Fig 1: Piwil1 increased tumor growth potential and enhanced tumor metastatic potential. a The effects of Piwil1 on the cell growth of transfected cells were determined by MTT assay (*P < 0.05, **P < 0.01, ***P < 0.001). b Plated colony forming assays were performed to measure cell proliferation of transfected cells. Representative images are shown. The colonies were counted and graphed (*P < 0.05). c Representative images of spheroid colonies were shown. Scale bars, 100 μm. Quantification of the absolute number of spheroids formed by IshikawashPiwil1 cells or HEC-1BexPiwil1 cells compared to the control cells after 5 days (*P < 0.05). d Cell migration and invasion was measured in transwell chambers. Quantification of migration after 24 h and quantification of invasion after 48 h. Data are reported as fold induction vs control cells. Scale bar, 100 μm. **P < 0.01 Representative images are shown
Fig 2: Expression of Piwil1 in human endometrial carcinoma tissue. a RT-qPCR analysis for Piwil1 mRNA in normal human endometrial tissues (n = 10) and endometrial cancer tissues (n = 18) (**P < 0.01). b Representative Piwil1 immunohistochemical staining of normal endometrial tissues, endometrial atypical hyperplasia and two endometrial cancer types. Original magnification 200×, scale bar, 100 μm (upper); 400×, scale bar, 50 μm (lower). c Immunohistochemistry scores (IS) of Piwil1 in normal endometrium (n = 44), atypical hyperplasia (n = 17), and endometrial carcinoma tissues (n = 83) (***P < 0.001)
Fig 3: Piwil1 led to increased acquisition of endometrial cancer stem cell markers. a RT-qPCR and western blot demonstrated expression level of CD44, ALDH1, CD133, Oct4 and Nanog in IshikawaNT and IshikawashPiwil1 cells (*P < 0.05). b RT-qPCR and western blot demonstrated expression level of CD44, ALDH1, CD133, Oct4 and Nanog in HEC-1BEV and HEC-1BexPiwil1 cells (*P < 0.05, **P < 0.01). c Representative immunofluorescence images showing CD44, ALDH1 and CD133 expression in IshikawaNT and IshikawashPiwil1 cells or in HEC-1BEV and HEC-1BexPiwil1 cells. Nuclei were stained with DAPI. Scale bars, 25 μm
Fig 4: Identification of PIWIL1-bound RNAs. (A) RNA immunoprecipitation (RIP) was performed in the cytosolic fraction of COLO 205 cells with two different antibodies (IP1 and IP2); the presence of PIWIL1 protein was assessed by western blotting in the cytosolic lysate (Lys), Input (In), IP1, and IP2; PIWIL1 was absent in negative controls (IgG and NoAb); IgG were used as a control for IP loading. (B) Pie chart representing the number of enriched molecules per category in both IP1 and IP2 samples; (C) Venn diagram summarizing the number of piRNAs (left) and unannotated molecules (right) identified with both antibodies; (D) heatmaps representing Z-score expression values for 317 piRNAs (on the left) and 225 unannotated RNAs (on the right) identified in PIWIL1 RIP-Seq with two different antibodies (IP1 and IP2), compared to the input RNA. (E) Network analysis and functional enrichment for 106 validated interactions revealed a group of 27 master-regulator genes targeted by the PIWIL1–piRNA complex and 20 correlated genes strongly related with molecular pathways crucial for CRC pathogenesis. Relations between genes (173 links, Table S9E) and association to pathways are represented with a color code. Abbreviations: ERBB, avian erythroblastosis oncogene B; IGF1R, insulin like growth factor 1 receptor; mTOR, mechanistic target of rapamycin kinase; PI3K, phosphoinositide 3-kinase; PPARA, peroxisome proliferator activated receptor alpha; SMAD2/3, mothers against decapentaplegic homolog 2/3; TGF, transforming growth factor; TNF, tumor necrosis factor; Wnt, wingless/int-1.
Fig 5: Characterization of main molecular features of the piRNA pathway in colorectal cancer (CRC) cell lines. (A) The expression level of PIWIL mRNAs in 8 CRC cell lines was determined by real-time qRT-PCR. The highest expression for PIWIL1 was observed in COLO 205 and SW-1417. PIWIL2 and PIWIL4 were identified in some cell lines but at lower levels, while PIWIL3 was undetectable in all cases. (B) DNA methylation profile of PIWIL1 promoter in 8 CRC cell lines; a marked hypomethylation, correlated to PIWIL1 expression, was observed for two cell lines (COLO 205 and SW-1417), while a strong methylation was present in the absence of PIWIL1. CpG sites highlighted in blue constitute a CpG island. (C) Western blotting analysis performed onto total protein lysate from 8 CRC cell lines confirmed the presence of abundant PIWIL1 levels in COLO 205 and medium-abundant levels in SW-1417; as a control to test the specificity of the antibody, total protein lysate from HCT 116 cells overexpressing PIWIL1 or transfected with empty vector were loaded in the same gel; β-actin was used as a loading control. (D) Gene expression (indicated as fragment per kilobase million, FPKM) was measured for known germline components of the piRNA pathway genes by RNA-Seq; genes were grouped according to their role in the piRNA pathway. (E) Pie-chart representing the relative abundance of small RNA categories identified in non-treated (left) and sodium-periodate (NaIO4)/β-elimination oxidized RNA (right) from COLO 205; small RNA-Seq analysis confirmed the presence of piRNAs in the COLO 205 cell line and their resistance to the sodium periodate/β-elimination treatment. (F) Scatterplot representing the effect of the sodium periodate (NaIO4)/β-elimination treatment on the piRNAs population; log2 CPM (count per million) values are displayed per each COLO 205 piRNA in treated (y-axis) and non-treated (x-axis) RNA; the treated (T) vs. non-treated (NT) log2 ratio of expression values (p value < 0.01) was used to identify 767 methylated piRNAs (log2(T/NT) > 2), 1514 partially methylated piRNAs (−2 < log2(T/NT) < 2), and 2078 non-methylated piRNAs (log2(T/NT) < −2) show that PIWIL1 transcripts were highly abundant in COLO 205 and medium-low expressed in SW-1417 cell lines, while PIWIL2 and PIWIL4 genes were expressed at low levels in 6 out of 8 cell lines and PIWIL3 was undetectable in all cases with HCT 116 and RKO cells negative for all PIWIL transcripts.
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