Increased Cell Apoptosis Rate Research Articles

AEBP1 restores osteoblastic differentiation under dexamethasone treatment by activating PI3K/AKT signalling.

Adipocyte enhancer-binding protein 1 (AEBP1) is closely implicated in osteoblastic differentiation and bone fracture; this research aimed to investigate the effect of AEBP1 on restoring osteoblastic differentiation under dexamethasone (Dex) treatment, and its interaction with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Pre-osteoblastic MC3T3-E1 cells were cultured in osteogenic medium and treated by Dex to mimic steroid-induced osteonecrosis cellular model. They were then further transfected with control or AEBP1-overexpressed lentiviral vectors. Finally, cells were treated with the PI3K inhibitor LY294002, with or without AEBP1-overexpressed lentiviral vectors. AEBP1 expression showed a downward trend in MC3T3-E1 cells under Dex treatment in a dose-dependent manner. AEBP1-overexpressed lentiviral vectors increased relative cell viability, alkaline phosphatase (ALP) staining, Alizarin red staining and osteoblastic differentiation markers including osteocalcin (OCN), osteopontin (OPN), collagen type I alpha 1 (COL1A1), runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2), but decreased cell apoptosis rate in MC3T3-E1 cells under Dex treatment; besides, AEBP1-overexpressed lentiviral vectors positively regulated p-PI3K and p-AKT expressions. Furthermore, LY294002 treatment decreased relative cell viability, Alizarin red staining, osteoblastic differentiation markers including OCN, OPN, RUNX2 and BMP, increased cell apoptosis rate and did not affect ALP staining in MC3T3-E1 cells under Dex treatment; meanwhile, LY294002 treatment weakened the effect of AEBP1 overexpression vectors on the above cell functions. AEBP1 restores osteoblastic differentiation under Dex treatment by activating the PI3K/AKT pathway.

Regorafenib enhances the efficacy of photodynamic therapy in hepatocellular carcinoma through MAPK signaling pathway suppression

Photodynamic therapy (PDT) is a promising and innovative approach for treating tumors. The synergistic effect of PDT and chemotherapy can enhance the anti-tumor efficacy by leveraging their complementing benefits. In this study, we created lipid vesicles to deliver a photosensitizer (chlorin e6, Ce6) and Regorafenib into tumors for the purpose of examining the effectiveness and mechanism of Lipo-Ce6@Rego-PDT (LCR-P) on Hepatocellular carcinoma (HCC) both in vitro and in vivo. We found that the cytotoxicity on HCC caused by LCR-P was significantly stronger than that caused by Lipo-Ce6-PDT (LC-P). Cellular ROS production in the LCR-P group was approximately higher than that in the LC-P group, and Regorafenib significantly inhibited the phosphorylation of JNK, ERK, and P38 of Lipo-Ce6-PDT group in vitro and in vivo. Furthermore, Regorafenib significantly downregulated the expression of Bcl-2 and upregulated the expression of Bax and cleaved caspase-3 of LC-P group in vitro and in vivo. Compared with LC-P, LCR-P significantly increased cell apoptosis rate. The body weight and HE staining of normal organs primarily indicated the safety of this combined strategy. These results indicate that the combination of Regorafenib and Lipo-Ce6 can significantly enhance the anti-tumor efficiency of PDT for HCC and exhibits good biosafety.

High STING expression exacerbates renal ischemia-reperfusion injury in mice by regulating the TLR4/NF-κB/NLRP3 pathway and promoting inflammation and apoptosis

To investigate renal expression level of STING in mice with renal ischemia-reperfusion injury (IRI) and its regulatory role in IRI. C57BL/6 mice were divided into sham operation group, IRI (induced by clamping the renal artery) model group, IRI+DMSO treatment group, and IRI+SN-011 treatment group. Serum creatinine and blood urea nitrogen of the mice were analyzed, and pathological changes in the renal tissue were assessed with PAS staining. RT-qPCR, ELISA, Western blotting, and immunohistochemistry were used to detect the expression levels of STING, KIM-1, Bcl-2, Bax, caspase-3, TLR4, P65, NLRP3, caspase-1, CD68, MPO, IL-1β, IL-6, and TNF-α in the renal tissues. In the cell experiment, HK-2 cells exposed to hypoxia-reoxygenation (H/R) were treated with DMSO or SN-011, and cellular STING expression levels and cell apoptosis were analyzed using RT-qPCR, Western blotting or flow cytometry. In C57BL/6 mice, renal IRI induced obvious renal tissue damage, elevation of serum creatinine and blood urea nitrogen levels and renal expression levels of KIM-1, STING, TLR4, P65, NLRP3, caspase-1, caspase-3, Bax, CD68, MPO, IL-1β, IL-6, and TNF-α, and reduction of Bcl-2 expression level. Treatment of the mouse models with SN-011 for inhibiting STING expression significantly alleviated these changes. In HK-2 cells, H/R exposure caused significant elevation of cellular STING expression and obviously increased cell apoptosis rate, which was significantly lowered by treatment with SN-011. Renal STING expression is elevated in mice with renal IRI to exacerbate renal injury by regulating the TLR4/NF-κB/NLRP3 pathway and promoting inflammation and apoptosis in the renal tissues.

Pristimerin enhances cisplatin-induced apoptosis in nasopharyngeal carcinoma cells via ROS-mediated deactivation of the PI3K/AKT signaling pathway

To explore the effect of pristimerin combined with cisplatin on proliferation and apoptosis of nasopharyngeal carcinoma cells. CCK-8 assay was used to examine the survival rate of HNE-1 and CNE-2Z cells following treatment for 24 h with different concentrations of pristimerin, cisplatin or their combination. The changes in colony formation ability, apoptosis, and intracellular reactive oxygen species (ROS) levels of the treated cells were analyzed using colony formation assay and flow cytometry. Western blotting was performed to detect the changes in protein expressions in the cells. The effects of pre-treatment with NAC on proliferation, apoptosis, and PI3K/AKT signaling pathway were observed in pristimerin- and/or cisplatin-treated cells. Both pristimerin and cisplatin significantly lowered the survival rate of HNE-1 and CNE-2Z cells (P < 0.05). Compared with pristimerin or cisplatin alone, their combination more strongly inhibited survival and colony formation ability of the cells, increased cell apoptosis rate and intracellular ROS levels, upregulated the protein expressions of Bax, cleaved caspase-3, and cleaved PARP, and downregulated the protein expressions of Bcl-2, Mcl-1, PARP and p-PI3K and p-AKT (P < 0.05). NAC pretreatment significantly attenuated proliferation inhibition and apoptosis-promoting effects of pristimerin combined with cisplatin, and partially restored the downregulated protein expressions of p-PI3K and p-AKT in HNE-1 and CNE-2Z cells with the combined treatment (P < 0.05). Pristimerin can enhance cisplatin-induced proliferation inhibition and apoptosis in nasopharyngeal carcinoma cells, the mechanism of which may involve ROS-mediated deactivation of the PI3K/AKT signaling pathway.

Effects of 4-hydroxy-2(3H)-benzoxazolone on proliferation and apoptosis of pancreatic cancer L3.6 cells

This study explored the effects of 4-hydroxy-2(3H)-benzoxazolone(HBOA) on the proliferation and apoptosis of pancreatic cancer cells and its molecular mechanism. The L3.6 cells cultured in vitro were treated with HBOA of 0-1.0 mmol·L~(-1). The cell viability was detected by the cell counting kit-8(CCK-8) method, and the half inhibitory concentration(IC_(50)) was analyzed to determine the drug concentration and time. The cell morphology was observed under an inverted microscope and by acridine orange(AO) staining. The ability of proliferation and self-renewal were evaluated through live cell counting and colony formation experiments. The cell cycle progression and cell apoptosis rate were detected by flow cytometry. The morphology of cell apoptosis was observed by scanning electron microscopy. The mRNA expression of proliferating cell nuclear antigen(PCNA), cyclinA1, cyclinA2, cyclin dependent kinase 2(CDK2), and cyclin dependent kinase inhibitor 1A(P21) were determined by qPCR. The level of reactive oxygen species(ROS), lipid peroxide, and mitochondrial membrane potential were measured by flow cytometry. The activity of protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathway was detected by Western blot. Compared with the control group, the cells treated with HBOA exhibited a significant decrease in viability. Then the optimal concentration and intervention time of HBOA were determined to be 0.4 mmol·L~(-1), 0.6 mmol·L~(-1), and 48 h. Compared with the control group, groups with HBOA of 0.4 mmol·L~(-1 )and 0.6 mmol·L~(-1) showed a significant suppression in cell proliferation and colony formation ability, down-regulated mRNA of PCNA, cyclinA1, cyclinA2, and CDK2, up-regulated P21 mRNA, S-phase cell cycle arrest, and increased cell apoptosis rate. There was an appearance of apoptotic bodies, increased ROS and lipid peroxide, decreased mitochondrial membrane potential(with a significant decrease in 0.6 mmol·L~(-1) group), and down-regulated p-Akt and p-mTOR proteins. The results show that HBOA inhibits the proliferation of pancreatic cancer L3.6 cells and induces cell apoptosis, which may be related to the increase in reactive oxygen species and the inhibition of the Akt/mTOR pathway.

Eukaryotic initiation factor 3a promotes the development of diffuse large B-cell lymphoma through regulating cell proliferation

BackgroundOne-third of diffuse large B-cell lymphoma (DLBCL) patients suffer relapse after standard treatment. Eukaryotic initiation factor 3a (eIF3a) is a key player in the initial stage of translation, which has been widely reported to be correlated with tumorigenesis and therapeutic response. This study aimed to explore the biological role of eIF3a, evaluate its prognostic and therapeutic potential in DLBCL.MethodsRNA-seq datasets from GEO database were utilized to detect the expression and prognostic role of eIF3a in DLBCL patients. Protein level of eIF3a was estimated by western blot and immunohistochemical. Next, DLBCL cells were transfected with lentiviral vector either eIF3a-knockdown or empty to assess the biological role of eIF3a. Then, samples were divided into 2 clusters based on eIF3a expression and differentially expressed genes (DEGs) were identified. Function enrichment and mutation analysis of DEGs were employed to detect potential biological roles. Moreover, we also applied pan-cancer and chemosensitivity analysis for deep exploration.ResultseIF3a expression was found to be higher in DLBCL than healthy controls, which was associated with worse prognosis. The expression of eIF3a protein was significantly increased in DLBCL cell lines compared with peripheral blood mononuclear cells (PBMCs) from healthy donors. eIF3a knockdown inhibited the proliferation of DLBCL cells and the expression of proliferation-related proteins and increase cell apoptosis rate. Besides, 114 DEGs were identified which had a close linkage to cell cycle and tumor immune. eIF3a and DEGs mutations were found to be correlated to chemosensitivity and vital signal pathways. Pan-cancer analysis demonstrated that high eIF3a expression was associated with worse prognosis in several tumors. Moreover, eIF3a expression was found to be related to chemosensitivity of several anti-tumor drugs in DLBCL, including Vincristine and Wee1 inhibitor.ConclusionsWe firstly revealed the high expression and prognostic role of eIF3a in DLBCL, and eIF3a might promote the development of DLBCL through regulating cell proliferation and apoptosis. eIF3a expression was related to immune profile and chemosensitivity in DLBCL. These results suggest that eIF3a could serve as a potential prognostic biomarker and therapeutic target in DLBCL.

Bone mesenchymal stem cell-derived exosomal miR-26a-3p promotes autophagy to attenuate LPS-induced apoptosis and inflammation in pulmonary microvascular endothelial cells.

Acute lung injury (ALI) is a serious lung disease. The apoptosis and inflammation of pulmonary microvascular endothelial cells (PMVECs) are the primary reasons for ALI. This study aimed to explore the treatment effect and regulatory mechanism of bone mesenchymal stem cell-derived exosomes (BMSC-expos) on ALI. PMVECs were stimulated by Lipopolysaccharide (LPS) to imitate ALI environment. Cell viability was determined by CCK-8 assay. Cell apoptosis was evaluated by TUNEL and flow cytometry. ELISA was utilized for testing the contents of TNF-α, IL-1β, IL-6, and IL-17. Western blot was applied for testing the levels of autophagy-related proteins LC3, p62, and Beclin-1. RNA interaction was determined by luciferase reporter assay. The ALI rat model was established by intratracheal injection of LPS. Evans blue staining was utilized for detecting pulmonary vascular permeability. Our results showed that LPS stimulation notably reduced cell viability, increased cell apoptosis rate, and enhanced the contents of inflammatory factors in PMVECs. However, BMSC-exo treatment significantly abolished the promoting effects of LPS on cell injury. In addition, we discovered that BMSC-exo treatment notably activated autophagy in LPS-induced PMVECs. Furthermore, BMSC-expos upregulated miR-26a-3p expression and downregulated PTEN in PMVECs. MiR-26a-3p was directly bound to PTEN. MiR-26a-3p overexpression reduced cell apoptosis, and inflammation and promoted autophagy by silencing PTEN. Animal experiments proved that miR-26a-3p overexpression effectively improved LPS-induced lung injury in rats. The results proved that BMSC-expos promotes autophagy to attenuate LPS-induced apoptosis and inflammation in pulmonary microvascular endothelial cells via miR-26a-3p/PTEN axis.

Xinfeng Capsule alleviates interleukin-1β-induced chondrocyte inflammation and extracellular matrix degradation by regulating the miR-502-5p/TRAF2/NF-κB axis

To investigate the mechanism that mediates the inhibitory effect of Xinfeng Capsule (XFC) on interleukin (IL)-1β-induced impairment of chondrocytes. XFC-medicated serum was collected from SD rats with XFC gavage, and its optimal concentration for chondrocyte treatment was determined using Cell Counting Kit-8 assay and flow cytometry. Dual luciferase reporter analysis was performed to analyze the targeting relationship between miR-502-5p and TRAF2. In cultured human chondrocytes induced with IL-1β, the effects of transfection with miR-502-5p inhibitor and XFC-medicated serum, alone or in combination, on expression levels of IL-1β, tumor necrosis factor-α (TNF-α), IL-4, and IL-10 were examined with ELISA, and the changes in the expressions of collagen type Ⅱ alpha 1 (COL2A1), matrix metalloproteinase 13 (MMP13), adisintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and miR-502-5p/TRAF2/NF-κB axis gene expression were detected using RT-qPCR, Western blotting, and immunofluorescence assay. In cultured human chondrocytes, treatment with IL-1β significantly decreased the cell viability, increased cell apoptosis rate, lowered miR-502-5p, IL-4, IL-10, and COL2A1 expressions, and enhanced IL-1β, TNF-α, ADAMTS5, MMP13, TRAF2, and NF-κB p65 expressions (P < 0.05), and these changes were significantly improved by treatment with XFC-medicated serum at the optimal concentration of 20% (P < 0.05). Transfection of the chondrocytes with miR-502-5p inhibitor resulted in elevated expressions of IL-1β, TNF-α, ADAMTS5, MMP13, TRAF2, and NF-κB p65 and lowered expressions of miR-502-5p, IL-4, IL-10, and COL2A1, and XFC-medicated serum obviously reversed the effects of miR-502-5p inhibitor. XFC can inhibit IL-1β-induced inflammatory response and ECM degradation in cultured human chondrocytes possibly by regulating the miR-502-5p/TRAF2/NF-κB axis.

Universal nanosonosensitizer for ROS-mediated reduction of various cancer cells.

Sonodynamic therapy (SDT) has attracted great attention due to its deep tissue penetration, uniform tissue energy distribution, and noninvasiveness features. Additionally, external triggers can precisely focus on the tumor site with good specificity and high controllability. In the past decade, numerous sonosensitizers have been designed and used for SDT. However, the research and development of universal sonosensitizers for many different types of tumors are equally important in clinical treatment. Herein, we synthesized and studied the universality of four MWO4-PEG nanoparticles (NPs). All of the four MWO4-PEG NPs exhibited highly efficient ultrasound (US)-triggered production of 1O2 and ˙OH, enabling effective decreased cell viability, increased cell apoptosis rate, and a destruction of mouse tumors under US stimulation. The US-triggered NPs indicated good sonosensitivity and low toxicity to nine kinds of cancer cells. After accomplishing its therapeutic functions, NiWO4-PEG could be metabolized by the mouse body without any long-term toxicity. The PEGylated MWO4-PEG NPs shown in this study would provide efficient and universal US-triggered cancer therapy with the advantages of a cost-effective, convenient, and noninvasive agent that is promising for noninvasive SDT cancer treatment.

Galectin-1 knockdown inhibits proliferation, migration, invasion and promotes apoptosis of lung adenocarcinoma cells in vitro

To investigate the effect of galectin-1 on biological behaviors of lung adenocarcinoma cells and the underlying mechanism. The expression levels of galectin-1 mRNA were detected in 8 pairs of lung adenocarcinoma tissues and adjacent normal tissues and in lung adenocarcinoma cell lines A549 and H1299 and normal bronchial epithelial cell line BEAS-2b using qRT-PCR. The effect of galectin-1 knockdown by RNA interference on the proliferation, invasion and migration abilities and apoptosis of lung adenocarcinoma cells were examined using CCK8 assay, Transwell assay, scratch assay and flow cytometry. Western blotting was performed to detect the expressions of apoptosis-related proteins BAX, BCL-2, and caspase-3 and the proteins involved in the AKT and ERK pathways. The mRNA expression of galectin-1 was significantly increased in lung cancer tissues and lung adenocarcinoma cell lines (P < 0.05). In lung adenocarcinoma cells, galectin-1 knockdown significantly inhibited cell proliferation, migration and invasion and obviously increased cell apoptosis rate (P < 0.05), causing also significant reduction of the phosphorylation level of ERK signaling pathway (P < 0.05). Galectin-1 knockdown inhibits proliferation, migration, and invasion and promotes apoptosis of lung adenocarcinoma cells, and this effect is mediated probably by inhibition of the phosphorylation levels of the ERK pathway.

MiR-16-5p regulates apoptosis and migration of drug-resistant breast cancer cells by targeting YWHAQ

To examine the role of miR-16-5p in regulating biological behaviors of paclitaxel- resistant breast cancer cells and its molecular mechanism. The expression of miR-16-5p was examined in 13 pairs of breast cancer and adjacent tissues and in parental SKBR-3 cells and paclitaxel-resistant SKBR-3/PR cells using qRT-PCR. The target genes of miR-16- 5p were predicted by bioinformatic analysis, and their targeted binding was tested using luciferase assay. The cells were transfected with a miR-16-5p mimics, a miR-16-5p inhibitor, a specific siRNA targeting YWHAQ (si-YWHAQ), or both the miR-16-5p mimics and si-YWHAQ, and the changes in cellular expressions of YWHAQ, Bcl-2 and Bax were detected using Western blot. The changes in proliferation and migration of the cells were evaluated with CCK-8 assay and Transwell assay, and the cell cycle changes and cell apoptosis were analyzed with flow cytometry. The expression of miR-16-5p was significantly lower in breast cancer tissues than in paired adjacent tissues (P < 0.01). Bioinformatic analysis predicted that YWHAQ was the target gene of miR-16-5p, which was confirmed by luciferase assay. Compared with parental SKBR- 3 cells, SKBR- 3/PR cells showed a lowered level of miR-16-5p expression and an increased expression of YWHAQ. Transfection with the miR-16-5p mimics significantly inhibited YWHAQ expression (P < 0.01), while miR-16-5p inhibitor promoted YWHAQ expression in SKBR-3/PR cells (P < 0.01). The miR-16-5p mimics caused cell cycle arrest in G0/G1 phase (P < 0.0l), suppressed proliferation and migration, and increased apoptosis rate of SKBR-3/PR cells (P < 0.0l). Knocking down YWHAQ also reduced the migration ability of SKBR-3/PR cells and increased cell apoptosis rate. Transfection with either miR-16-5p mimics or si-YWHAQ resulted in increased Bax expression and lowered expressions of YWHAQ and Bcl-2 in the cells. The cells transfected with both miR-16-5p mimics and si-YWHAQ showed obviously suppressed cell migration (P < 0.01) and significantly increased apoptosis rate (P < 0.01). miR-16-5p can modulate the expressions of Bcl- 2 and Bax by targeted regulation of YWHAQ to modify the biological behaviors of paclitaxel-resistant breast cancer cells.

Interference of CTPS gene promotes toosendanin-induced apoptosis of human gastric cancer MKN-45 cells

To investigate the effect of interference of CTPS gene on toosendanin-induced apoptosis of gastric cancer MKN-45 cells. Bioinformatic analysis was used to analyze CTPS gene expression in human gastric cancer tissues and the overall survival of gastric cancer patients with high CTPS gene expression. Human gastric cancer MKN-45 cells were transfected with a short hairpin interfering RNA targeting CTPS gene, and 48 h later, qRT-PCR and Western blotting were used to detect cellular expression CTPS at both the mRNA and protein levels. MKN-45 cells with CTPS knockdown were treated with 80 nmol/L toosendanin for 48 h, and the cell viability was assessed with MTT assay; the cell morphology was observed using laser confocal microscope, and the expression of γH2AX was detected with immunofluorescence assay. Bioinformatic analysis suggested that CTPS was highly expressed in human gastric cancer tissues, and gastric cancer patients with high CTPS gene expression had a shorter overall survival. MKN-45 cells transfected with Sh-CTPS interference vector showed significantly lowered cell survival rate (P < 0.01) with obvious cell shrinkage, irregular morphology, typical apoptotic changes, and increased cell apoptosis rate (P < 0.05). Treatment of the transfected cells with 80 nmol/L toosendanin for 48 h resulted in further reduction of the cell survival rate (P < 0.001), and the cells showed an increased apoptotic rate (P < 0.05) with appearance of apoptotic bodies. Interference of CTPS gene can promote TSN-induced apoptosis of gastric cancer MKN-45 cells.

Forkhead box A2 transcriptionally activates hsa-let-7 g to inhibit hypoxia-induced epithelial-mesenchymal transition by targeting c14orf28 in colorectal cancer

Background and Study AimsThis study aimed to investigate the effect of Forkhead Box A2 (FOXA2) on migration, invasion, and epithelial-mesenchymal transition (EMT) of colorectal cancer (CRC) cells in hypoxia and explore its related molecular mechanisms. Patients and MethodsA cellular hypoxia model was established, and the FOXA2 overexpression vector was transfected into SW480 and HCT116 cells. Cell apoptosis, migration, and invasion were examined by flow cytometry, scratch test, and transwell-invasion assay. Next, the hsa-let-7 g gene expression was detected by quantitative reverse transcription-polymerase chain reaction. Relative protein levels of HIF-1, FOXA2, c14orf28, E-cadherin, N-cadherin, and Vimentin were detected by western blot. ResultsHypoxia-exposed CRC cells showed a significantly increased cell apoptosis rate, as well as enhanced cell invasion and migration abilities compared with the cells in normoxia. FOXA2 overexpression induced apoptosis and inhibited hypoxia-exposed CRC cell migration and invasion. Additionally, FOXA2 overexpression led to the significantly increased hsa-let-7 g and E-cadherin expression, as well as the decreased c14orf28, N-cadherin, and Vimentin expression in hypoxic CRC cells. ConclusionsThis study demonstrated that FOXA2 could affect the apoptosis, migration, invasion, and EMT of CRC cells under hypoxia conditions. FOXA2 transcriptionally activates hsa-let-7 g to inhibit hypoxia-induced EMT by targeting c14orf28.

Hyaluronic acid-modified, IR780-conjugated and doxorubicin-loaded reduced graphene oxide for targeted cancer chemo/photothermal/photodynamic therapy

We used reduced graphene oxide (rGO), which has two times higher photothermal conversion efficiency than graphene oxide (GO), as a photothermal agent for cancer photothermal therapy (PTT). By conjugating a photosensitizer IR780 to rGO, the IR780-rGO could be endowed with reactive oxygen species (ROSs) generation ability for concurrent photodynamic therapy (PDT). The IR780-rGO was coated with hyaluronic acid (HA) by electrostatic interaction to facilitate its intracellular uptake by U87 glioblastoma cells. The IR780-rGO/HA was loaded with doxorubicin (DOX) for chemotherapy (CT), to develop a pH-responsive drug delivery nano-platform for targeted multimodal cancer CT/PTT/PDT. We fully characterized the properties of all nanocomposites during the synthesis steps. The high loading efficiency of DOX on IR780-rGO-HA provides 3mg/mg drug loading, while IR780-rGO-HA/DOX shows 3 times higher drug release at endosomal pH value (pH5) than at pH7.4. The mechanism for PTT/PDT was confirmed from the ability of IR780-rGO-HA to induce time-dependent temperature rise, synthesis of heat shock protein 70 (HSP70) and generation of intracellular ROSs, after exposure to 808nm near infrared (NIR) laser light. The nano-vehicle IR780-rGO-HA shows high biocompatibility toward 3T3 fibroblast and U87 cancer cell lines, as well as enhanced intracellular uptake by U87 through active targeting. This translates into increased cytotoxicity of IR780-rGO-HA/DOX, by lowering the drug half-maximal inhibitory concentration (IC50) from 0.7 to 0.46μg/mL. This IC50 is further decreased to 0.1μg/mL by irradiation with NIR laser for 3min at 1.5W/cm2. The elevated cancer cell killing mechanism was supported from flow cytometry analysis, where the highest cell apoptosis/necrosis rate was observed in combination CT/PTT/PDT. Using xenograft tumor model created by subcutaneous implantation of U87 cells in nude mice, IR780-rGO-HA/DOX delivered through intravenous (IV) injection and followed with 808nm laser treatment for 5min at 1.5W/cm2 results in the lowest tumor growth rate, with negligible change of tumor volume from its original value at the end 20-day observation period. The therapeutic efficacy was supported from inhibited cell proliferation rate, increased cell apoptosis rate, and increased production of HSP70 from immunohistochemical staining of tumor tissue slices. The safety of the NIR-assisted multimodal cancer treatment could be confirmed from non-significant change of body weight and hematological parameters of blood sample. Taken together, we conclude that IV delivery of IR780-rGO-HA/DOX plus NIR laser treatment is an effective nanomedicine approach for combination cancer therapy.

Bone Marrow Mesenchymal Stem Cell (BMSC)-Exosomes Overexpressing miR-141 Inhibit the Malignant Biological Behavior of Glioma Cells via Wnt Signaling

Our study explores whether BMSC-exosomes overexpressing miR-141 can regulate Wnt signal to inhibit the malignant biological behavior of glioma cells. Thirty healthy mice were selected to construct a glioma mouse model and assigned randomly into the control group, miR-141 NC group, and miR-141 mimic group followed by analysis of cell proliferation, apoptosis, protein expression and mRNA expression by MTT method, flow cytometry, Western blot and RT-PCR methods. Compared with the other two groups, miR-141 mimic group showed reduced number of cell proliferation at 24 h and 48 h, decreased cell migration and invasion ability, and the increased cell apoptosis rate (P &lt; 0.05). In miR-141 mimic group, the protein expression of miR-141 was the highest, while the protein expression of β-catenin, survivin and c-myc was the lowest (P &lt; 0.05). In conclusion, BMSC-exosomes overexpressing miR-141 can inhibit the malignant biological behavior of GC cells possibly by inhibiting the activation of Wnt signaling pathway.

MiR-22-5p regulates the self-renewal of spermatogonial stem cells by targeting EZH2.

MiRNAs play an important role in spermatogonial stem cells (SSCs). The purpose of this study was to investigate the basic function of miR-22-5p in cryptorchidism. The results of RT-PCR, western blot, and immunohistochemistry showed that miR-22-5p was increased while EZH2 decreased in the testicular tissues of patients with cryptorchidism. Overexpression of miR-22-5p inhibited the proliferation of SSCs, increased cell apoptosis rate, and reduced expression of SSC marker proteins (GDNF and DAZL); however, knockout of miR-22-5p has the opposite effect. The Luciferase reporter gene assays demonstrated that EZH2 is a direct target of miR-22-5p. Moreover, EZH2 overexpression could reverse the effect of miR-22-5p mimic on SSCs’ proliferation, apoptosis, and expression of SSC marker proteins. Our results demonstrated that miR-22-5p regulates SSCs’ self-renewal by targeting EZH2, which indicated that miR-22-5p may serve as a biological marker for the treatment of infertility caused by cryptorchidism.

The microRNA-520a-3p inhibits invasion and metastasis by targeting NF-kappaB signaling pathway in non-small cell lung cancer.

To identify the expression of miR-520a-3p and AKT1 in non-small cell lung cancer cells (NSCLC) and the mechanism in inhibiting cell invasion and metastasis by targeting NF-kappaB signaling pathway. Bioinformatics analysis and dual luciferase reporter gene assay were used to predict and verify the targeting relationship between miR-520a-3p and AKT1. EdU assay was used to detect the proliferation of NSCLC cells. Flow cytometry detected the apoptosis of NSCLC cells. Transwell assay tested the invasion ability of NSCLC cells. qRT-PCR measured the expression of miR-520a-3p and AKT1 mRNA in NSCLC cells; while western blotting was adopted to detect the protein expressions of AKT1, Ki67, CyclinD1, Bax, Bcl-2, MMP-2, MMP-9, NF-kB p65, IkBs kinase (IKK), NF-kB inducing kinase (NIK). Bioinformatics analysis suggested that miR-520a-3p could target AKT1. miR-520a-3p could regulate the expression of AKT1 negatively. Compared to mimic-NC group, miR-520a-3p mimic group had increased expressions of miR-520a-3p and Bax, while decreased expressions of AKT1, Ki67, CyclinD1, Bcl-2, MMP-2, MMP-9, NF-kB p65, IKK and NIK, reduced cell proliferation, invasion, and increased cell apoptosis rate (all P < 0.05). Compared to inhibitor NC group, miR-520a-3p inhibitor group had decreased expressions of miR-520a-3p and Bax, but increased expressions of AKT1, Ki67, CyclinD1, Bcl-2, MMP-2, MMP-9, NF-kB p65, IKK and NIK, promoted cell proliferation, invasion, and suppressed cell apoptosis rate (all P < 0.05). Overexpression of miR-520a-3p can target and downregulate the expression of AKT1 to inhibit the invasion and metastasis of NSCLC via suppressing the activation of NF-kappaB signaling pathway.

AMG-510 Plus Cetuximab Enhance Radiosensitivity in KRAS p.G12C Mutant Colorectal Carcinoma Cell Lines via Increasing Apoptosis and Inducing G1/S Arrest

<h3>Purpose/Objective(s)</h3> KRAS p.G12C mutation is rare (∼3%) but with a dismal prognosis in colorectal carcinoma (CRC). AMG-510 is a first-in-class KRAS p.G12C inhibitor, which shows clinical efficacy in KRAS p.G12C mutant solid tumors including non-small cell lung cancer and CRC. The addition of cetuximab could revert resistance to AMG-510 in CRC. Herein, we evaluate the anti-cancer effect of AMG-510 plus cetuximab in combination with radiation in CRC. <h3>Materials/Methods</h3> Two KRAS p.G12C mutant CRC cell lines were treated with AMG-510, AMG-510 plus cetuximab, irradiation (IR), and the combination of IR and AMG-510 plus cetuximab. Clonogenic assays were used to study the radiosensitizing effect of AMG-510 combined with cetuximab. Immunofluorescence staining of rH2AX was used to detect double-strand break (DSB) repair. Cell proliferation was performed using a cell counting kit. Then, we performed flow cytometry analysis to detect cell apoptosis rate and cell cycle distribution. <h3>Results</h3> First, we found that AMG-510 plus cetuximab enhanced the radiosensitivity of X-ray in the SW837 cell line (sensitization enhancement ratio: 1.32). The combination of AMG-510 and cetuximab reduced cell proliferation and increased cell apoptosis rate and DNA damage, with or without irradiation. The flow cytometry analysis of the cell cycle showed G1/S phase arrest after AMG-510 monotherapy and AMG-510 plus cetuximab treatment. Moreover, the P53/Rb/P21 pathway was activated after the addition of AMG-510 with or without cetuximab. <h3>Conclusion</h3> The combination of AMG-510 and cetuximab might be a potent treatment as a radiosensitizer. However, more studies in vivo and clinical trials should be performed to further evaluate this regimen in the future.

Cryopreserved-pollen viability is regulated by NO-induced programmed cell death.

After cryopreservation, the NO content in pollen increased, inducing programmed cell death as a key reason for reduced viability. Low recovery of biomaterials after cryopreservation is a bottleneck that limits the application of this technology. At present, the mechanism of viability decline after cryopreservation is not fully understood. In this study, the effects of nitric oxide (NO) on programmed cell death (PCD) and its relationship with viability were investigated, using Paeonia lactiflora 'Fen Yu Nu' pollen with significantly decreased viability after cryopreservation. The results showed that: the activity of caspase-3-like and caspase-9-like protease and the apoptosis rate of pollen cells were significantly increased, the expression level of the promoting PCD (pro-PCD) genes was up-regulated, while the expression level of the inhibiting PCD (anti-PCD) genes was down-regulated after preservation in liquid nitrogen (LN); the NO content in pollen cells increased significantly after LN exposure. The correlation analysis showed that NO was significantly correlated with pollen viability and all indicators of PCD. The addition of a NO carrier SNP after LN storage reduced pollen viability, increased endogenous NO content, decreased mitochondrial membrane potential level, activated caspase-3-like and caspase-9-like protease in pollen cells, and increased cell apoptosis rate. The expression levels of pro-PCD genes PDCD2 and ATG8CL were significantly up-regulated, while the expression levels of anti-PCD genes DAD1, BI-1 and LSD1 were significantly down-regulated. The addition of NO scavenger c-PTIO improved pollen viability, and produced the opposite effect of sodium nitroferricyanide (III) dihydrate (SNP), but did not change the mitochondrial membrane potential. These results suggest that NO induced PCD during the cryopreservation of pollen, which was one of the reasons for the significant decrease of pollen viability after cryopreservation.

Silencing LncRNA SNHG7 alleviates hypoxia/reoxygenation-induced cardiomyocyte damage by regulating the expression of miR-181b-5p

To study the effect of silencing LncRNA SNHG7 on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and its targeted regulation on miR-181b-5p. Rat cardiomyocytes H9c2 were cultured in vitro and randomly divided into control group, H/R group, H/R + si-NC group, H/R + si-SNHG7 group, H/R + si-SNHG7 + anti-miR-NC group and H/R + si-SNHG7 + anti-miR-181b-5p group. The content of lactate dehydrogenase (LDH), malondialedhyde (MDA) and the activity of superoxide dismutase (SOD) were detected. Flow cytometry was carried out to detect the rate of apoptosis. qRT-PCR was used to detect the expression of SNHG7 and miR-181b-5p. Dual luciferase report experiment was used to verify the targeting relationship between SNHG7 and miR-181b-5p. Western blotting was used to detect the expression of Bax and Bcl-2. Compared with the control group, the H/R group showed significantly increased SNHG7 expression in cardiomyocytes, reduced miR-181b-5p expression, higher levels of LDH and MDA, reduced activity of SOD, increased cell apoptosis rate, higher level of Bax protein, and reduced level of Bcl-2 protein (all P< 0.05). Compared with the H/R and H/R + si-NC groups, the H/R + si-SNHG7 group had significantly reduced level of LDH and MDA, increased activity of SOD, reduced apoptosis rate, reduced level of Bax protein, increased level of Bcl-2 protein (all P< 0.05). The dual luciferase report experiment confirmed that SNHG7 could target miR-181b-5p. Interference with the expression of miR-181b-5p could reduce the effect of silencing SNHG7 on H/R-induced cardiomyocyte oxidative stress and apoptosis. Silencing SNHG7 may inhibit H/R-induced cardiomyocyte oxidative stress and apoptosis by up-regulating the expression of miR-181b-5p, thereby exerting a protective effect on cardiomyocytes.