SIRT3 Regulates the ROS-FPR1/HIF-1α Axis under Hypoxic Conditions to Influence Lung Cancer Progression.

Hypoxia-inducible factor (HIF-1α) is a therapeutic target in lung cancer, and the deacetylase sirtuin 3 (SIRT3) is closely associated with tumorigenesis. Formyl peptide receptor 1 (FPR1) is involved in a wide range of physiopathological processes in various tumor cells. We explored whether SIRT3 affects the development of lung cancer by regulating the reactive oxygen species (ROS)-FPR1/HIF-1α axis under hypoxic conditions. The effects of SIRT3 overexpression on the levels of FPR1, HIF-1α, ROS, inflammatory factors, and cell proliferation and migration in A549 cells under hypoxic conditions were assessed in combination with the FPR1 inhibitor. BALB/c nude mice were subcutaneously injected with cancer cells transfected/untransfected with SIRT3 overexpressing lentiviral vectors. Immunohistochemistry and enzyme-linked immunosorbent assay were performed to detect SIRT3 expression and the expression levels of IL-1ß, TNF-α, and IL-6, respectively, in tumor tissues. Cell proliferation, invasion, migration, and IL-1ß, TNF-α, IL-6, and ROS levels were significantly higher in the Hypoxia group than in the Control group. Moreover, the mRNA and protein expression levels of SIRT3 were significantly down-regulated, whereas they were significantly up-regulated for FPR1 and HIF-1α. In contrast, SIRT3 overexpression in a hypoxic environment inhibited cell proliferation, invasion, and migration, decreased IL-1ß, TNF-α, IL-6, and ROS levels, up-regulated the mRNA and protein expression levels of SIRT3, and down-regulated the mRNA and protein expression levels of FPR1 and HIF-1α. In addition, we found the same results in tumorigenic experiments in nude mice. SIRT3 in hypoxic environments may affect tumor cell proliferation, invasion, migration, and inflammation levels via the ROS-FPR1/HIF-1α axis, thereby inhibiting tumor cell development.

Influence of Lacidophilin Vaginal Capsules plus rh-IFN-α2b on Efficacy, Vaginal Microecology, and Safety of Patients with HPV Infection.

Background: Human papillomavirus (HPV) is a self-limiting disease, and there is no specific antiviral drug at present. Purpose: Here, we analyzed the influence of lacidophilin vaginal capsules plus recombinant human interferon α-2b (rh-IFN-α2b) on efficacy, vaginal microecology, and safety of patients with HPV infection. Two hundred cases of HPV infection admitted between January 2019 and December 2020 were retrospectively collected. Of them, 90 cases receiving rh-IFN-α2b intervention were assigned to the control group (CG), and 110 cases given lacidophilin vaginal capsules in addition to rh-IFN-α2b were included in the research group (RG). Baseline data, efficacy, vaginal microecology, microecological restoration recovery, and incidence of adverse events (AEs) were compared between the two groups. Results: The analyses revealed nonsignificant difference in baseline data between RG and CG, indicating comparability. In terms of efficacy, RG showed a statistically higher negative conversion ratio (NCR) than CG (57.27% vs. 47.78%), as well as an obviously higher overall response rate (ORR) (90.90% vs. 72.22%). As far as the vaginal microecology was concerned, the incidence rates of catalase-positive, sialidase-positive, abnormal microbial density, and abnormal microbial diversity of RG were significantly lower compared with CG, but no evident differences were determined in Trichomonas vaginalis-positive and Candida-positive. As for microecological restoration, RG had an obviously higher vaginal microecological recovery rate than CG (90.00% vs. 65.56%), as well as notably lower vaginal secretion pH and Nugent score. On the other hand, RG and CG showed no statistical significance in the incidence of AEs (12.73% vs. 13.33%). Conclusions: The main contributions of this study are as follows: first, it is confirmed that lacidophilin vaginal capsules plus rh-IFN-α2b has better clinical effects than rh-IFN-α2b alone in HPV-infected patients; second, it demonstrates that the combination therapy can significantly improve NCR and ORR, without increasing the incidence of AEs, and is beneficial to improve patients' vaginal microecology and promote its restoration from the multidimensional aspects of efficacy, safety, and vaginal microecology and its recovery. Our findings provide valuable clinical evidence for the drug treatment of HPV-infected patients.

CTB-193M12.5 Promotes Hepatocellular Carcinoma Progression via Enhancing NSD1-Mediated WNT10B/Wnt/ß-Catenin Signaling Activation.

Background: Hepatocellular carcinoma (HCC) is the second lethal malignancy among all cancers. Many molecular alterations have been found in HCC. However, the interactions and modulatory mechanisms among these molecules in HCC are still unclear. Methods: CTB-193M12.5 expression in tissues and cells were detected by quantitative polymerase chain reaction (qPCR). In vitro experiments were conducted to evaluate the function of CTB-193M12.5 in cell proliferation, apoptosis, migration and invasion. The interaction between CTB-193M12.5 and nuclear receptor binding SET domain-containing protein 1 (NSD1) was assessed by RNA-protein pull-down and RNA immunoprecipitation assays. The roles of CTB-193M12.5 on WNT10B and Wnt/ß-catenin signaling was detected by chromatin immunoprecipitation assay, qPCR, Western blot, and dual luciferase reporter assay. Results: We identified a novel prognosis-related long noncoding RNA (lncRNA) CTB-193M12.5 in HCC. CTB-193M12.5 was upregulated in HCC and its high expression was correlated with alpha fetoprotein, large tumor size, aggressive clinical characteristics, and poor survival. Functional experiments showed that CTB-193M12.5 enhanced HCC cellular proliferation, suppressed HCC cellular apoptosis, and promoted HCC cellular migration and invasion. CTB-193M12.5 knockdown exerted opposite effects in HCC. Mechanistic investigation demonstrated that CTB-193M12.5 was mainly distributed in nucleus. Histone methyltransferase NSD1 was identified as a CTB-193M12.5 interactor. CTB-193M12.5 bound and recruited NSD1 to the promoter of WNT10B, leading to an increase in di-methylation of histone H3 at lysine 36 (H3K36me2) and the reduction of tri-methylation of histone H3 at lysine 27 (H3K27me3) at WNT10B promoter. Therefore, CTB-193M12.5 epigenetically activated WNT10B transcription. Through upregulating WNT10B, CTB-193M12.5 further activated Wnt/ß-catenin signaling. Functional rescue experiments demonstrated that overexpression of WNT10B reversed the tumor suppressive roles of CTB-193M12.5 knockdown, while Wnt/ß-catenin signaling inhibitor ICG-001 abolished the oncogenic roles of CTB-193M12.5 overexpression. Conclusion: CTB-193M12.5 was a highly expressed and poor prognosis-related lncRNA in HCC. CTB-193M12.5 functioned as an oncogenic lncRNA through promoting NSD1-mediated WNT10B/Wnt/ß-catenin signaling activation.

Metformin suppresses lung adenocarcinoma by downregulating long non-coding RNA (lncRNA) AFAP1-AS1 and secreted phosphoprotein 1 (SPP1) while upregulating miR-3163.

AFAP1-AS1 plays a pro-tumor role in lung cancer. However, no investigation has focused on whether it is involved in the anticancer activity of metformin (Met) in the treatment of lung adenocarcinoma (LUAD). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect the expression of long non-coding (lnc)RNA AFAP1-AS1, the microRNA (miR)-3163, and secreted phosphoprotein 1 (SPP1) in LUAD tissues, or of A549 and H3122 cells. Cell Counting Kit-8, wound scratch, and cell invasion assays were performed to evaluate the effect of the overexpression of lncRNA AFAP1-AS1, miR-3163, and SPP1 on the malignant behaviors of A549 and H3122 cells. Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway-related proteins were detected by Western blot analysis. Dual luciferase reporter or RIP assays were used to determine the interplay between AFAP1-AS1 and miR-3163, or of miR-3163 and SPP1. Met inhibits the malignant characteristics of A549 and H3122 cells in vitro. GEPIA database analysis showed that AFAP1-AS1 is a highly expressed lncRNA in LUAD tissues, which was validated by RT-qPCR. Overexpression of AFAP1-AS1 suppressed the met-mediated anti-tumor activity in A549 and H3122 cells, while AFAP1-AS1 silencing promoted it. Met inhibited AFAP1-AS1 expression, which resulted in reduced proliferation, migration, and invasion in A549 and H3122 cells. This led to AFAP1-AS1-mediated suppression of miR-3163 and, subsequently, the upregulation of SPP1. Met exerts its antitumor activities by regulating the AFAP1-AS1/miR-3163/SPP1/PI3K/Akt/mTOR axis. Our findings deepen our understanding of mechanisms underlying anti-tumor effect of Met in LUAD.

lncRNA MT1JP-overexpression abolishes the silencing of PTEN by miR-32 in hepatocellular carcinoma.

Previous studies have shown that long non-coding RNA (lncRNA) MT1JP plays a role as a tumor suppressor in several types of cancer. The present study aimed to explore the role of MT1JP in hepatocellular carcinoma (HCC). Paired HCC and non-tumor tissues from 64 patients with HCC were subjected to RNA isolation and reverse transcription-quantitative PCR (RT-qPCR) to analyze the differential expression of MT1JP, microRNA (miR)-32 and phosphatase and tensin homolog (PTEN) in HCC. Cell transfections, followed by RT-qPCR and western blotting, were carried out to investigate the interactions among MT1JP, miR-32 and PTEN. The role of MT1JP, miR-32 and PTEN in regulating HCC cell proliferation was assessed using a Cell Counting Kit-8 assay. It was found that MT1JP was downregulated in HCC cancer tissues compared with that in non-cancer tissues. Survival analysis showed that patients with low MT1JP expression levels exhibited a significantly higher 5-year overall survival rate compared with patients with high MT1JP levels. The expression of MT1JP in HCC tissues was positively associated with PTEN and negatively associated with miR-32. Overexpression of MT1JP increased the expression levels of PTEN and decreased the expression levels of miR-32. Overexpression of miR-32 did not affect the expression of MT1JP but decreased the expression levels of PTEN and attenuated the effect of overexpression of MT1JP on the expression of PTEN. Overexpression of MT1JP and PTEN decreased the proliferation of HCC cells. Overexpression of miR-32 played an opposite role and attenuated the effects of overexpression of MT1JP. Therefore, MT1JP may upregulate PTEN by downregulating miR-32 to regulate HCC cell proliferation.

KB-68A7.1 Inhibits Hepatocellular Carcinoma Development Through Binding to NSD1 and Suppressing Wnt/ß-Catenin Signalling.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies with extremely poor prognosis. Therefore, revealing the critical molecules involved in HCC progression and prognosis is urgently needed. In this study, through combining public dataset and our cohort, we found a novel prognosis-related long non-coding RNA KB-68A7.1 in HCC. KB-68A7.1 was lowly expressed in HCC, whose low expression was associated with large tumour size, aggressive clinical characteristic, and poor survival. Gain- and loss-of-function assays demonstrated that KB-68A7.1 restricted HCC cellular proliferation, induced HCC cellular apoptosis, and suppressed HCC cellular migration and invasion in vitro. Xenograft assays demonstrated that KB-68A7.1 suppressed HCC tumour growth and metastasis in vivo. These functional assays suggested KB-68A7.1 as a tumour suppressor in HCC. Histone methyltransferase nuclear receptor binding SET domain-containing protein 1 (NSD1) was found to bind to KB-68A7.1. KB-68A7.1 was mainly distributed in the cytoplasm. The binding of KB-68A7.1 to NSD1 sequestrated NSD1 in the cytoplasm, leading to the reduction in nuclear NSD1 level. Through decreasing nuclear NSD1 level, KB-68A7.1 reduced di-methylation of histone H3 at lysine 36 (H3K36me2) and increased tri-methylation of histone H3 at lysine 27 (H3K27me3) at the promoter of WNT10B, a target of NSD1. Thus, KB-68A7.1 repressed WNT10B transcription. The expression of WNT10B was negatively correlated with that of KB-68A7.1 in HCC tissues. Through repressing WNT10B, KB-68A7.1 further repressed Wnt/ß-catenin signalling. Functional rescue assays showed that overexpression of WNT10B reversed the tumour-suppressive roles of KB-68A7.1, whereas the oncogenic roles of KB-68A7.1 depletion were abolished by Wnt/ß-catenin signalling inhibitor. Overall, this study identified KB-68A7.1 as a lowly expressed and prognosis-related lncRNA in HCC, which suppressed HCC progression through binding to NSD1 and repressing Wnt/ß-catenin signalling.

Inhibition of formyl peptide receptor 1 activity suppresses tumorigenicity in vivo and attenuates the invasion and migration of lung adenocarcinoma cells under hypoxic conditions in vitro.

BACKGROUND: Tumor hypoxia has been widely reported to promote metastasis. However, the molecular mechanisms underlying metastasis-associated hypoxia remain unclear. Formyl peptide receptor 1 (FPR1) has been reported to be highly expressed under hypoxic conditions. This study aimed to explore the role of FPR1 in tumor cells under hypoxic conditions. METHODS: The expressions of FPR1 and hypoxia-inducible factor 1α (HIF-1α) in A549 cells under hypoxic conditions were detected using western blot. The expression of FPR1 in A549 cells under hypoxic conditions was suppressed using the FPR1 antagonist Boc2. Wound-healing and Transwell assays were performed to investigate the migration and invasion of cells. Furthermore, the tumorigenicity of A549 cells was evaluated by constructing a hypoxic mouse model of lung adenocarcinoma. The expression levels of HIF-1α and FPR1 in tumors were measured by real-time polymerase chain reaction (PCR) and western blot. RESULTS: The expression levels of FPR1 and HIF-1α increased in a time-dependent manner after exposure to hypoxic conditions. Wound-healing and Transwell assays showed that hypoxia promoted the migration and invasion abilities of A549 cells, whereas downregulation of FPR1 blocked the effects of hypoxia on A549 cells. Our in vivo results demonstrated that the tumor volumes and weights of mice exposed to hypoxic conditions were significantly higher than those of untreated mice. Furthermore, the downregulation of FPR1 blocked the effects of hypoxia in the mice. Meanwhile, the expressions of HIF-1α and FPR1 at the protein and mRNA levels were increased after hypoxic exposure, whereas FPR1 antagonist Boc2 suppressed the effect of hypoxia on the expression of FPR1. CONCLUSIONS: Our results suggest that FPR1 could be a therapeutic target for suppressing the invasion and tumorigenicity of lung adenocarcinoma cells.

CRISPR/Cas9-mediated knockout of NSD1 suppresses the hepatocellular carcinoma development via the NSD1/H3/Wnt10b signaling pathway.

BACKGROUND: The NSD family of histone lysine methyltransferases have emerged as important biomarkers that participate in a variety of malignancies. Recent evidence has indicated that somatic dysregulation of the nuclear receptor binding SET domain-containing protein 1 (NSD1) is associated with the tumorigenesis in HCC, suggesting that NSD1 may serve as a prognostic target for this malignant tumor. However, its mechanism in human hepatocellular carcinoma (HCC), the major primary malignant tumor in the human liver, remains unclear. Hence, we investigated how NSD1 regulated HCC progression via regulation of the Wnt/ß-catenin signaling pathway. METHODS: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis was performed to identify the expression of NSD1 in HCC cells and clinically obtained tissues. The relationship between NSD1 expression and prognosis was analyzed by Kaplan-Meier survival curve. Further, a NSD1 knockout cell line was constructed by CRISPR/Cas9 genomic editing system, which was investigated in a battery of assays such as HCC cell proliferation, migration and invasion, followed by the investigation into NSD1 regulation on histone H3, Wnt10b and Wnt/ß-catenin signaling pathway via ChIP. Finally, a nude mouse xenograft model was conducted in order to assess tumorigenesis affected by NSD1 knockout in vivo. RESULTS: NSD1 was overexpressed in HCC tissues and cell lines in association with poor prognosis. Knockout of NSD1 inhibited the proliferation, migration and invasion abilities of HCC cells. CRISPR/Cas9-mediated knockout of NSD1 promoted methylation of H3K27me3 and reduced methylation of H3K36me2, which inhibited Wnt10b expression. The results thereby indicated an inactivation of the Wnt/ß-catenin signaling pathway suppressed cell proliferation, migration and invasion in HCC. Moreover, these in vitro findings were reproduced in vivo on tumor xenograft in nude mice. CONCLUSION: In conclusion, the study provides evidence that CRISPR/Cas9-mediated NSD1 knockout suppresses HCC cell proliferation and migration via the NSD1/H3/Wnt10b signaling pathway, suggesting that NSD1, H3 and Wnt10b may serve as potential targets for HCC.

Downregulation of long noncoding RNA LINC00460 expression suppresses tumor growth in vitro and in vivo in gastric cancer.

BACKGROUND: Long noncoding RNA have been indicated to be involved in tumor development. However, the role of LINC00460 in gastric cancer (GC) still remains large unknown. The current study is designed aiming at determining the effects of LINC00460 on GC progression. PATIENTS AND METHODS: The expression of LINC00460 in GC tissues and cells were detected by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). The cell proliferation, cell cycle distribution and cell invasion were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), flow cytometry analysis and transwell cell invasion assays. Western blot analysis was used to detect the WNT signaling related protein expression. Tumor xenograft assay was used to detect the effects of LINC00460 in vivo. RESULTS: In the study, we demonstrated that LINC00460 expression was higher in gastric cancer tissues compared to adjacent normal tissues. Higher LINC00460 expression associated with lymph node metastasis and advanced TNM stage. Furthermore, we showed that higher LINC00460 expression predicted a poor disease-free survival (DFS) and overall survival (OS) time of gastric cancer. Multivariate analysis showed that LINC00460 expression was an independent risk factor of GC prognosis. Furthermore, in vitro, we demonstrated that inhibition of LINC00460 expression suppressed cell proliferation, S phase cell number and cell invasion of gastric cancer cells compared to the control groups. In addition, we showed that downregulation of LINC00460 inhibited the Wnt/ß-catenin signaling by downregulating c-Myc and ß-catenin expression, which indicated LINC00460 could promote cell proliferation and invasion by activating Wnt/ß-catenin signaling. In vivo, we also demonstrated that LINC00460 knockdown significantly suppressed cell proliferation. CONCLUSIONS: LINC00460 is a new type of molecule involved in the development of GC, which may become a potential target for the treatment of GC.