In Aerodynamics, L. Clancy writes: "To distinguish it from the total and dynamic pressures, the actual pressure of the fluid, which is associated not with its motion but with its state, is often referred to as the static pressure, but where the term pressure alone is used it refers to this static pressure. Examples are aircraft in flight, and ships moving in open bodies of water. If the fluid flow at some point along a streamline is brought to rest, this point is called a stagnation point, and at this point the total pressure is equal to the stagnation pressure. If both the gas pressure and volume change simultaneously, then work will be done on or by the gas.
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Abstract Although several studies indicate that RNA-binding proteins RBPs contribute to key steps in a variety of physiological processes and cancer, the detailed biological functions and mechanisms remain to be determined. Despite intensive efforts to improve early detection and develop novel therapeutic strategies, patients with HCC still face a high incidence of postoperative recurrence and unsatisfactory survival rates 2 , 3.
Therefore, it is critical to elucidate the molecular mechanisms underlying hepatocarcinogenesis to develop novel therapeutics targeting HCC. RBPs are dynamic, often multi-functional, modulators acting on several layers of post-transcriptional gene expression.
Most studies show that RBPs can lead to different diseases, including muscular atrophies, neurological disorders, and cancer, because of a significant change or disturbance in RBPs regulating some essential cellular functions 6—8. By performing bioinformatics analysis in well-established datasets, we identified a set of candidate RBPs implicated in HCC progression, and RPS3 was selected for further analysis due to its abundant expression and uncharacterized role in HCC.
Human ribosomal protein S3 RPS3 , a component of the 40S ribosomal subunit, is mainly involved in ribosomal maturation and initiation of translation through association with initiation factors 9. RPS3 also has various extra-ribosomal functions, including DNA repair 10—13 , apoptosis modulation, cell signalling 14 , 15 , transcriptional regulation, and transformation 16— In addition, both up-regulation and intrinsic dysfunctions in ribosomes result in an increasing incidence of tumors, and RPS3 is involved in radioresistance or invasion of tumor cells 19 , Accumulating evidence indicates that SIRT1 is involved in many biological processes, including transcriptional silencing, DNA repair, circadian clock maintenance, cellular metabolism, stress response, ageing, and tumorigenesis 23 , Based on recent reports, traditional Chinese herbal medicine CHM is emerging as an intriguing and viable choice because of its multilevel, multi-targeted, and coordinated intervention effects against HCC.
The extensive application of phytochemical and molecular biological approaches in many CHM-derived compounds has shown great potential in the development of natural anti-HCC products Thirty-six normal liver, 31 para-tumor and HCC tissue samples were used to construct tissue microarrays.
HCC and para-tumor tissue samples were surgically resected from HCC patients who underwent hepatectomy at Peking Union Medical College Hospital PUMCH, Beijing, China between and , and normal liver tissues were collected from patients with hepatolithiasis who were treated at the same hospital. All diagnoses were confirmed by pathology. Complete clinicopathological and follow-up data were available for the HCC samples.
Forty-eight hours after transfection, cells were harvested and lysed to evaluate the transfection efficiency. Briefly, transfected cells were plated in six-well plates at a density of cells per well.
The medium was changed every 3 days. After 2 weeks, colonies were fixed with methanol and stained with crystal violet for 20 min. Each experiment was repeated at least three times. Cell cycle analysis Cells with different treatments were washed three times with phosphate-buffered saline PBS , detached with 0. After treatment with 2. Wound-healing migration assay To perform migration assays, we seeded cells in confluent monolayers in six-well plates after transfection.
Phase contrast images were acquired at an identical location at 0, 24, 48, and 72 h after scratching, and the width W of the scratch wound was measured. The rate of closure of the open wounds was calculated. All measurements were performed in triplicate at least three times. Then, the cells on the upper surface of the filters were removed using cotton wool swabs. RNA was quantified by absorbance at nm. Primer sequences are listed in supplementary data List of primer sequences. At various time points thereafter, cells were lysed and subjected to immunoblotting analysis as described below.
The cells were then incubated with the puromycin for one hour before cell lysates were collected and subjected to immunoblotting analysis with anti-puromycin antibodies. After three washes with TBST, the membrane was incubated with a secondary antibody at room temperature for 2 h. The primer sequences are listed in supplementary data list of primer sequences. The biotinylated RNAs were then extracted with a phenol—chloroform mixture, precipitated with ethanol and rehydrated in DEPC-treated water.
The pull-down materials were subsequently analysed by western blotting by probing the membranes successively with RPS3-specific and GAPDH-specific antibodies.
Forty-eight hours later, cell lysates were collected, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System Promega and normalized to Renilla luciferase activity. The mice were sacrificed after 4—6 weeks, and tumors were removed for assessment.
After washing, each slide was incubated with the appropriate HRP-labeled secondary antibody, and signals were developed with DAB solution before counterstaining with hematoxylin. Samples were sequenced on an Illumina Hiseq instrument. Raw Data was processed with Perl scripts to ensure the quality of data used in further analysis. The adopted filtering criteria were as follows: i remove the adaptor-polluted reads reads containing more than five adapter-polluted bases were regarded as adaptor-polluted reads and would be filtered out ; ii remove the low-quality reads.
Bowtie2 was used for building the genome index, and Clean Data was mapped to the reference genome using TopHat v2.
Fragments Count for each gene in each sample was counted by HTSeq v0. Each assay was performed in three independent replicates.
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