Screening miRNAs for early diagnosis of colorectal cancer by small RNA deep sequencing and evaluation in a Chinese patient population

Purpose: This study aims to screen microRNAs (miRNAs), for an early diagnosis of colorectal cancer, by deep sequencing and evaluation of total miRNAs using clinical samples from a Chinese patient population. Methods: Total small RNAs from normal colonic mucosa, colonic adenomas, and colorectal cancer tissues were prepared for miRNA analysis by deep sequencing. The sequencing data were then analyzed by bioinformatics for candidate diagnostic miRNAs, which were further validated for their up- or downregulation status. Results: Comparison of cancer tissues with normal mucosa identified 99 upregulated and 90 downregulated miRNAs. Comparison of adenomas and normal mucosa found 114 upregulated and 107 downregulated miRNAs. Comparison of cancer and adenoma tissues found 70 upregulated and 27 downregulated miRNAs. Selected up- and downregulated miRNAs were validated for their expressions in 12 cases of patients with cancer and polyps. Specifically, for the upregulated miRNAs, miR-18a-5p and miR-21-3p were significantly upregulated in adenomas and cancer tissues, compared with the normal mucosa; miR-135b-5p, miR-17-5p, miR-182-5p, miR-200a-5p, and miR-200c-3p were significantly upregulated in cancer tissues compared to the normal mucosa, but their differential expression was not significant in adenoma tissues when compared with the normal mucosa. miR-183-5p and miR-96-5p were significantly upregulated in adenoma tissues when compared with normal mucosa, but these differences were not significant in cancer tissues when compared to normal mucosa. For the downregulated miRNAs, miR-133a-3p was significantly downregulated in both adenoma and cancer tissues when compared to normal mucosa; miR-204-5p, miR-125b-5p, miR-139-5p, miR-100-5p, and miR-30a-5p were significantly downregulated in cancer tissues compared to the normal mucosa, but their differential expression was not significant in adenoma tissue compared to normal mucosa. Conclusion: The findings of this study show that a number of miRNAs might be important in the diagnosis and prognosis of colorectal cancer in Chinese patients using the method of small RNA deep sequencing. Upregulation of miR-18a-5p and miR-21-3p or downregulation of miR-133a-3p in adenoma and cancer tissues may serve as an index for early screening of colorectal cancer. Other miRNAs, such as miR-135b-5p, miR-17-5p, miR-182-5p, miR-200a-5p, miR-200c-3p, miR-183-5p, and miR-96-5p, which were either up- or downregulated, in cancer tissues, but not in adenoma tissues, have limited significance in early diagnosis. Further study is needed to determine a screening index with diagnostic value

Bioactive Peptides from Angelica sinensis

Since excessive reactive oxygen species (ROS) is known to be associated with aging and age-related diseases, strategies modulating ROS level and antioxidant defense systems may contribute to the delay of senescence. Here we show that the protein hydrolyzate from Angelica sinensis was capable of increasing oxidative survival of the model animal Caenorhabditis elegans intoxicated by paraquat. The hydrolyzate was then fractionated by ultrafiltration, and the antioxidant fraction (<3 kDa) was purified by gel filtration to obtain the antioxidant A. sinensis peptides (AsiPeps), which were mostly composed of peptides with <20 amino acid residues. Further studies demonstrate that AsiPeps were able to reduce the endogenous ROS level, increase the activities of the antioxidant enzymes superoxide dismutase and catalase, and decrease the content of the lipid peroxidation product malondialdehyde in nematodes treated with paraquat or undergoing senescence. AsiPeps were also shown to reduce age pigments accumulation and extend lifespan but did not affect the food-intake behavior of the nematodes. Taken together, our results demonstrate that A. sinensis peptides (AsiPeps) are able to delay aging process in C. elegans through antioxidant activities independent of dietary restriction

Screening miRNAs for early diagnosis of colorectal cancer by small RNA deep sequencing and evaluation in a Chinese patient population

Purpose: This study aims to screen microRNAs (miRNAs), for an early diagnosis of colorectal cancer, by deep sequencing and evaluation of total miRNAs using clinical samples from a Chinese patient population. Methods: Total small RNAs from normal colonic mucosa, colonic adenomas, and colorectal cancer tissues were prepared for miRNA analysis by deep sequencing. The sequencing data were then analyzed by bioinformatics for candidate diagnostic miRNAs, which were further validated for their up-or downregulation status. Results: Comparison of cancer tissues with normal mucosa identified 99 upregulated and 90 downregulated miRNAs. Comparison of adenomas and normal mucosa found 114 upregulated and 107 downregulated miRNAs. Comparison of cancer and adenoma tissues found 70 upregulated and 27 downregulated miRNAs. Selected up-and downregulated miRNAs were validated for their expressions in 12 cases of patients with cancer and polyps. Specifically, for the upregulated miRNAs, miR-18a-5p and miR-21-3p were significantly upregulated in adenomas and cancer tissues, compared with the normal mucosa; miR-135b-5p, miR-17-5p, miR-182-5p, miR-200a-5p, and miR-200c-3p were significantly upregulated in cancer tissues compared to the normal mucosa, but their differential expression was not significant in adenoma tissues when compared with the normal mucosa. miR-183-5p and miR-96-5p were significantly upregulated in adenoma tissues when compared with normal mucosa, but these differences were not significant in cancer tissues when compared to normal mucosa. For the downregulated miRNAs, miR-133a-3p was significantly downregulated in both adenoma and cancer tissues when compared to normal mucosa; miR-204-5p, miR-125b-5p, miR-139-5p, miR-100-5p, and miR-30a-5p were significantly downregulated in cancer tissues compared to the normal mucosa, but their differential expression was not significant in adenoma tissue compared to normal mucosa. Conclusion: The findings of this study show that a number of miRNAs might be important in the diagnosis and prognosis of colorectal cancer in Chinese patients using the method of small RNA deep sequencing. Upregulation of miR-18a-5p and miR-21-3p or downregulation of miR-133a-3p in adenoma and cancer tissues may serve as an index for early screening of colorectal cancer. Other miRNAs, such as miR-135b-5p, miR-17-5p, miR-182-5p, miR-200a-5p, miR-200c-3p, miR-183-5p, and miR-96-5p, which were either up-or downregulated, in cancer tissues, but not in adenoma tissues, have limited significance in early diagnosis. Further study is needed to determine a screening index with diagnostic value.National Science Foundation of China [30572447, 30973837, 81273944]; Jiangsu National Science Foundation [BK20151081]; Nanjing Science Fundation [201402041]; Nanjing Medical Science Foundation [YKK14140]SCI(E)[email protected]

The tumor suppressive role of CAMK2N1 in castration-resistant prostate cancer.

Prostate cancer at advanced stages including metastatic and castration-resistant cancer remains incurable due to the lack of effective therapies. The CAMK2N1 gene, cloned and characterized as an inhibitor of CaMKII (calcium/calmodulin-dependent protein kinase II), has been shown to affect tumorigenesis and tumor growth. However, it is still unknown whether CAMK2N1 plays a role in prostate cancer development. We first examined the protein and mRNA levels of CAMK2N1 and observed a significant decrease in human prostate cancers comparing to normal prostate tissues. Re-expression of CAMK2N1 in prostate cancer cells reduced cellular proliferation, arrested cells in G0/G1 phases, and induced apoptotic cell death accompanied by down-regulation of IGF-1, ErbB2, and VEGF downstream kinases PI3K/AKT, as well as the MEK/ERK-mediated signaling pathways. Conversely, knockdown of CAMK2N1 had a significant opposite effects on these phenotypes. Our analyses suggest that CAMK2N1 plays a tumor suppressive role in prostate cancer cells. Reduced CAMK2N1 expression correlates to human prostate cancer progression and predicts poor clinical outcome, indicating that CAMK2N1 may serve as a biomarker. The inhibition of tumor growth by expressing CAMK2N1 established a role of CAMK2N1 as a therapeutic target

CAMK2N1 inhibits prostate cancer progression through androgen receptor-dependent signaling.

Castration resistance is a major obstacle to hormonal therapy for prostate cancer patients. Although androgen independence of prostate cancer growth is a known contributing factor to endocrine resistance, the mechanism of androgen receptor deregulation in endocrine resistance is still poorly understood. Herein, the CAMK2N1 was shown to contribute to the human prostate cancer cell growth and survival through AR-dependent signaling. Reduced expression of CAMK2N1 was correlated to recurrence-free survival of prostate cancer patients with high levels of AR expression in their tumor. CAMK2N1 and AR signaling form an auto-regulatory negative feedback loop: CAMK2N1 expression was down-regulated by AR activation; while CAMK2N1 inhibited AR expression and transactivation through CAMKII and AKT pathways. Knockdown of CAMK2N1 in prostate cancer cells alleviated Casodex inhibition of cell growth, while re-expression of CAMK2N1 in castration-resistant cells sensitized the cells to Casodex treatment. Taken together, our findings suggest that CAMK2N1 plays a tumor suppressive role and serves as a crucial determinant of the resistance of prostate cancer to endocrine therapies

Autophosphorylation Mechanism of the Ser/Thr Kinase Stk1 From Staphylococcus aureus

The eukaryotic-like Ser/Thr kinase Stk1 is crucial for virulence, cell wall biosynthesis, and drug susceptibility in methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA). Importantly, MRSA lacking Stk1 become sensitive to β-lactam antibiotics, implying that Stk1 could be an alternative target for combination therapy. However, the autophosphorylation mechanism of Stk1 remains elusive. Using a phosphoproteomic study, we identified six in vivo phosphorylated activation loop residues (Ser159, Thr161, Ser162, Thr164, Thr166, and Thr172) of Stk1, which are also phosphorylated in vitro. We further showed that cis autophosphorylation of Thr172 in the GT/S motif is essential for self-activation and kinase activity of Stk1 kinase domain (Stk1-KD), whereas the trans autophosphorylation of other activation loop serines/threonines are required for the optimal kinase activity of Stk1-KD. Moreover, substitution of the activation loop serines/threonines impaired in vivo autophosphorylation activity of kinase variants, while T172A and T172D variants were unable to autophosphorylate in the cellular content, underlining the essential role of Thr172 for Stk1 activity in vivo. This study provides insights into molecular basis for regulation of Stk1 activity from S. aureus

Portulaca oleracea extract can inhibit nodule formation of colon cancer stem cells by regulating gene expression of the notch signal transduction pathway

© The Author(s) 2017. To investigate whether Portulaca oleracea extract affects tumor formation in colon cancer stem cells and its chemotherapy sensitivity. In addition, to analyze associated genetic changes within the Notch signal transduction pathway. Serum-free cultures of colon cancer cells (HT-29) and HT-29 cancer stem cells were treated with the chemotherapeutic drug 5-fluorouracil to assess sensitivity. Injections of the stem cells were also given to BALB/c mice to confirm tumor growth and note its characteristics. In addition, the effect of different concentrations of P. oleracea extract was tested on the growth of HT-29 colon cancer cells and HT-29 cancer stem cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The effects of P. oleracea extract on the expression of β-catenin, Notch1, and Notch2 in the HT-29 cells were studied using reverse transcription polymerase chain reaction and Western blotting. The tumor volume of the HT29 cells was two times larger than that of HT29 cancer stem cells. Treatment with P. oleracea extract inhibited the proliferation of both HT-29 cancer cells and HT-29 cancer stem cells at doses from 0.07 to 2.25 μg/mL. Apoptosis of HT-29 cancer cells and HT-29 cancer stem cells was assessed by flow cytometry; it was enhanced by the addition of P. oleracea extract. Finally, treatment with P. oleracea extract significantly downregulated the expression of the Notch1 and β-catenin genes in both cell types. The results of this study show that P. oleracea extract inhibits the growth of colon cancer stem cells in a dose-dependent manner. Furthermore, it inhibits the expression of the Notch1 and β-catenin genes. Taken together, this suggests that it may elicit its effects through regulatory and target genes that mediate the Notch signal transduction pathway