Molecular and Cellular Probes最新文献

Breast cancer has become the number one cancer in the world, and intestinal flora may be closely linked to it. Geographic location also has an important impact on human intestinal flora. We conducted the first study on the intestinal flora of breast cancer patients and non-breast cancer patients in a tropical region - Hainan Province in China. At the same time, Pacbio platform based on third-generation sequencing was used for the first time to conduct 16S full-length sequencing of fecal microorganism DNA. We completed the species diversity analysis and differential species analysis of the intestinal flora between the two groups, inferred their functional genetic composition and performed functional difference analysis. There were statistically significant differences in alpha diversity between the two groups in Hainan Province. By species composition difference analysis, at the phylum level, Bacteroidales (P = 0.006) and Firmicutes (P = 0.002) was differed between the two groups, and at the genus level, 17 breast cancer-related differential species such as Bacteroides were screened. According to the five grouping methods including ER level, PR level, HER2 status, Ki67 index and histological grade of breast cancer patients, 4, 1, 9, 6, 5 differential microbiota were screened out respectively, which were in total 25 (P < 0.05 for all subgroups) . The functional prediction and difference analysis revealed two functional metabolisms with significant differences between the two groups of microbes (P < 0.05). These results suggest that breast cancer is associated with changes in the composition and function of intestinal flora. These microflora and functional differences may become biomarkers or new targets for diagnosis and treatment of breast cancer.

Background

Cancer stem cells (CSCs) are different from regular cancer cells because of their self-renewal feature and differentiation potential, which establishes the backbone of the vital role of CSCs in the progress and drug resistance of hepatocellular carcinoma (HCC). The objective of this study was to evaluate the effects of blood exosome-derived miRNA-30d-5p on the stemness and gemcitabine resistance of HCC cells and the underlying mechanisms.

Methods

The expression data of HCC-related miRNAs and mRNAs were downloaded from TCGA database and analyzed for differences. Employing the databases of starBase, TargetScan, miRDB, and mirDIP, we conducted target gene prediction upstream of mRNA. The expression of miRNA-30d-5p and SOCS3 mRNA was assayed by qRT-PCR, and the binding between them was validated by dual luciferase assay. CCK-8 was employed to evaluate cell viability and the IC₅₀ value of gemcitabine. Cells were subjected to a sphere-forming assay to assess their ability to form spheres. Western blot was applied to evaluate the levels of cell surface marker proteins (Nanog, CD133, and Oct4) and exosome markers (CD9, CD81, and FLOT1).

Results

Bioinformatics analysis found that SOCS3 expression was down-regulated in HCC. qRT-PCR showed that SOCS3 expression was notably lower in HCC cell lines than in normal liver cell WRL68. At the cellular functional level, SOCS3 overexpression inhibited the viability, sphere-forming ability, stemness, and gemcitabine resistance of HCC cells. Bioinformatics analysis demonstrated that miRNA-30d-5p was the upstream regulator of SOCS3 and highly expressed in HCC tissues and cells. Dual luciferase assay demonstrated that miRNA-30d-5p could bind SOCS3. Rescue experiments showed that upregulating SOCS3 could reverse the effects of miRNA-30d-5p overexpression on the viability, sphere-forming ability, and gemcitabine sensitivity of HCC cells.

Conclusions

Blood exosome-derived miRNA-30d-5p promoted the stemness and gemcitabine resistance of HCC cells by repressing SOCS3 expression. Hence, the miRNA-30d-5p/SOCS3 axis might be a therapeutic target for chemotherapy resistance and a feasible marker for the prognosis of HCC patients.

Background

Formin-related protein-1(FRL1) has reportedly been overexpressed in a variety of malignancies, such as clear cell renal cell carcinoma (ccRCC). However, the clinical value and molecular mechanisms underlying ccRCC tumorigenesis and progression in association with FRL1 remain poorly understood.

Methods

Immunohistochemical analysis was performed on 119 paraffin-embedded RCC tissue samples to detect FRL1 expression and analyze its prognostic value. Colony formation, the CCK-8 assay, flow cytometry, and in vivo nude mice subcutaneous experiments were used to identify the effects of FRL1 on growth and proliferation. In vitro tests for wound healing, migration, and invasion were used to assess the involvement of FRL1 in invasion and metastatic potential. The process of epithelial-mesenchymal transition process (EMT) and the MMP2 expression were detected in stably transfected RCC cells via western blotting, as well as in tumor tissue paraffin sections from xenograft model.

Results

Both FRL1 mRNA and protein levels were noticeably elevated in ccRCC cell lines and samples. Aberrant overexpression of FRL1 was associated with unfavorable clinicopathological features of ccRCC and indicated poor prognosis. Ectopic overexpression of FRL1 increased the growth-promoting traits of ccRCC cells as well as the migratory and invasive capacity of RCC cells, whereas FRL1-silencing caused the opposite results. In addition, FRL1 promoted epithelial-mesenchymal transition (EMT) and upregulated the expression of matrix metalloproteinase 2 (MMP2). Finally, overexpression of FRL1 upregulated phosphorylation level of ERK1/2 with no effect on total level of ERK1/2 in the RCC cells. MAPK/ERK inhibitor reversed the promotional effects of FRL1.

Conclusion

FRL1 was overexpressed in ccRCC tissues and predicted poor prognosis. FRL1 contributes to invasion and aggressive phenotype of ccRCC by facilitating EMT through MAPK/MMP2 axis.

Background

COTE-1 has been found to promote the proliferation and invasion of non-small cell lung cancer. However, the mechanism of COTE-1 in SCLC is still unclear. Exploring the role of COTE-1 in SCLC is expected to provide a potential target for the prognosis and treatment of SCLC.

Methods

The expression of COTE-1 and ki-67 was detected by immunohistochemical staining. PCR detected COTE-1 expression level. Cell proliferation activity was detected by CCK8 assay. A wound healing test detected cell migrative ability. Transwell invasion assay detected cell invasive ability. The numbers of autophagosomes were observed by transmission electron microscopy. WB detected the expression levels of autophagy-related proteins and AMPK/mTOR pathway-related proteins. The effect of COTE-1 expression level on the proliferation of SCLC tumor tissues was investigated by establishing a mouse SCLC xenograft tumor model.

Results

The expression of COTE-1 in SCLC tissues and cells was higher than that in normal tissues and cells. In SCLC cells with high COTE-1 expression, the expression level of autophagy proteins was notably increased, the number of intracellular autophagosomes increased, and the proliferative activity, migration and invasion abilities were enhanced. COTE-1 promotes autophagy, proliferation, and invasion of SCLC cells under nutrient deprivation by activating the AMPK/mTOR signaling pathway. Activation of autophagy by COTE-1 promotes the proliferation and development of xenograft tumors in a mouse model of SCLC.

Conclusion

COTE-1 promotes the proliferation, migration and invasion of small cell lung cancer by mediating autophagy based on the AMPK/mTOR pathway.

Purpose

Given the high incidence and mortality rates of colorectal cancer (CRC) and the inadequacy of existing treatments for many patients, this study aimed to explore the potential of Capping Actin Protein (CAPG), a protein involved in actin-related movements, as a novel therapeutic target for CRC.

Methods

Bioinformatic analysis of gene expression was conducted using the UALCAN website. Cell proliferation was measured using the CCK-8 kit. Cell cycle, apoptosis, and ferroptosis were analyzed using flow cytometry. Tumorigenesis was evaluated by the subcutaneous inoculation of CRC cells into BALB/c nude female mice. Differentially expressed genes and signaling pathways were identified using RNA sequencing.

Results

CAPG was significantly overexpressed in human CRC tissues and its upregulation was correlated with poor overall survival. CAPG knockdown led to notable inhibition of CRC cells in vitro and in vivo. Interference with CAPG blocked the cell cycle at the G1 phase and triggered apoptosis and ferroptosis by upregulating the P53 pathway in CRC cells.

Conclusion

CRC patients with higher CAPG levels have a poorer prognosis. CAPG inhibits apoptosis and ferroptosis, while promoting CRC cell proliferation by repressing the P53 pathway. Our study suggests that CAPG may be a potential therapeutic target for CRC prognosis and treatment.

Background

Non-alcohol fatty liver disease (NAFLD) is the most prevalent hepatopathy in China, with few effective cures currently. This work aimed to confirm the effect of DHM in vivo/vitro and explore the potential mechanism based on a network pharmacology-based approach.

Methods

The rats were fed using a high-fat diet (HFD) to accumulate lipid. DHM at different concentrations was used to treat the HFD rats. The serum total cholesterol (TC), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were detected using ELISA kits. The target genes of DHM against NAFLD were screened by online databases. Then, the cytotoxicity of DHM in primary hepatocytes and HepG2 cells was determined by MTT reagent. qRT-PCR was used to quantify the expression level of PPAGR and CASP3 mRNA. Cell apoptosis and intracellular triglyceride (TG) were detected.

Results

HFD diet increased rat liver weight/body weight ratio, serum TC, ALT, and AST. But DHM treatment can reduce these elevated indicators. DHM targeted 14 potential genes in NAFLD. PPARG and CASP3 were two hub genes for DHM against NAFLD, with score factor coefficients of −7.1 and −6.8 kcal/mol. DHM reduced the increased PPARG mRNA level and intracellular TG induced by palmitic acid. DHM can reduce the increased CASP3 mRNA level and cell apoptosis induced by palmitic acid.

Conclusion

This work demonstrates a mechanism of DHM that alleviates lipid metabolism disorder and cell apoptosis for the treatment of NAFLD, evidencing the potential application of DHM in NAFLD.

Background

Breast cancer (BC) has been identified as a significant contributor to the rising number of female cancer deaths. As, it has become clear that breast cancer development depends on the interplay of several biological factors against a single molecule. This research aimed to use proteomics to gain a regulatory and metabolic understanding of BC pathophysiology.

Method

For the study, a breast cancer proteomics dataset was downloaded from ProteomeXchange and then analyzed by employing MaxQuant and Perseus. Functional enrichment analysis through Metascape and Cytoscape software showed DEPs related biomedical phenomena with potential abruption. The expression of selected lncRNA in terms of the highest connectivity parameters was then quantitatively assessed through RT-PCR in 30 tumor tissues of breast cancer patients, as compared to the adjacent healthy ones.

Result

The results indicated that among the 3048 identified proteins, 1149 were differentially expressed, which could be mainly enriched in several key terms. Furthermore, the obtained findings revealed that ITGB1-DT was significantly overexpressed in tumor tissues. Moreover, we found five potential compounds that could be attributed to ITGB1-DT targets (ATN-161, Firategrast, SB-683698, dabigatran-etexilate, and tranexamic-acid).

Conclusion

These analyses proposed that ITGB1-DT could be employed as a differentiated factor to identify breast tumor tissues in healthy samples. Besides this, Firategrast could be introduced as a potential remedial agent for breast cancer patients. Overall, from the analysis of a proteomics dataset, an integrative map was generated, and a novel biomarker that may have been implicated in the early detection of BC was introduced.

Toll-like receptor 4 (TLR4) plays a critical role in various human diseases, and was associated with pyroptotic cell death and inflammatory responses. DNA methylation, which has stable and reversible properties, has been reported to alter the expression of target genes, including TLR4. However, the role of methylated TLR4 in osteomyelitis (OM) and the underlying molecular mechanisms remain unclear. RNA sequencing was used to identify differentially expressed genes and associated signaling pathways. RT-qPCR, Western blot, emzyme-linked immunosorbent assay (ELISA), cell counting kit-8 (CCK-8) and LDH assay kit were used to detect mRNA and protein expression of relevant genes, cell viability and the LDH activity, respectively. TLR4 methylation was detected by methylation-specific PCR (MSP) and verified by Chromatin immunoprecipitation (ChIP). Here, we found that DNA methyltransferase-1 (DNMT1)-mediated TLR4 demethylation significantly suppressed lipopolysaccharides (LPS)-induced pyroptosis and inflammatory response by inhibiting the TLR4/nuclear transcription factor-kappa B (NF-κB) axis. First, we confirmed TLR4 as the study target by mRNA transcriptome sequencing analysis, and TLR4 was observably high-expressed in both OM patients and LPS-treated osteoblastic MC3T3-E1. Then, we found that downregulation of DNMT1 blocked TLR4 promoter methylation modification, resulting in upregulation of TLR4. Simultaneously, functional experiments indicated that suppression of TLR4 or overexpression of DNMT1 promoted cell proliferation and inhibited cell pyroptosis and inflammation in LPS-induced MC3T3-E1, while upregulation of TLR4 restored the effects of DNMT1 silencing on OM progression. In addition, TLR4 elevated phosphorylation of IκB-α and NF-κB p65 in the NF-κB signal pathway, and inhibition of TLR4 or the NF-κB inhibitor PDTC reversed the influence of inhibition of DNMT1. In conclusion, our study demonstrated that DNMT1-mediated TLR4 DNA methylation alleviated LPS-induced OM by inhibiting the NF-κB signaling pathway.

Background

Aberrant expression of circRNAs is involved in the progression of hepatocellular carcinoma (HCC). This study aimed at screening the pro-tumorigenic circular RNAs (circRNAs) in HCC and the mechanisms of circCPSF6 expression influencing HCC characteristics.

Method

circCPSF6 was identified in HCC tissues using high-throughput sequencing data, and its expression was verified in both HCC tissues and cell lines using quantitative real-time PCR (qRT-PCR). CCK-8 and Transwell assays were used to evaluate the effects of circCPSF6 on HCC proliferation and migration. A xenograft mouse model was used to investigate the effects of circCPSF6 on HCC progression in vivo, and the significance of circCPSF6 in HCC was verified both in vivo and in vitro. circCPSF6-associated miRNAs and mRNAs were identified using bioinformatic analyses. Luciferase reporter, RNA pull-down, Fluorescence in situ hybridization, and RNA immunoprecipitation assays were performed to elucidate the circCPSF6 regulatory axis in HCC.

Result

CircCPSF6 expression was increased in HCC cell lines and tissues, and the expression of its parental mRNA was positively correlated with tumor severity and negatively correlated with survival. Mechanistic analyses of HCC cell lines showed that tumorigenesis was inhibited by circCPSF6 knockdown and promoted by its overexpression. Functional analyses revealed that circCPSF6 mediated HCC development by sponging miR-145-5p as a competing endogenous RNA. Furthermore, this sponging upregulated the miR-145-5p target gene MAP4K4, a classical pro-tumorigenic gene.

Conclusion

Our findings reveal a regulatory network that includes the circCPSF6–miR-145-5p–MAP4K4 axis. Elements of this axis are potential HCC biomarkers, as well as targets for HCC treatment.

Lung cancer is one of the most common malignant tumors and has a poor prognosis and a low survival rate. Traditional treatments, such as radiotherapy and chemotherapy, still face some challenges because of high drug resistance and toxicity. Therefore, it is necessary to discover a new kind of targeted drug with low toxicity and high efficiency. CDK12 is a cell cycle-dependent kinase whose main function is to activate RNA polymerase II (RNAPII) and promote the transcriptional extension of RNA. However, the role and molecular mechanism of CDK12 in lung cancer are still unclear.

In this study, the mutation and RNA-Seq data of CDK12 in lung adenocarcinoma and squamous cell carcinoma were downloaded from The Cancer Genome Atlas (TCGA) database and analyzed with the custom scripts. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) and cell colony formation assays. A subcutaneous tumor experiment in nude mice was used to examine the effects of CDK12 knockdown on the in vivo tumor growth of NSCLC cells. The cell cycle distribution and the apoptosis rate of lung cancer cells were assessed by flow cytometry. Regulation of TANK-binding kinase 1 (TBK1) by CDK12 was evaluated by quantitative PCR, immunoprecipitation and Western blot analysis.

In this study we have analyzed the mutation and expression data of The Cancer Genome Atlas (TCGA) database and found that CDK12 is highly expressed in lung cancer tissues. Clinical correlation analysis showed that high expression of CDK12 in NSCLC reduces patient survival, but its high expression is only related to early tumor progression and has no significant correlation with late tumor progression and metastasis. Furthermore, we present evidence that CDK12 depletion in lung cancer cell lines not only leads to the inhibition of cell growth and induces apoptosis but also inhibits tumor growth of NSCLC cells in vivo. CDK12 positively regulates the expression of the oncogene TBK1 in lung cancer cells. These results revealed that CDK12 affects the progression of non-small cell lung cancer through positive regulation of TBK1 expression, suggesting that CDK12 might be a potential molecular target for the treatment of non-small cell lung cancer.