RESUMO
Angiotensin-converting enzyme 2 (ACE2) and several proteins have been identified as entry factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether long noncoding RNAs are involved in SARS-CoV-2 entry remains unknown. In this study, we investigated the role of small nucleolar RNA host gene 15 (SNHG15) in SARS-CoV-2 entry using a SARS-CoV-2 spike pseudotyped lentivirus with a luciferase reporter. Overexpression of SNHG15 promoted but SNHG15 knockdown limited SARS-CoV-2 entry in a dose- and time-dependent manner. SNHG15 interacted with Rab-like protein 2A (RABL2A). Overexpression and knockdown of RABL2A produced similar effects on SARS-CoV-2 entry as those of SNHG15. Furthermore, RABL2A knockdown abolished the SNHG15-mediated increase in SARS-CoV-2 entry. In conclusion, SNHG15 is a critical regulatory factor that aids SARS-CoV-2 entry through RABL2A.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , COVID-19/genética , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Receptores Virais/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
In this study, we examined the regulatory role of CCDC34 in the sorafenib sensitivity of hepatocellular carcinoma (HCC) and its functional partners. Wide-type Huh7 and Hep3B and induced sorafenib-resistant (SR) Huh7/SR and Hep3B/SR cells were used as in vitro cell models. Immunofluorescent staining and coimmunoprecipitation were performed to check protein-protein interaction. Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL), PI/Annexin V staining, and western blot analysis were performed to assess cell response to sorafenib. The results showed that CCDC34 upregulation in HCC was associated with poor survival. Huh7/SR and Hep3B/SR cells had significantly higher CCDC34 expression than the parental cell lines. RABL2A expression was significantly upregulated in SR HCC cells and interacted with CCDC34 in its GTP-bound state in Huh7/SR and Hep3B/SR cells. RABL2A depletion sensitized Huh7/SR and Hep3B/SR cells to sorafenib. RABL2A Q80L mutant (GTP-bound state locked), but not S35N mutant (GDP-bound state locked) overexpression increased sorafenib IC50 of Huh7 and Hep3B cells. CCDC34 depletion nearly abrogated the protective effects of RABL2A Q80L overexpression both in vitro and in vivo. RABL2A Q80L overexpression significantly increased the expression of p-p38 and p-JNK, the effects of which were significantly attenuated by CCDC34 depletion. In summary, we infer that the RABL2A-CCDC34 axis plays an important role in mediating p38/MAPK and JNK/MAPK signaling, thereby contributing to acquired sorafenib resistance in HCC.
Assuntos
Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/metabolismo , Proteínas de Neoplasias/metabolismo , Sorafenibe/metabolismo , Antígenos de Neoplasias/fisiologia , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , China , Resistencia a Medicamentos Antineoplásicos/fisiologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/fisiologia , Transdução de Sinais/efeitos dos fármacos , Sorafenibe/farmacologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
OBJECTIVE: To define RABL2A localization in human sperm and to assess any potential association between RABL2A variants and male infertility associated with oligoasthenospermia. DESIGN: Genetic association study. SETTING: Public university. PATIENT(S): Australian men: 110 oligoasthenospermic infertile and 105 proven fertile. INTERVENTION(S): Human semen samples processed by immunostaining with high-throughput-sequencing platform to screen the entire protein-coding and flanking exon/intron regions of the RABL2A gene. MAIN OUTCOME MEASURE(S): Presence of RABL2A in human sperm and frequencies of RABL2A genetic variants in fertile and infertile men. RESULT(S): RABL2A localization in sperm was highly conserved between mouse and human, being localized to the tail. Direct DNA sequencing revealed 23 RABL2A genetic variants, including 16 intronic, 6 untranslated region (UTR), and one exonic missense variants. Of these, eight variants have not been previously reported. Although the majority of these variants showed no significant association with fertility status, allelic frequency of the intronic variant 114391996 delC was significantly increased in oligoasthenospermic men. Bioinformatics analysis suggested that the 114391996 delC allele would alter the splicing of RABL2A pre-mRNA. CONCLUSION(S): Our data suggest the 114391996 delC allele in the RABL2A gene may act as a risk factor for oligoasthenospermic infertility in Australian men.