells and decreased further in DDP-resistant A549 (A549/DDP) cells. Importantly, use of the distal PAS (dPAS) and XBP1-UL level were gradually reduced in A549 cells under increasing concentrations of DDP, which was attributed to DDP-induced endoplasmic reticulum (ER) stress. Furthermore, XBP1 transcripts with shorter 3'UTR (XBP1-US) were more stable and presented stronger potentiation on DDP resistance. The choice of proximal PAS (pPAS) was attributed to CPSF6 elevation, which was caused by BRCA1-distrupted R-loop accumulation in CPSF6 5'end. DDP-induced nuclear LINC00221 also facilitated CPSF6-induced pPAS choice in the pre-XBP1 3'end. Finally, we found that unlike the unspliced XBP1 protein (XBP1-u), the spliced form XBP1-s retarded p53 degradation to facilitate DNA damage repair of LUAD cells. The current study provides new insights into tumor progression and DDP resistance in LUAD, which may contribute to improved LUAD treatment.

Genome-wide screening for differentially methylated long noncoding RNAs identifies LIFR-AS1 as an epigenetically regulated lncRNA that inhibits the progression of colorectal cancer.

BACKGROUND: Aberrant DNA methylation is an epigenetic marker that has been linked to the pathogenesis of colorectal cancer (CRC). Long noncoding RNAs (lncRNAs) have been increasingly identified to be associated with tumorigenic processes of CRC. Identifying epigenetically dysregulated lncRNAs and characterizing their effects during carcinogenesis are focuses of cancer research. METHODS: Differentially methylated loci and expressed lncRNAs were identified by integrating DNA methylome and transcriptome analyses using The Cancer Genome Atlas database. Bisulfite sequencing PCR (BSP) was performed to analyze LIFR-AS1 promoter methylation status. The functional roles of LIFR-AS1 in CRC were determined by in vitro and in vivo experiments. RESULTS: We identified a novel hypermethylated lncRNA, LIFR-AS1, that was downregulated and associated with tumorigenesis, metastasis, and poor prognosis in CRC. High methylation burden of LIFR-AS1 indicated a poor survival of CRC patients. Promoter hypermethylation of LIFR-AS1 in tumor tissues was confirmed by BSP. Functional assays revealed that LIFR-AS1 could competitively bind to hsa-miR-29b-3p, and repressed colon cancer cell proliferation, colony formation and invasion. LIFR-AS1 also inhibited tumor growth in a mouse xenograft model of CRC. CONCLUSIONS: Our results showed that the identified DNA methylation-dysregulated lncRNAs may be potential biomarkers and highlighted a role for LIFR-AS1 as a tumor suppressor in CRC.

Tumor-Associated Macrophages Promote Metastasis of Oral Squamous Cell Carcinoma via CCL13 Regulated by Stress Granule.

M2 tumor-associated macrophages (TAMs) have been a well-established promoter of oral squamous cell carcinoma (OSCC) progression. However, the mechanisms of M2 TAMs promoting OSCC metastasis have not been elucidated clearly. This study illustrated the regulatory mechanisms in which M2 TAMs enhance OSCC malignancy in a novel point of view. In this study, mass spectrometry was utilized to analyze the proteins expression profile of M2 type monocyte-derived macrophages (MDMs-M2), whose results revealed the high expression of G3BP1 in M2 macrophages. RNA sequencing analyzed the genome-wide changes upon G3BP1 knockdown in MDMs-M2 and identified that CCL13 was the most significantly downregulated inflammatory cytokines in MDMs-M2. Co-immunoprecipitation and qualitative mass spectrometry were used to identify the proteins that directly interacted with endogenous G3BP1 in MDMs-M2. Elevated stress granule (SG) formation in stressed M2 TAMs enhanced the expression of CCL13, which promoted OSCC metastasis both in vitro and in vivo. For mechanisms, we demonstrated SG formation improved DDX3Y/hnRNPF-mediated CCL13 mRNA stability, thus enhancing CCL13 expression and promoting OSCC metastasis. Collectively, our findings demonstrated for the first time the roles of CCL13 in improving OSCC metastasis and illustrated the molecular mechanisms of CCL13 expression regulated by SG, indicating that the SG-CCL13 axis can be the potential targets for TAM-navigated tumor therapy.

Clinical Results Evaluation of Patients With Submental Mass Through Endoscopy-Assisted Transoral Surgery.

BACKGROUND: Surgical resection through extraoral approach is the first choice for submental mass but leaves a visible scar. This study introduces an endoscopy-assisted transoral approach to resect submental mass and evaluates the clinical results. PATIENTS AND METHODS: From September 2018 to December 2019, 5 patients with submental mass underwent surgical resection through endoscopy-assisted transoral approach. The swallowing, speech, and appearance domains of the University of Washington Quality of Life questionnaire were assessed preoperatively and at 3 months postoperatively. RESULTS: Each mass was completely removed without rupture. No patient developed any permanent postoperative complications. The function and aesthetic outcomes were excellent without recurrence. CONCLUSIONS: Endoscopy-assisted transoral approach for resection of submental mass is a reliable technique that achieves excellent postoperative aesthetics and functional results.

Two-Lines-Four-Regions: A New Concept in Endoscopic-Assisted Surgery of Parotid Gland Tumors.

PURPOSE: Endoscopic-assisted surgery of parotid gland tumor is challenging due to the complex anatomic structures. This study compared an innovative endoscopic-assisted operation called the two-lines-four-regions method to a traditional endoscopic-assisted surgery in the treatment of parotid gland tumors. METHODS: In this retrospective cohort study, patients were assigned to the traditional endoscopic group (Trad-En group) and the two-lines-four-regions endoscopic group (Tlfr-En group) according to whether two-lines-four-regions concept was used before the surgery. The primary outcomes are operation time (minutes) and postoperative complications present or absent. Other outcomes including intraoperative blood loss (milliliter), whether to increase auxiliary incision (yes/no), postoperative drainage (milliliter), and retention time (day) are the secondary outcomes. χ2 analysis or Fisher exact test was used to compare the statistical differences of those variables in 2 groups, and a P value of less than .05 was considered to indicate statistical significance. RESULTS: A total of 121 patients with parotid gland tumors underwent endoscopic-assisted surgery; 59 patients were assigned to the Trad-En group, and 62 patients were assigned to the Tlfr-En group. The mean operation times (65.24 ± 14.82 min), blood loss (9.85 ± 3.38 mL), and the amount of drainage (10.52 ± 3.17 mL) in the Tlfr-En group were shorter than the values in the Trad-En group (75.75 ± 14.16 min, 10.52 ± 3.17 mL, and 16.54 ± 3.21 mL, respectively; P < .05). The median follow-up time was 34 months (range 4 to 70 months). No tumor recurrence was found in all patients. CONCLUSIONS: This study demonstrated that the new concept of two-lines-four-regions of parotid gland makes the endoscopic-assisted surgery of parotid gland tumors more simplified, efficient, and minimally invasive.

The circular RNA hsa_circ_0001394 promotes hepatocellular carcinoma progression by targeting the miR-527/UBE2A axis.

Circular RNAs (circRNAs) have been recognized as significant participants in the progression of different cancers; however, the detailed mechanisms of circRNAs in hepatocellular carcinoma (HCC) remain unclear. In this study, hsa_circ_0001394 was identified by RNA-seq analysis, and hsa_circ_0001394 was determined to be highly expressed in HCC specimens and cell lines. Patients with high expression of hsa_circ_0001394 tended to exhibit poor survival. Increased hsa_circ_0001394 expression in plasma was closely correlated with clinicopathological features including elevated vascular invasion and an advanced TNM stage, as indicated by alpha-fetoprotein (AFP) analysis. Hsa_circ_0001394 promoted the proliferation, migration, and invasion of HCC cells, whereas knockdown of hsa_circ_0001394 inhibited HCC tumorigenesis in vivo. In addition, mechanistic studies showed that miR-527 negatively interacted with hsa_circ_0001394. Furthermore, UBE2A was revealed to serve as a target of miR-527. Overall, the present study suggested that hsa_circ_0001394 may function as a sponge to promote HCC progression by regulating the miR-527/UBE2A pathway. Thus, hsa_circ_0001394 may become a promising biomarker and potential therapeutic target in HCC treatment.

Novel design for local full-thickness skin graft: optimizing donor sites of radial forearm free flap.

OBJECTIVE: Donor site deformities remain the primary issues after radial forearm free flap (RFFF) translocations. This study introduced a local full-thickness skin graft (FTSG) from an adjacent cutaneous area to manage RFFF donor sites and evaluated the esthetic and functional outcomes post-operatively. PATIENTS AND METHODS: This was a retrospective study among patients undergoing ablative surgery for head and neck tumors followed by defect reconstruction using a RFFF. The RFFF donor site defects were covered by local FTSGs from the adjacent skins whose harvesting was assisted by a forearm model or traditional FTSGs from abdomen. The color matching degree, surgical scars, ranges of wrist movement, and grip strengths in donor forearms were assessed in two groups at twelve months after surgery. RESULTS: Patients undergoing local FTSGs recovery exhibited better color matching than those repaired by traditional FTSGs from the abdomen. In forearm model, the local FTSGs width should be less than 22% of the forearm circumference located 5 cm away from the center of Fossa cubitalis. All the local FTSGs transplanted with this technique showed primary or secondary healing. CONCLUSIONS: This study demonstrated using this innovatively designed local FTSGs to close RFFF donor sites was more advantageous than traditional methods.

Engineered extracellular vesicles for concurrent Anti-PDL1 immunotherapy and chemotherapy.

Immune checkpoint inhibitors (ICI) targeting PD-1/PD-L1 have been approved for the treatment of a variety of cancers. However, the efficacy of antibody-based ICIs could be further improved by mitigating anti-drug antibodies, proteolytic cleavage, and on-target off-tumor toxicity. One strategy for accomplishing this is through the use of extracellular vesicles (EVs), cell derived submicron vesicles with many unique properties. We constructed an engineered MDA-MB-231 cell line for harvesting EVs. This was accomplished by overexpressing a high-affinity variant human PD-1 protein (havPD-1), while simultaneously knocking out intrinsic PD-L1 and beta-2 microglobulin. The engineered havPD-1 EVs reduced PD-L1 overexpressing cancer cell proliferation and induced cellular apoptosis. Moreover, the EVs were shown to efficiently block PD-L1 mediated T cell suppression. Meanwhile antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity were not observed. The havPD-1 EVs treatment resulted in robust anti-tumor activity in both preventative co-implantation and therapeutic xenograft tumor models reconstituted with human T cells. The efficacy of the havPD-1 EVs was shown to be comparable to clinical anti-PD1 monoclonal antibodies. Additionally, loading the havPD-1 EVs with a potent PARP inhibitor was shown to further augment treatment efficacy. In brief, the engineered universal EVs harboring havPD-1 proteins can be used for cancer concurrent immunotherapy and chemotherapy.

Circulating Exosomal miR-96 as a Novel Biomarker for Radioresistant Non-Small-Cell Lung Cancer.

Patients with non-small-cell lung cancer (NSCLC) frequently develop radioresistance, resulting in poor response to radiation and unfavourable prognosis. Early detection of radioresistance hence can guide the adjustment of treatment regimens in time. Exosomes are lipid bilayer-enclosed vesicles with sub-micrometer size that are released by various cells. Exosomes contain a tissue-specific signature wherein a variety of proteins and nucleic acids are selectively packaged. Growing evidence shows exosomes are involved in cancer pathophysiology and exosomes as the latest addition to the liquid biopsy portfolio have been used in cancer diagnosis. Compared to cell free RNA, exosomal lipid envelope can effectively protect RNA cargo against degradation. Therefore, exosomes may hold great promise for the identification of radioresistance. Here, we report six plasma exosomal miRNAs could be used to distinguish radioresistant NSCLC patients from radiosensitive NSCLC patients and to evaluate the prognosis of NSCLC. Samples were obtained from 52 NSCLC patients with or without radioresistance and 45 age-matched healthy volunteers. Exosomes in 1 ml plasma were isolated followed by extraction of small RNA. The expression levels of miRNAs were determined by quantitative real-time PCR. Potential miRNA markers were further evaluated in additional 52 NSCLC patients. We found exosomal miR-1246 and miR-96 are significantly overexpressed in NSCLC patients. Moreover, exosomal miR-96 in patients with radioresistant NSCLC is significantly higher than that of controls. Exosomal miR-96 also demonstrates a significant correlation with vascular invasion and poor overall survival. Altogether, our results indicate that exosomal miR-96 could be a non-invasive diagnostic and prognostic marker of radioresistant NSCLC.

Metformin enhances osteogenic differentiation of stem cells from human exfoliated deciduous teeth through AMPK pathway.

Stem cells from human exfoliated deciduous teeth (SHEDs) are ideal seed cells in bone tissue engineering. As a first-line antidiabetic drug, metformin has recently been found to promote bone formation. The purpose of this study was to investigate the effect of metformin on the osteogenic differentiation of SHEDs and its underlying mechanism. SHEDs were isolated from the dental pulp of deciduous teeth from healthy children aged 6 to 12, and their surface antigen markers of stem cells were detected by flow cytometry. The effect of metformin (10-200 µM) treatment on SHEDs cell viability, proliferation, and osteogenic differentiation was analyzed. The activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation Thr172 (p-AMPK) was determined by western blot assay. SHEDs were confirmed as mesenchymal stem cells (MSCs) on the basis of the expression of characteristic surface antigens. Metformin (10-200 µM) did not affect the viability and proliferation of SHEDs but significantly increased the expression of osteogenic genes, alkaline phosphatase activity, matrix mineralization, and p-AMPK level expression in SHEDs. Compound C, a specific inhibitor of the AMPK pathway, abolished metformin-induced osteogenic differentiation of SHEDs. Moreover, metformin treatment enhanced the expression of proangiogenic/osteogenic growth factors BMP2 and VEGF but reduced the osteoclastogenic factor RANKL/OPG expression in SHEDs. In conclusion, metformin could induce the osteogenic differentiation of SHEDs by activating the AMPK pathway and regulates the expression of proangiogenic/osteogenic growth factors and osteoclastogenic factors in SHEDs. Therefore, metformin-pretreated SHEDs could be a potential source of seed cells during stem cell-based bone tissue engineering.