Current predictive tools and imaging modalities are not accurate enough for preoperative diagnosis of lymph node metastatic prostate cancer (LNM PCa). Proteomic analysis is introduced to screen potential biomarkers for early detection of LNM PCa. In our initial study, protein samples from localized and LNM PCa as well as benign prostatic hyperplasia tissues were analyzed using two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) coupled with MALDI-TOF/TOF MS. We identified 58 proteins that were differentially expressed in the LNM PCa group relative to the localized PCa group. Six of these proteins, e-FABP5, MCCC2, PPA2, Ezrin, SLP2, and SM22, are functionally relevant to cancer metastasis. Expression of these proteins was therefore further validated in tissue samples from the original cohort and also from a larger, independent cohort of patients using real time PCR, Western blotting, and immunohistochemistry staining. In addition, the serum levels of e-FABP5 were also examined by ELISA. Relative to localized PCa tissues, LNM PCa tissues had increased expression of e-FABP5, MCCC2, PPA2, Ezrin, and SLP2 and decreased expression of SM22. Patients with LNM PCa had significantly higher levels of serum e-FABP5. This study presents evidence that increased expression of e-FABP5, MCCC2, PPA2, Ezrin, and SLP2 and decreased expression of SM22 are useful diagnostic markers for the existence of LNM PCa.
BackgroundAlthough the serological antibody responses induced by SARS-CoV-2 vaccines are well characterized, little is known about their ability to elicit mucosal immunity.ObjectivesThis study aims to examine and compare the mucosal and systemic responses of recipients of two different vaccination platforms: mRNA (Comirnaty) and inactivated virus (CoronaVac).MethodsSerial blood and nasal epithelial lining fluid (NELF) samples were collected from the recipients of either Comirnaty or CoronaVac. The plasma and NELF immunoglobulins A and G (IgA and IgG) specific to SARS-CoV-2 S1 protein (S1) and their neutralization effects were quantified.ResultsComirnaty induced nasal S1-specific immunoglobulin responses, which were evident as early as 14 ± 2 days after the first dose. In 64% of the subjects, the neutralizing effects of NELF persisted for at least 50 days. Moreover, 85% of Comirnaty recipients exhibited S1-specific IgA and IgG responses in plasma by 14 ± 2 days after the first dose. By 7 ± 2 days after the booster, all plasma samples possessed S1-specific IgA and IgG responses and were neutralizing. The induction of S1-specific plasma antibodies by CoronaVac was IgG dominant, and 83% of the subjects possessed S1-specific IgG by 7 ± 2 days after the booster, with neutralizing effects.ConclusionComirnaty induces S1-specific IgA and IgG responses with neutralizing activity in the nasal mucosa; a similar response is not seen with CoronaVac.Clinical ImplicationThe presence of a nasal response with mRNA vaccine may provide additional protection compared with inactivated virus vaccine. However, whether such widespread immunological response may produce inadvertent adverse effects in other tissues warrants further investigation.
Backgrounds: Colorectal carcinoma (CRC) is a common malignant tumor.Increasing evidences indicated that CRC showed a resistance to 5-fluorouracil (5-FU) and further resulted in a poor prognosis. In this study, we aim to investigate the effect of long noncoding RNA nuclear paraspeckle assembly transcript 1 (LncRNA NEAT1) on cell viability, sensitivity to 5-FU, and autophagy of CRC cell lines. Methods: MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide) was used to detect cell viability, immunofluorescent staining was used to detect autophagy puncta, and luciferase reporter system was used to determine binding ability between miR-34a and NEAT1 or putative targets. Additionally, indicated mRNAs and protein expressions were determined by qRT-PCR or western blotting, respectively. Results: We found that NEAT1 expression was increased in CRC tissues and cells, which showed a negative correlation with miR-34a expression. In addition, NEAT1 knockdown noticeably inhibited the proliferation of CRC cells and enhanced 5-FU sensitivity. It revealed that NEAT1 knockdown suppressed the LC3 puncta and the expressions of Beclin-1, ULK1, and ratio of LC3II/I. Overexpression of miR-34a showed similar trends with NEAT1 knockdown. miR-34a was validated to target the putative binding sites in 3′-UTR of HMGB1, ATG9A, and ATG4B, which are involved in the activation of autophagy. Inhibition of miR-34a or overexpression of HMGB1 could effectively reverse elevated 5-FU sensitivity upon NEAT1 knockdown. In addition, 3-MA reversed NEAT1 overexpression-induced resistance in HT29 cells. Conclusion: These findings indicate that LncRNA NEAT1 could target miR-34a and promote autophagy to facilitate 5-FU chemoresistance in CRC.
K E Y W O R D Sautophagy, colorectal carcinoma, HMGB1, LncRNA NEAT1, miR-34a
MicroRNAs (miRNAs) are small non-coding RNAs that participate in the spatiotemporal regulation of messenger RNA and protein synthesis. Aberrant miRNA expression leads to developmental abnormalities and diseases. The miR-135a is considered to be oncogenic; however, the functions and mechanisms of miR-135a in colorectal cancer (CRC) are largely unknown. Thus, we investigated the functions and mechanisms of miR-135a, especially its relationship with the metastasis suppressor 1 (MTSS1) gene in CRC. The expression of miR-135a was determined by real-time polymerase chain reaction, while its effect on cell proliferation, migration, and invasion was determined by MTT, without and with matrigel, respectively. The expression of MTSS1 was detected by western blot analysis. It was found that miR-135a expression was higher in human CRC samples than in non-tumor control tissue. Using SW480 and SW620 CRC cell lines, increased proliferation was observed in response to miR-135a. We also demonstrated that miR-135a promoted mobility and invasion via transwell assay with and without Matrigel, respectively, of CRC cells. In contrast, inhibition of miR-135a reduced their proliferative and invasive capability. MTSS1 was identified as a candidate target gene of miR-135a by luciferase report assay. Western blot analysis showed that the expression of MTSS1 was regulated by miR-135a overexpression and knockdown. Similarly, miR-135a-mediated cell mobility and invasion were reduced after MTSS1 was knocked down by small interfering RNA. These data indicated that miR-135a promotes the growth and invasion of CRC cells, at least partially, through targeting MTSS1.
Melanocortin-4 receptor (MC4R) plays important roles in regulation of multiple physiological processes, and interaction of MC4R and melanocortin receptor accessory protein 2 (MRAP2) is suggested to play pivotal role in energy balance of vertebrates. Topmouth culter (Culter alburnus) is an economically important freshwater fish in China. Herein we cloned culter mc4r, mrap2a, and mrap2b. Culter mc4r consisted of a 981 bp open reading frame encoding a protein of 326 amino acids. qRT-PCR revealed that mc4r, mrap2a, and mrap2b were primarily expressed in the central nervous system. In the periphery, mc4r and mrap2b were expressed more widely in the male, while mrap2a was expressed more widely in the female. Culter MC4R could bind to four peptide agonists and increase intracellular cAMP production dose dependently. Culter MC4R was constitutively active in both cAMP and ERK1/2 pathways, which was differentially regulated by culter MRAP2a and MRAP2b. Culter MRAP2a significantly increased B max and decreased agonist-stimulated cAMP, while MRAP2b increased cell surface and total expression but did not affect B max and agonist-stimulated cAMP. These results will aid the investigation of the potential physiological processes that MC4R might be involved in topmouth culter.
Background: GSK-3 is a key pro-apoptotic kinase, and its activity is strictly regulated. Results: GSK-3 is cleaved at both N and C termini by calpain. Conclusion: N-or C-terminal truncation activates GSK-3, and Ser-9/Ser-389 phosphorylation protects GSK-3 from calpain cleavage. Significance: The GSK-3 C terminus functions as an autoinhibitory domain, and Ser-9/Ser-389 phosphorylation and calpainmediated cleavage operate together in regulating GSK-3 activity.
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