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  • br RNA extraction and qRT PCR experiment br

    2020-08-12


    RNA extraction and qRT-PCR experiment
    Total RNA was isolated from PCa and BPH tissues using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. PrimeScript RT reagent Kit (Takara) was used for RNA transcription. The primers (Qing Ke Company) used in this study are presented in Table 1.
    Western blot analysis
    Total protein was extracted from the tissues using RIPA lysis buffer (P0013C, Beyotime) according to the manufacturer’s
    + MODEL
    ARHGEF38 as a novel biomarker 3
    Table 1 List of primers used for reverse transcription-quantitative polymerase chain reaction analysis.
    Gene Primer sequence
    ARHGEF38 F: 50 -ACCTTGAGGAGGAGCCAATC-30
    R: 50 -TCTTTTCCCGCTTTGCCATC-30 GADPH F: 50 -ACCTGACCTGCCGTCTAGAA-30
    R: 50 -TCCACCACCCTGTTGCTGTA-30
    Abbreviation: F, forward; R, Reverse.
    Statistical analyses
    Mean and standard deviation (SD) were used to indicate the degree of dispersion of the data. Chi-square test was used
    to assess the significance of ARHGEF38 expression when grouped using mRNA expression, age, PSA, and GS. Pearson test was used for pathological grading, correlation analysis of GS, and ARHGEF38 expression. The survival analyses were performed using the R survival analysis package. The analyses were performed with respect to the expression of ARHGEF38 and Ki67. The statistical significance for the data was analyzed using SPSS 19.0 software. Two-tailed Stu-dent’s t-test was used to compare two independent groups. p < 0.05 was considered statistically significant.
    Results
    Differential expression analysis and enrichment analysis
    After analyzing the consistency of the data and standardi-zation, a total of 243 DEGs were selected from 3 datasets, of which, 32 genes were upregulated and 211 genes were downregulated. Fold change values of at least 2.0 and p < 0.001 were regarded as significant (Fig. 1A). BPH and normal tissues served as controls. Enrichment analysis was
    Figure 1 DEGs and enrichment analysis. (A) In total of 243 DEGs were selected from the 3 datasets, including 32 up-regulated genes and 211 down-regulated genes. (B) Enrichment analysis showed that the DEGs were related to biological regulation, multicellular organismal process and other biological processes (BP), membrane, nucleus, vesicle and cell components (CC), protein binding, ion binding, nucleic CP-456773 binding and molecular function (MF).
    + MODEL
    performed by WebGestaltR to further identify the functions of DEGs (Fig. 1B). The results indicated that the DEGs were related to biological regulation, multicellular organismal process, and biological processes in the cell such as protein binding, ion binding, nucleic acid binding, and other mo-lecular functions.
    ARHGEF38 expression is significantly increased in human PRAD
    The primary aim of this study was to identify a diagnostic and prognostic biomarker for PCa. Two gene expression
    Table 2 Differential expression analysis of ARHGEF38 inPRAD in GEO and TCGA databases.
    TCGA
    symbol
    FC p Value
    FC p Value
    FC p Value
    In bold are significant differences (*p < 0.05). BPH and normal tissues were considered as control group (N), while PCa tissues were regarded as experimental group (C).
    profile datasets (GSE21034 and GSE54808) from GEO database were selected.17,18 Meanwhile, we also further verified the difference in mRNA expression level of the ARHGEF38 gene in PRAD in TCGA datasets. The differen-tial expression analysis of ARHGEF38 using the data ob-tained from GEO and TCGA databases are summarized in Table 2. BPH and normal tissues were considered as the control group (N), while PCa tissues were regarded as the experimental group (C). Furthermore, the expression of ARHGEF38 in six cancers colon adenocarcinoma (COAD), lung squamous cell carcinoma (LUSC), PRAD, kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), and bladder urothelial carcinoma (BLCA) were analyzed using TCGA and Genotype-Tissue Expression (GTEx) sequencing data. The results showed that ARH-GEF38 and Ki67 were upregulated in COAD, PRAD, KICH, and BLCA tissues (Fig. 2A and D). Meanwhile, compared to normal tissues, the expression of ARHGEF38 and Ki67 were significantly increased in PRAD (Fig. 2B and E). The expression of ARHGEF38 mRNA in the GS8 group was significantly higher than that in the GS6 and GS7 groups (Fig. 2C). Furthermore, A positive correlation between ARHGEF38 expression and Ki67 expression in TCGA PRAD tumor data set indicated the increase in ARHGEF38 expression with an increase in proliferation (r Z 0.443, Fig. 2F).
    Figure 2 ARHGEF38 and Ki67 is overexpressed in PCa tissues using TCGA and GTEx sequencing data. (A) ARHGEF38 expression levels in COAD, LUSC, PRAD, KICH, KIRC and BLCA. (B) ARHGEF38 expression was analyzed by RNA-seq in PCa (n Z 499) and normal tissues (n Z 52). (C) ARHGEF38 expression in PRAD by GS categories. (D) Ki67 expression levels in COAD, LUSC, PRAD, KICH, KIRC and BLCA. (E) Ki67 expression was analyzed by RNA-seq in PCa (n Z 499) and normal tissues (n Z 52). (F) The correlation between ARHGEF38 and ki67 expression was detected by analyzing TCGA data set.