Identification of colon adenocarcinoma necroptosis subtypes and tumor antigens for the development of mRNA vaccines.

Background: Colon adenocarcinoma (COAD) is a serious public health issue due to high incidence and mortality rate. This study aimed to identify possible tumor antigens and necroptosis subtypes of COAD for the development of mRNA vaccines and the selection of appropriate patients for precision therapy. Methods: Gene expression profiles and clinical information for COAD were obtained from The Cancer Genome Atlas and Gene Expression Omnibus, respectively. We comprehensively studied the alterations in necroptosis-related genes (NRGs) using cBioPortal, and screened the hub NRGs associated with the prognosis of patients with COAD using Gene Expression Profiling Interactive Analysis 2. Consensuses clustering analysis was performed to identify necroptosis subtypes. Weighted gene co-expression network analysis (WGCNA) was used to identify the co-expression modules of the NRGs. The necroptosis landscape of COAD was assessed using graph learning-based dimensionality reduction. Finally, a drug sensitivity analysis of the two necroptosis subtypes was performed. Findings: Two tumor antigens, BLC-2-associated X protein (BAX) and interleukin 1 beta (IL1B) were identified based on their associations with prognosis of patients and antigen presenting cell infiltration. Two necroptosis subtypes (N1 and N2) were distinguished in patients with COAD, and they were characterized by their differential survival status and molecular expression levels of immune checkpoint proteins and immunogenetic cell death modulators. Furthermore, the necroptosis landscape of COAD indicated that individual patients had obvious heterogeneity. Co-expression modules were identified using WGCNA, and the hub NRGs were found to be involved in various immune processes. Drug sensitivity analysis indicated that there were significant differences in drug sensitivity between the N1 and N2 subtypes. Cell experiments suggested that both overexpression of BAX and IL1B promoted necroptosis of COAD cells and enhanced the cytotoxicity of CD8+ T cells. Interpretation: BAX and IL1B are potential antigens for the development of anti-COAD mRNA vaccines, specifically for patients with the N2 subtype. Consequently, this study will guide the development of more effective immunotherapeutic approaches and the identification of appropriate patients.

Prognostic value of autophagy-related genes based on single-cell RNA-sequencing in colorectal cancer.

Background: Colorectal cancer (CRC) is the second most common cancer in China. Autophagy plays an important role in the initiation and development of CRC. Here, we assessed the prognostic value and potential functions of autophagy-related genes (ARGs) using integrated analysis using single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) and RNA sequencing (RNA-seq) data from The Cancer Genome Atlas (TCGA). Methods: We analyzed GEO-scRNA-seq data from GEO using various single-cell technologies, including cell clustering, and identification of differentially expressed genes (DEGs) in different cell types. Additionally, we performed gene set variation analysis (GSVA). The differentially expressed ARGs among different cell types and those between CRC and normal tissues were identified using TCGA-RNA-seq data, and the hub ARGs were screened. Finally, a prognostic model based on the hub ARGs was constructed and validated, and patients with CRC in TCGA datasets were divided into high- and low-risk groups based on their risk-score, and immune cells infiltration and drug sensitivity analyses between the two groups were performed. Results: We obtained single-cell expression profiles of 16,270 cells, and clustered them into seven types of cells. GSVA revealed that the DEGs among the seven types of cells were enriched in many signaling pathways associated with cancer development. We screened 55 differentially expressed ARGs, and identified 11 hub ARGs. Our prognostic model revealed that the 11 hub ARGs including CTSB, ITGA6, and S100A8, had a good predictive ability. Moreover, the immune cell infiltrations in CRC tissues were different between the two groups, and the hub ARGs were significantly correlated with the enrichment of immune cell infiltration. The drug sensitivity analysis revealed that the patients in the two risk groups had difference in their response to anti-cancer drugs. Conclusion: We developed a novel prognostic 11-hub ARG risk model, and these hubs may act as potential therapeutic targets for CRC.

Increased expression of NOP14 is associated with improved prognosis due to immune regulation in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third most common of cancer-related deaths. Nucleolar protein 14 (NOP14) is known to play different roles in diverse types of cancers. However, little is known about its roles in CRC. Here, we assessed the prognostic value and functions of NOP14 in CRC using the data from The Cancer Genome Atlas (TCGA) and validated them based on the data from Gene Expression Omnibus (GEO). METHODS: NOP14 mRNA and protein data in CRC was obtained from the TCGA, GEO, human protein atlas (HPA), and UALCAN databases. Survival and Cox regression analysis was performed to assess the prognostic value of NOP14 in CRC patients. Next, to evaluate the potential functions of NOP14, a protein-protein interaction (PPI) network was constructed and gene set enrichment analysis (GSEA) of differential expression genes (DEGs) associated with dysregulated NOP14 was performed. Finally, to investigate the immune response associated with NOP14 expression in CRC, we analyzed the correlations between immune cells infiltration and NOP14 expression level. Additionally, the correlations between immune molecule expression levels with NOP14 expression level were analyzed. RESULTS: High NOP14 mRNA expression was observed in CRC tissues based on the data from TCGA and GEO datasets. Similarly, high NOP14 protein levels were found in CRC tissues according to the immunohistochemical images from HPA. Interestingly, high NOP14 expression level was associated with an improved prognosis in CRC patients. Univariate and multivariate Cox regression analysis indicated that high NOP14 expression level was an independent protective factor for CRC patients. With the support of PPI network analysis, we found several risk genes interacted with NOP14. GSEA revealed that high NOP14 expression inhibited several signal pathways involved in tumor formation and development. Additionally, high NOP14 expression was positively associated with most kinds of immune cell infiltrations and the expression levels of some molecules related to immune activation. CONCLUSION: Altogether, these results indicated that high NOP14 expression leads to improved prognosis in CRC patients by inhibiting the signaling pathways involved in tumor growth and promoting the immune responses.

Troglitazone inhibits hepatic oval cell proliferation by inducing cell cycle arrest through Hippo/YAP pathway regulation.

Hepatic oval cells have strong proliferation and differentiation capabilities and are activated when chronic liver injury occurs or when liver function is severely impaired. Peroxisome proliferation-activated receptors (PPARs) are ligand-dependent, sequence-specific nuclear transcription factors. PPARγ is closely related to liver diseases (such as liver cancer, liver fibrosis and non-alcoholic fatty liver disease). As the main effector downstream of the Hippo signaling pathway, YAP can activate the hepatic progenitor cell program, and different expression or activity levels of YAP can determine different liver cell fates. We found that troglitazone (TRO), a classic PPARγ activator, can inhibit the growth of hepatic oval cells, and flow cytometry results showed that TRO inhibited the growth of WB-F344 cells by arresting the cells in the G0/1 phase. Western blot results also confirmed changes in G0/1 phase-related protein expression. Further experiments showed that PPARγ agonists induced hepatic oval cell proliferation inhibition and cell cycle G0/1 phase arrest through the Hippo/YAP pathway. Our experiment demonstrated, for the first time, the relationship between PPARγ and the Hippo/YAP pathway in liver oval cells and revealed that PPARγ acts as a negative regulator of liver regeneration by inhibiting the proliferation of oval cells.

Dihydromyricetin-mediated inhibition of the Notch1 pathway induces apoptosis in QGY7701 and HepG2 hepatoma cells.

AIM: To investigate whether Dihydromyricetin (DHM) inhibits cell proliferation and promotes apoptosis by downregulating Notch1 expression. METHODS: The correlation between Notch1 and Hes1 (a Notch1 target molecule) expression in hepatoma samples was confirmed by qRT-PCR. In addition, MTT assays, flow cytometry and TUNEL analysis showed that DHM possessed strong anti-tumor properties, evidenced not only by reduced cell proliferation but also by enhanced apoptosis in QGY7701 and HepG2 hepatocellular carcinoma (HCC) cells. The expressions of Notch1, Hes1, Bcl-2 and Bax were determined by Western blot. RESULTS: Among the tested samples (n = 64), the expression levels of Notch1 (75% of patients) and Hes1 (79.7% of patients) mRNA in tumor tissues were higher than in the normal liver tissues. There was a negative correlation between the expression of Notch1 and the degree of differentiation and positively correlated with the Alpha Fetal Protein concentration. The viability of HCC cells treated with DHM was significantly inhibited in a dose and time-dependent manner. Apoptosis was induced in HepG2 and QGY7701 cell lines following 24 h of DHM treatment. After treatment with DHM, the protein expression of Notch1 was downregulated, the apoptosis-related protein Bax was upregulated and Bcl2 was downregulated. Notch1 siRNA further enhanced the anti-tumor properties of DHM. CONCLUSION: Notch1 is involved in the development of HCC and DHM inhibits cell proliferation and promotes apoptosis by down-regulating the expression of Notch1.

Expression of Wnt3a in hepatocellular carcinoma and its effects on cell cycle and metastasis.

Invasion and metastasis are the primary causes of mortality from hepatocellular carcinoma (HCC). Effective inhibition against participants in the tumourigenesis and metastasis process is critical for treatment of HCC. Wnt3a is involved in the development and metastasis of many malignant tumours. However, the specific mechanisms of Wnt3a-mediated cell proliferation, invasion and metastasis in HCC remain unclear. In this study, we found that Wnt3a and its target gene c­Myc showed higher expression in tumour tissues than normal liver tissues in HCC patients; 71.8% of the cases studied had high Wnt3a and c­Myc expression levels (n=32); Wnt3a expression positively correlated with its target genes MMP­7 and c­Myc. Intriguingly, the expression of Wnt3a, MMP­7 and c­Myc is significantly correlated with Notch3 and Hes1 expression. In vitro experiments showed that Wnt3a was highly expressed in MHcc97H and SK­Hep­1 cells. Therefore, Wnt3a expression was silenced with siRNA, and then, MTT, flow cytometry, wound healing and Transwell assays were performed to analyse cell proliferation, cycle, migration and invasion. The results demonstrated that downregulation of Wnt3a expression inhibited cell viability and induced G0/G1 cell cycle arrest via decreased expression of cyclin D1 and c­Myc and increased expression of p21 and p27. In addition, deletion of Wnt3a significantly inhibited migration and invasion by downregulating MMP­2/-7/-9 expression via the MAPK (p38, ERK1/2 and JNK) pathway. In conclusion, our data show that Wnt3a is involved in HCC development. Wnt3a may be an effective target for treatment of HCC.

Diosmetin Induces Cell Apoptosis by Regulating CYP1A1/CYP1A2 Due to p53 Activation in HepG2 Cells.

It was explored that CYP1 family of cytochromes P450 were over-expressed in several types of cancer. Our study aimed to characterize anti-proliferative activity and metabolism of the natural flavonoid diosmetin in the human hepatoma cell HepG2, expressing CYP1 family. Diosinduced cell apoptosis could be reversed due to p53 blockade and the cellular P53 and CYP1A1/CYP1A2 proteins levels were examined. P53 and CYP1A1/CYP1A2 proteins were upregulated by Dios; when PFT-α was added into cells, the P53 levels were down-regulated accompanied with up-regulated CYP1A1/CYP1A2. Meanwhile, when cells were co-treated with Dios and PFT-α, P53 was down-regulated and CYP1A1/CYP1A2 up-regulated controlled with that of Dios treated cells. The data reveal the new evidence that cytochrome P450 CYP1A regulation by P53 enzyme plays an important role in Diosmetin anti-cancer activity of HepG2 cells.

Dihydromyricetin Enhances the Chemo-Sensitivity of Nedaplatin via Regulation of the p53/Bcl-2 Pathway in Hepatocellular Carcinoma Cells.

Chemotherapy is an effective weapon in the battle against cancer. Nedaplatin (NDP) is an improved platinum-containing drug with lower cytotoxicity than other similar drugs. However, the repeated use of NDP results in substantial hepatocyte damage as well as drug resistance in hepatocellular carcinoma (HCC) cases. Therefore, the development of effective chemotherapy strategies that enhance tumor sensitivity to chemotherapeutics and reduce the secondary damage to liver cells is urgently needed. Dihydromyricetin (DHM), a natural flavonoid compound, has been shown to have antitumor activity with no obvious toxicity to normal cells in vitro and in vivo. In this study, DHM and NDP were combined to treat liver cancer cells; we found that DHM functions as a protector of normal cells compared with the use of NDP alone. In addition, the synergy of DHM with NDP enhanced the effect of NDP on the induction of HCC cell apoptosis. We found that the combination caused clear changes in the level of reactive oxygen species (ROS). Furthermore, we demonstrated that the combination of DHM and NDP activated the p53/Bcl-2 signaling pathway, which resulted in mitochondrial dysfunction and induced cell death and growth inhibition in HCC cells.

Notch3 functions as a regulator of cell self-renewal by interacting with the ß-catenin pathway in hepatocellular carcinoma.

The Notch signaling pathway plays a role in cell proliferation, differentiation. Emerging data have revealed aberrant Notch3 expression in hepatocellular carcinoma (HCC). However, whether Notch3 plays a role in tumorigenesis or tumor progression is unclear. In this study, we found that over 71.8% of the cases studied had high Notch3 expression levels (n = 32); Notch3 expression positively correlated with alpha-fetoprotein (AFP) levels (p = 0.0311) and negatively correlated with the differentiation grade (p = 0.042). We demonstrated that the patients with higher levels of Notch3 expression commonly had a poor prognosis. We discovered that Notch3 expression is inversely correlated with ß-catenin content but positively associated with the protein level of Nanog. In parallel, we found that Notch3 attenuation resulted in the upregulation of ß-catenin and the downregulation of Nanog in the hepatoma cell lines QGY7701 and HepG2. The downregulation of Notch3 enhanced the sensitivity to cisplatin in the QGY7701 and HepG2 cells and inhibited the ability of QGY7701 cells to form tumors. The Notch3-positive cells had higher levels of aldehyde dehydrogenase (ALDH) activity, and a tendency to differentiate into Notch3-negative cells. In conclusion, our study demonstrated that Notch3 plays a role in modulating the stemness of tumor cells via the inactivation of the Wnt/ß-catenin pathway.

Mir-152 inhibits cell proliferation and colony formation of CD133(+) liver cancer stem cells by targeting KIT.

miR152 is involved in diverse biological functions and development of disease. This study investigates the role of mir-152 in cell proliferation and colony formation of liver cancer stem cells. We show that exogenous overexpression of mir-152 suppresses cell proliferation and colony formation in CD133(+) hep3B cells. We also show that KIT is a direct target of miR-152 and miR-152 downregulates protein expression of KIT by directly binding to 3' untranslated region of KIT. Downregulation of KIT by specific siRNAs inhibits proliferation and colony formation of CD133(+) hep3B cells, which is similar to inhibitory effects of miR-152. Moreover, exogenous expression of KIT compromises inhibitory effects of miR-152 on cell proliferation and colony formation. Our findings suggest that mir-152 inhibits cell proliferation and colony formation of CD133(+) hep3B cells by targeting KIT.

Dihydromyricetin promotes hepatocellular carcinoma regression via a p53 activation-dependent mechanism.

The development of antitumor chemotherapy drugs remains a key goal for oncologists, and natural products provide a vast resource for anti-cancer drug discovery. In the current study, we found that the flavonoid dihydromyricetin (DHM) exhibited antitumor activity against liver cancer cells, including primary cells obtained from hepatocellular carcinoma (HCC) patients. In contrast, DHM was not cytotoxic to immortalized normal liver cells. Furthermore, DHM treatment resulted in the growth inhibition and remission of xenotransplanted tumors in nude mice. Our results further demonstrated that this antitumor activity was caused by the activation of the p53-dependent apoptosis pathway via p53 phosphorylation at serine (15Ser). Moreover, our results showed that DHM plays a dual role in the induction of cell death when administered in combination with cisplatin, a common clinical drug that kills primary hepatoma cells but not normal liver cells.