The N6-methyladenosine modification of circALG1 promotes the metastasis of colorectal cancer mediated by the miR-342-5p/PGF signalling pathway.

BACKGROUND: Previous studies have shown that the N6-methyladenosine (m6A) modification enhances the binding ability of mRNAs/long noncoding RNAs (lncRNAs) to microRNAs (miRNAs), but the impact of this modification on the competitive endogenous RNA (ceRNA) function of circular RNAs (circRNAs) is unclear. METHODS: We used a human circRNA microarray to detect the expression profiles of circRNAs in 3 pairs of cancer and paracancerous tissues from patients with colorectal cancer (CRC) and 3 pairs of peripheral blood specimens from patients with CRC and healthy individuals. The circRNAs highly expressed in both peripheral blood and tumour tissues of patients with CRC, including circALG1, were screened. A quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of an expanded sample size was performed to detect the expression level of circALG1 in peripheral blood and tumour tissues of patients with CRC and determine its correlation with clinicopathological features, and circRNA loop-forming validation and stability assays were then conducted. Transwell assays and a nude mouse cancer metastasis model were used to study the function of circALG1 in CRC and the role of altered m6A modification levels on the regulation of circALG1 function. qRT-PCR, western blot (WB), Transwell, RNA-binding protein immunoprecipitation (RIP), RNA antisense purification (RAP), and dual-luciferase reporter gene assays were performed to analyse the ceRNA mechanism of circALG1 and the effect of the m6A modification of circALG1 on the ceRNA function of this circRNA. RESULTS: CircALG1 was highly expressed in both the peripheral blood and tumour tissues of patients with CRC and was closely associated with CRC metastasis. CircALG1 overexpression promoted the migration and invasion of CRC cells, and circALG1 silencing and reduction of the circALG1 m6A modification level inhibited CRC cell migration and invasion. In vivo experiments further confirmed the prometastatic role of circALG1 in CRC. Further mechanistic studies showed that circALG1 upregulated the expression of placental growth factor (PGF) by binding to miR-342-5p and that m6A modification enhanced the binding of circALG1 to miR-342-5p and promoted its ceRNA function. CONCLUSION: M6A modification enhances the binding ability of circALG1 to miR-342-5p to promote the ceRNA function of circALG1, and circALG1 could be a potential therapeutic target in and a prognostic marker for CRC.

Circular RNA RHBDD1 regulates tumorigenicity and ferroptosis in colorectal cancer by mediating the ELAVL1/SCD mRNA interaction.

Circular RNAs (circRNAs) are covalently closed noncoding RNA molecules that play multiple roles in tumorigenesis and metastasis. Ferroptosis is an iron-dependent, regulated form of cell death and has emerged as a promising target for cancer treatment. However, whether and how circRNAs regulate ferroptotic cell death in colorectal cancer (CRC) remains largely unknown. Three circRNA microarrays were used to screen differentially expressed circRNAs in CRC tissues. A series of functional experiments were conducted to investigate the effects of circRNA on CRC cell proliferation, migration and ferroptosis. We found that hsa_circ_0058495 (circRHBDD1), a novel circRNA, was significantly upregulated in colorectal cancer tissues and cells. The expression levels of circRHBDD1 in serum samples were strongly associated with the advancement of CRC. Silencing of circRHBDD1 remarkably suppressed the proliferation and migration of CRC cells in vitro. Moreover, the depletion of circRHBDD1 notably increased ferroptotic cell death and enhanced RSL3-induced ferroptosis in CRC cells. Mechanistically, circRHBDD1 upregulated the expression of stearoyl-CoA desaturase (SCD), a ferroptosis suppressor mediating lipid remodelling, by enhancing the ELAVL1/SCD mRNA interaction. Finally, circRHBDD1 knockdown repressed the tumorigenesis and ferroptosis of CRC cells in vivo. In conclusion, circRHBDD1 facilitates tumour progression and obstructs ferroptosis in CRC by regulating SCD expression in an ELAVL1-dependent manner.

The differences in virus shedding time between the Delta variant and original SARS-CoV-2 infected patients.

Background: The worldwide epidemic of Coronavirus Disease 2019 (COVID-19) has evolved into multiple variants. The Delta variant is known for its ability to spread and replicate, while data are limited about the virus shedding time in patients infected by the Delta variant. Methods: 56 Delta variant and 56 original SARS-CoV-2 infected patients from Hunan, China, matched according to age and gender divided into two groups and compared the baseline characteristics and laboratory findings with appropriate statistical methods. Results: Patients infected with the Delta variant had significantly fewer symptoms of fever (p < 0.001), fatigue (p = 0.004), anorexia (p < 0.001), shortness of breath (p = 0.004), diarrhea (p = 0.006), positive pneumonia rate of chest CT (p = 0.019) and chest CT ground glass opacities (p = 0.004) than those of patients with the original SARS-CoV-2. Patients of the Delta variant group had a significantly longer virus shedding time [41.5 (31.5, 46.75) vs. 18.5 (13, 25.75), p < 0.001] compared with the original SARS-CoV-2 group. The correlation analyses between the virus shedding time and clinical or laboratory parameters showed that the virus shedding time was positively related to the viral strain, serum creatinine and creatine kinase isoenzyme, while negatively correlated with lymphocyte count, total bilirubin and low-density lipoprotein. Finally, the viral strain and lymphocyte count were thought of as the independent risk factors of the virus shedding time demonstrated by multiple linear regression. Conclusion: COVID-19 patients infected with the Delta variant exhibited fewer gastrointestinal symptoms and prolonged virus shedding time than those infected with the original SARS-CoV-2. Delta variant and fewer lymphocyte were correlated with prolonged virus shedding time.

The Association Between Alveolar-Arterial Oxygen Tension Difference and the Severity of COVID-19 in Patients.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) emerged as a global pandemic and resulted in a significantly high death toll. Therefore, there is an urgent need to find a potential biomarker related to the disease severity that can facilitate early-stage intervention. METHODS: In the present study, we collected 242 laboratory-confirmed COVID-19-infected patients. The patients were grouped according to the alveolar to arterial oxygen tension difference (PA-aO2) value of COVID-19 infection after admission. RESULTS: Among the 242 laboratory-confirmed COVID-19- infected patients, 155 (64.05%) had an abnormal PA-aO2 value on admission. Compared with the normal PA-aO2 group, the median age of the abnormal PA-aO2 group was significantly older (p = 0.032). Symptoms such as fever, cough, and shortness of breath were more obvious in the abnormal PA-aO2 group. The proportion of severe events in the abnormal PA-aO2 group was higher than the normal PA-aO2 group (10.34% vs. 23.23%, p = 0.013). The abnormal PA-aO2 group had a higher possibility of developing severe events compared with the normal PA-aO2 group (HR 2.622, 95% CI 1.197-5.744, p = 0.016). After adjusting for age and common comorbidities (hypertension and cardiovascular disease), the abnormal PA-aO2 group still exhibited significantly elevated risks of developing severe events than the normal PA-aO2 group (HR 2.986, 95% CI 1.220-7.309, p = 0.017). Additionally, the abnormal PA-aO2 group had more serious inflammation/coagulopathy/fibrinolysis parameters than the normal PA-aO2 group. CONCLUSION: Abnormal PA-aO2 value was found to be common in COVID-19 patients, was strongly related to severe event development, and could be a potential biomarker for the prognosis of COVID-19 patients.

Intergenic CircRNA Circ_0007379 Inhibits Colorectal Cancer Progression by Modulating miR-320a Biogenesis in a KSRP-Dependent Manner.

Circular RNAs (circRNAs) are covalently closed RNA structures that play multiple roles in tumorigenesis and progression. Compared with exon‒intron circRNAs, the biological functions and implications of intergenic circRNAs in human cancer are still poorly understood. Here, we performed circRNA microarray analysis and identified an intergenic circRNA, circ_0007379, that was significantly downregulated in patients with colorectal cancer (CRC). The biogenesis of circ_0007379 was mediated by reverse complementary matches (RCMs) and was negatively regulated by the RNA helicase DHX9. Functionally, circ_0007379 suppressed CRC cell growth and metastasis in cell culture as well as in patient-derived organoid and xenograft models. Mechanistically, circ_0007379 acted as a scaffold to facilitate the processing of both pri-miR-320a and pre-miR-320a in a KSRP-dependent manner, leading to miR-320a maturation and subsequent repression of transcription factor RUNX1 expression. Thus, our findings establish a previously unrecognized function of circRNA in inhibiting CRC progression.

Encoding gene RAB3B exists in linear chromosomal and circular extrachromosomal DNA and contributes to cisplatin resistance of hypopharyngeal squamous cell carcinoma via inducing autophagy.

Cisplatin (DDP) resistance is an important factor that decreases the effect of chemotherapy, thus leading to local recurrence and lymph node metastasis of hypopharyngeal squamous cell carcinoma (HSCC). We aimed to explore the role and mechanism of extrachromosomal circular DNA (eccDNA) in the DDP resistance of HSCC. In our research, the HSCC cell line FaDu and the DDP-resistant cell line FaDu/DDP were used as subjects. eccDNA sequencing and whole transcriptome sequencing were conducted, followed by a combined analysis of the two sequencing profiles. Outward PCR, inward PCR and Sanger sequencing were used to verify sequences of the eccDNAs. Bioinformatics analysis based on TCGA/GEO was performed in addition to plasmid transfection, RNA interference, qRT-PCR and Western blot experiments to verify the expression level of RAB3B amplified from eccDNA. mRFP-GFP-LC3 adenoviral particle transfection and transmission electron microscopy were used to detect autophagic flux. Finally, we evaluated the role of RAB3B in FaDu/DDP cells and patient-derived organoids. Our results showed that we purified and sequenced more than 10 thousand eccDNAs from the two cell lines, and the size of the eccDNAs was distributed from 0.01 kb to 1000 kb. The combined analysis between eccDNA and transcript sequencing indicated that there were some highly expressed genes that were completely or partially transcribed from related sequences of eccDNAs and not from genome linear DNA. We further screened and verified the encoding gene RAB3B using full-length sequences that might be amplified from eccDNA [chr1circle 46219-52682 kb]. Finally, we confirmed that RAB3B could promote DDP resistance in HSCC by inducing autophagy. The eccDNA might play significant roles in DDP resistance in HSCC by amplifying related functional genes. Further study is needed to explore the novel mechanisms of eccDNA in the drug resistance of HSCC.

PTBP3 modulates P53 expression and promotes colorectal cancer cell proliferation by maintaining UBE4A mRNA stability.

The RNA binding protein PTBP3 was recently reported to play a critical role in multiple cancers, and the molecular mechanisms involved RNA splicing, 3' end processing and translation. However, the role of PTBP3 in colorectal cancer (CRC) remains poorly explored. Herein, PTBP3 was upregulated in CRC and associated with a poor prognosis. PTBP3 knockdown in colorectal cancer cell lines restricted CRC proliferative capacities in vitro and in vivo. Mechanistically, PTBP3 regulated the expression of the E3 ubiquitin ligase UBE4A by binding the 3' UTR of its mRNA, preventing its degradation. UBE4A participated in P53 degradation, and PTBP3 knockdown in colorectal cancer cell lines showed increased P53 expression. UBE4A overexpression rescued PTBP3 knockdown-induced inhibition of CRC cell proliferation and P53 expression. Our results demonstrated that PTBP3 plays an essential role in CRC cell proliferation by stabilizing UBE4A to regulate P53 expression and may serve as a new prognostic biomarker and effective therapeutic target for CRC.

The long noncoding RNA LUCAT1 promotes colorectal cancer cell proliferation by antagonizing Nucleolin to regulate MYC expression.

The long noncoding RNA (lncRNA) LUCAT1 was recently reported to be upregulated and to play an essential role in multiple cancer types, especially colorectal cancer (CRC), but the molecular mechanisms of LUCAT1 in CRC are mostly unreported. Here, a systematic analysis of LUACT1 expression is performed with data from TCGA database and clinic CRC samples. LUCAT1 is identified as a putative oncogene, which is significantly upregulated in CRC and is associated with poor prognosis. Loss of LUCAT1 restricts CRC proliferative capacities in vitro and in vivo. Mechanically, NCL is identified as the protein binding partner of LUCAT1 by using chromatin isolation by RNA purification coupled with mass spectrometry (ChIRP-MS) and RNA immunoprecipitation assays. We also show that NCL directly binds to LUCAT1 via its putative G-quadruplex-forming regions from nucleotides 717 to 746. The interaction between LUCAT1 and NCL interferes NCL-mediated inhibition of MYC and promote the expression of MYC. Cells lacking LUCAT1 show a decreased MYC expression, and NCL knockdown rescue LUCAT1 depletion-induced inhibition of CRC cell proliferation and MYC expression. Our results suggest that LUCAT1 plays a critical role in CRC cell proliferation by inhibiting the function of NCL via its G-quadruplex structure and may serve as a new prognostic biomarker and effective therapeutic target for CRC.

miR-133b down-regulates ABCC1 and enhances the sensitivity of CRC to anti-tumor drugs.

Multidrug resistance (MDR) is the main cause of failed chemotherapy treatments. Therefore, preventing MDR is pivotal in treating colorectal cancer (CRC). In a previous study miR-133b was shown to be a tumor suppressor. Additionally, in CRC cells transfected with miR-133b, ATP-binding cassette (ABC) subfamily C member 1(ABCC1) was shown to be significantly down regulated. Whether miR-133b also enhances the chemosensitivity of drugs used to treat CRC by targeting ABCC1 is still unclear. Here, we utilized flow cytometry and high-performance liquid chromatography (HPLC) analysis to identify the ability of miR-133b to reserve MDR in CRC. We then used a dual-luciferase reporter assay to validate that miR-133b targets ABCC1. Further in vivo experiments were designed to validate the method in which miR-133b reversed MDR in CRC cells. The results demonstrated that the level of miR-133b was down-regulated and the expression of ABCC1 was up-regulated in drug-resistant CRC cells compared to non-drug-resistant CRC cells. The restoration of miR-133b expression in CRC drug-resistant cells in vitro resulted in reduced IC50s to chemotherapeutic drugs, significantly induced G1 accumulation, inhibited growth and promoted necrosis in combination with either 5-fluorouracil (5-FU) or vincristine (VCR), and decreased the expression of ABCC1. The dual-luciferase assay demonstrated that miR-133b directly targets ABCC1. The combination of agomiRNA-133b with chemotherapeutic drugs in vivo inhibited tumor growth induced by CRC drug-resistant cells. A xenograft from the in vivo model resulted in up-regulated levels of miR-133b and down-regulated levels of ABCC1. Therefore, miR-133b enhances the chemosensitivity of CRC cells to anti-tumor drugs by directly down-regulating ABCC1. This discovery provides a therapeutic strategy in which miR-133b is used as a potential sensitizer for drug-resistant CRC.