Ⅰ.MIRNA-377-3p Inhibits Hepatocellular Carcinoma Growth And Metastasis Through Negative Regulation Of CPT1C-mediated Fatty Acid Oxidation Ⅱ.SARS-CoV-2 Spike L452R Mutation Increases Omicron Variant Fusogenicity And Infectivity As Well As Host Glycolysis

Ⅰ.miRNA-377-3p inhibits hepatocellular carcinoma growth and metastasis through negative regulation of CPT1C-mediated fatty acid oxidationLiver cancer is one of the most common malignant tumors in the world,and its morbidity and mortality are still rising year by year.Seventy-five to eighty-five percent of primary liver cancer is defined as hepatocellular carcinoma(HCC).The occurrence and development of HCC is a complex process of multi-factor,multi-stage and multichannel regulation.Since HCC is almost asymptomatic in the early stage,most patients are diagnosed as advanced,which brings great difficulties to the treatment of HCC.Although some HCC-related risk factors are relatively clear,such as viral hepatitis,liver cirrhosis and alcohol abuse,the molecular mechanism underlying the development and progression of HCC remains largely unknown.Thus,further understanding of HCC tumorigenesis needs to be clarified.Recent studies have demonstrated that altered lipid metabolism,which is among the most vital metabolic alterations in HCC,plays a critical role in tumor occurrence and development.Continuous fatty acid oxidation(FAO)is frequently activated in HCC cells to produce extra energy,thus maintaining the rapid cell proliferation.Carnitine palmitoyltransferase 1(CPT1),a key enzyme in FAO,is a rate-limiting FAO enzyme that catalyzes the acylation of long chain fatty acids and their entry into mitochondria for β-oxidation.As the last identified member of the CPT1 family,the location and carnitine acyltransferase activity of CPT1 C remained controversial.A micro RNA(miRNA)is a small single-stranded non-coding RNA molecule containing about 22 nucleotides,which functions in RNA silencing and posttranscriptional regulation of gene expression through binding to its 3’-untranslated region(UTR)of m RNA.Emerging evidence demonstrates that dysregulation of miRNA leads to alteration of lipid metabolism and takes an active role in various human cancers.However,whether there are miRNAs as upstream regulators of CPT1 C to regulate its expression and thus modulate the function of CPT1 C in HCC remains unclear.First,we confirmed the expression of CPT1 C in HCC cells and HEK-293 T cells.The immunofluorescence assay indicated that CPT1 C colocalized with mitochondria in HCC cells,suggesting that CPT1 C may be involved in the fatty acid oxidation pathway.Next,six miRNAs that might target CPT1 C were predicted by Target Scan and miRanda databases,including miR-339-5p,miR-432-5p,miR-377-3p,miR-592,miR-342-3p,and miR-4267.Western blot analysis showed that miR-377-3p significantly inhibited CPT1 C expression by directly targeting the 3’-UTR of CPT1 C.The luciferase reporter assay showed that increased miR-377-3p expression significantly reduced CPT1 C 3’-UTR activity,but produced a smaller or no change in the luciferase activity of mutant CPT1 C 3’-UTR luciferase reporter.Through tracing of CY3-labeled palmitate,fatty acid oxidation analysis,flow cytometry,oil red O staining and droplet immunofluorescence,it was proven that miR-377-3p regulates fatty acid transport andβ oxidation in hepatocellular carcinoma by targeting CPT1 C.With CCK8 and colony formation assay,wound healing assays and transwell assay,we demonstrated that miR-377-3p inhibits the proliferation,migration and invasion of HCC cells by inhibiting CPT1 C expression and confirmed that the miR-377-3p/CPT1 C axis mainly regulates the proliferation,migration and invasion of HCC cells by regulating fatty acid oxidation.Furthermore,we demonstrated that the miR-377-3p/CPT1 C axis regulates HCC growth and metastasis in vivo using xenograft mouse models and metastasis models.Statistical results of clinical specimens showed that miR-377-3p expression was significantly downregulated,and conversely,CPT1 C expression was significantly upregulated in HCC patients,compared with normal tissues.Kaplan-Meier survival analysis indicated that high miR-377-3p expression was associated with longer overall survival(OS),and high CPT1 C expression was associated with shorter OS.Through miRNA in situ hybridization(MISH)and tissue immunofluorescence staining,we found that miR-377-3p was negatively correlated with CPT1 C expression,CPT1 C expression was negatively correlated with lipid drop levels,and miR-377-3p was positively correlated with lipid drop levels.Taken together,the miR-377-3p/CPT1 C axis has great clinical significance in HCC.In this study,we demonstrated for the first time the function of CPT1 C in the fatty acid oxidation pathway of HCC,identified miR-377-3p as a key regulator of CPT1 C expression and lipid metabolism,and confirmed the key role of the miR-377-3p/CPT1 C axis in the progression of HCC.Therefore,the miR-377-3p/CPT1 C axis may be a promising tumor therapeutic target and prognostic marker for HCC.Ⅱ.SARS-Co V-2 spike L452 R mutation increases Omicron variant fusogenicity and infectivity as well as host glycolysisAn acute respiratory disease caused by severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)that surfaced in China in late 2019,continues to spread rapidly across the globe 22 causing serious concerns.SARS-Co V-2 is highly transmissible and has a huge impact on the entire world and human health systems.Increasing evidence has demonstrated human-to-human transmission mainly through the upper respiratory tract,causing damage to the lower respiratory tract and leading to severe pneumonia.Mutations in the SARS-Co V-2 virus occur spontaneously during replication and thousands of mutations have accumulated and continue to since the emergence of the virus.Mutations in the SARS-Co V-2 receptor binding domain(RBD)may affect the infectivity,pathogenicity and immune escape of the virus.The Delta variant,which contains many mutants,is highly infectious and pathogenic.The Omicron variant has rapidly displaced the Delta variant,and shows less pathogenicity and more transmission than the Delta variant.However,the mechanism by which the Omicron variant reduces pathogenicity is not well elucidated.The L452 R mutation,one of the most frequent mutations and critical in Delta mutants,is also the only RBD domain mutation that emerges in the Delta variant but is absent in the Omicron variant.Since the L452 R mutation plays a critical role in SARSCOV-2 fusion and infectivity,we constructed an Omicron mutant with the L452 R mutation(Omicron-L452R)to study the effect of L452 R on Omicron variant.Here,we found that the L452 R mutation rescued fusogenicity by enhancing cleavage of the spike protein and further strengthened the high infectivity of the Omicron variant.Moreover,the L452 R mutation restored the ability of Omicron to infect lung tissues of humanized ACE2 mice.Furthermore,the Omicron-L452 R variant dramatically enhanced host glycolysis.Taken together,the L452 R mutation dramatically increases the risk of the Omicron variant.Our study elucidates the low pathogenicity mechanism of Omicron variant and suggests that the L452R-integrated Omicron variant should be closely monitored,and specific therapeutic antibodies and vaccines targeting the OmicronL452 R variant should be developed.Moreover,the research suggest that the use of glycolysis inhibitors may provide a new perspective for the discovery of novel treatments for COVID-19.