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. 2018 Oct 6;8(19):5213-5230.
doi: 10.7150/thno.28001. eCollection 2018.

PIWI-interacting RNA-54265 Is Oncogenic and a Potential Therapeutic Target in Colorectal Adenocarcinoma

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Free PMC article

PIWI-interacting RNA-54265 Is Oncogenic and a Potential Therapeutic Target in Colorectal Adenocarcinoma

Dongmei Mai et al. Theranostics. .
Free PMC article

Abstract

Although PIWI-interacting RNAs (piRNAs) have recently been linked to human diseases, their roles and functions in malignancies remain unclear. This study aimed to investigate the significance of some piRNAs in colorectal cancer (CRC). Methods: We first analyzed the expression profile of piRNAs in CRC using the TCGA and GEO databases. The top 20 highly expressed piRNAs were selected and tested in our CRC tumor and non-tumor tissue samples. We then examined the relevance of the significantly differentially expressed piRNA to the CRC outcomes in 218 patients receiving postoperative chemotherapy and 317 patients receiving neoadjuvant chemotherapy. A series of biochemical and molecular biological assays were conducted to elucidate the functional mechanism of a piRNA of interest in CRC. Furthermore, experiments with mice xenografts were performed to evaluate the therapeutic effect of an inhibitor specific to the piRNA. Results: We found that among the examined 20 piRNAs, only piRNA-54265 was overexpressed in CRC compared with non-tumor tissues and higher levels in tumor or in serum were significantly associated with poor survival in patients. Functional assays demonstrated that piRNA-54265 binds PIWIL2 protein and this is necessary for the formation of PIWIL2/STAT3/phosphorylated-SRC (p-SRC) complex, which activates STAT3 signaling and promotes proliferation, metastasis and chemoresistance of CRC cells. Treatment with a piRNA-54265 inhibitor significantly suppressed the growth and metastasis of implanted tumors in mice. Conclusion: These results indicate that piRNA-54265 is an oncogenic RNA in CRC and thus might be a therapeutic target.

Keywords: STAT3; biomarker; colorectal cancer; piRNA; therapeutic target.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
piR-54265 is overexpressed in CRC and associated with clinical outcomes. (A) Expression levels of the top 20 highly expressed piRNAs in CRC and paired non-tumor tissue samples from 110 individuals recruited at SYSUCC. Only piR-54265 shows significantly higher levels in CRC than in normal tissues (mean ± SEM; ***, P<0.001). (B) DNA copy number alterations of piR-54265 gene locus in CRC from TCGA database. (C) Lower methylation status of piR-54265 gene locus in CRC than in normal tissues based on TCGA database. (D) piR-54265 expression levels in CRC with different tumor stages (mean ± SEM). P-values by one-way ANOVA show significant difference among four groups. (E) Tumor piR-54265 expression levels of CRC in stage I-III and stage IV by RT-qPCR (mean ± SEM). P-values by Student's t-test. (F-G) Kaplan-Meier estimates of progression-free survival (PFS) time (F) and overall survival (OS) time (G) in CRC patients with different piR-54265 levels in tumor. HR: hazard ratio; CI: confidence interval.
Figure 2
Figure 2
piR-54265 promotes CRC cell proliferation and invasiveness. (A-B) Overexpression of piR-54265 (A) promoted but knockdown of piR-54265 (B) suppressed CRC cell proliferation (mean ± SEM, N=6; *, P<0.05; **, P<0.01 and ***, P<0.001). (C) Effects of piR-54265 expression on the colony formation ability of CRC cells. Representative images (left panel) and quantification (right panel) of colony formation ability (mean ± SEM). (D-E) Effects of piR-54265 expression on subcutaneous xenograft growth in mice. Images of xenograft tumors (upper panels) and tumor growth curves as a function of time (lower panels). (F) Effects of piR-54265 on CRC cell apoptosis. Flow cytometry images (left panel) and quantitative statistics (mean ± SEM, N=3; **, P<0.01; ***, P<0.001) (right panel). (G-H) Effects of piR-54265 on the abilities of migration (G) and invasion (H) of CRC cells in vitro. Representative images of transwell assays (left panels) and quantitative statistics (mean ± SEM, N=3; **, P< 0.01; ***, P<0.001; ****, P<0.0001) (right panels). Scale bars, 100 μm. (I-L) Effects of piR-54265 expression on metastasis of CRC cells in mice. Luminescence imaging of metastases (I) and quantification (J-K) of radiance intensity (mean ± SEM, N=6 in each group). (L) H&E staining images of pathological sections of metastatic cancers in the lung, brain and bone in mice (scale bars, 500 μm). *, P<0.05; **, P<0.01; ***, P<0.001 and ****, P<0.0001 by Student's t-test. OE: overexpression; KD: knockdown.
Figure 3
Figure 3
piR-54265 interacts with PIWIL2, facilitating PIWIL2/STAT3/p-SRC formation. (A) RNA immunoprecipitation assays showed specific bind of piR-54265 to PIWIL2. Results are mean ± SEM of piR-54265 enrichment relative to input from three independent experiments. (B) Biotin-labeled piR-54265 RNA pulldown coupled with western blot analysis revealed interaction of piR-54265 with PIWIL2, STAT3 and p-SRC. (C) Schematic of the domain structure of PIWIL2 protein. (D) Biotin-labeled piR-54265 RNA pulldown from lysates containing FLAG-tagged full-length or truncated PIWIL2 protein coupled with Western blot analysis revealed the interaction of piR-54265 with PIWIL2 via the PIWI domain in PIWIL2. (E) Co-immunoprecipitation assays revealed the interaction of STAT3 with PIWIL2 via the PAZ domain in PIWIL2. (F) RNA immunoprecipitation assays showed specific association of piR-54265 with STAT3 and p-SRC in CRC cells. Results are mean ± SEM of piR-54265 enrichment relative to input from three independent experiments. (G) Reciprocal immunoprecipitation assays showed that the interaction among PIWIL2, STAT3 and SRC in CRC cells was affected by piR-54265 expression. (H) Effects of overexpression or knockdown of piR-54265 on the expression levels of STAT3 and SRC and their phosphorylated forms detected by western blot.
Figure 4
Figure 4
piR-54265 enhances oncogenic STAT3 signaling. (A) Effects of piR-54265 on expression and/or activation of proliferation and metastasis-related STAT3 downstream modules in CRC cells detected by western blot. (B-C) Immunohistochemical (IHC) staining of proliferation- and metastasis-related STAT3 downstream modules in mouse xenograft tumors of CRC cells with overexpression or knockdown of piR-54265. IHC staining of proliferation-related molecules (B) and metastasis-related molecules (C). 200×; scale bars, 50 μm. (D) Knockdown of PIWIL2 or STAT3 affects piR-54265-induced CRC cell proliferation (each point in the curve represents mean ± SEM; N=6; **, P<0.01; ***, P<0.001). (E) Knockdown of PIWIL2 or STAT3 affects the piR-54265-induced migration (upper panel) and invasion (lower panel) abilities of CRC cells (mean ± SEM; N=6. ***, P<0.001; ****, P<0.0001). See also Figure S8B. (F) Knockdown of PIWIL2 or STAT3 affects piR-54265-induced CRC cell apoptosis (mean ± SEM; N=3; ***, P<0.001). See also Figure S8C. OE: overexpression; KD: knockdown.
Figure 5
Figure 5
piR-54265 is a therapeutic target in mouse xenograft tumors. (A-B) Effect of piR-54265 overexpression (OE) or knockdown (KD) on chemosensitivity of CRC cells to 5-FU (A) or oxaliplatin (B) as presented in statistic histograms of IC50 values. (mean ± SEM, N=6; ***, P<0.001; ****, P<0.0001). (C-D) Effects of piR-54265 overexpression (OE) or knockdown (KD) on apoptosis of CRC cells induced by 5-FU (C) or oxaliplatin (D). Results are mean ± SEM (N=3). **, P<0.01; ***, P<0.001. See also Figure S8A. (E-F) Effects of piR-54265 on sensitivity of mouse subcutaneous xenograft tumors to chemotherapeutic agents. Overexpression of piR-54265 (OE) conferred xenograft tumor tolerance toward oxaliplatin (oxal) and 5-FU (E). Knockdown of piR-54265 (KD) conferred xenograft tumor sensitivity to oxaliplatin and 5-FU (F). Arrows indicate the time of drug treatment. All P<0.05 for repeated measurement of a general linear model. (G) Timeline schematic for intra-tumor treatment of mouse subcutaneous xenografts with antagopiR54265. Colored arrows indicate the times when different events occurred. (H-I) Injection of antagopiR54265 into xenograft tumors significantly suppressed tumor growth. Images of xenograft tumors with or without injection of antagopiR54265 (H) and the growth curves of xenografts (I). Each point in the curves presents mean ± SEM (N=5). *, P<0.05; **, P<0.01 compared with each control. (J) Timeline schematic for treatment of mouse metastatic tumors with antagopiR54265 alone or combination with 5-FU. Colored arrows indicate the times when different events occurred. (K-L) Injection of antagopiR54265 via tail vein significantly suppressed tumor spread and metastasis. Radiant images of metastatic tumors in mice (K) and quantitation of tumor metastasis indicated by radiance intensity (L). Each point in the curve is mean ± SEM of radiance (N=5). *, P<0.05; **, P<0.01 and ***, P<0.001. (M) Survival times of mice with metastatic tumors treated with or without antagopiR54265 or antagopiR54265 plus 5-FU. All log-rank P<0.05 compared with control group.
Figure 6
Figure 6
Serum piR-54265 predicts clinical outcomes of CRC patients. (A) Northern blot of piR-54265 in randomly selected serum samples from individuals with CRC confirmed its existence and expected molecular size of 29 nucleotides. (B) Stability of serum piR-54265 under different storage conditions. piR-54265 levels in sera of 8 randomly selected individuals with CRC were detected: 1, at day 1 (2017/04/16); 2, after storage at -80 °C for about 7 months (2017/11/21); and 3, after storage at room temperature for 80 h (mean ± SEM, N=3; all differences are not significant). (C) Serum piR-54265 levels in tumors of different stages (mean ± SEM). P-value by one-way ANOVA shows difference among four groups, Stage I, II, III and IV (left panel). P-value by Student's t-test shows the significant difference between Stage I-III and IV (right panel). (D) The levels of piR-54265 in serum were positively correlated with the levels of piR-54265 in tumors from patients recruited at SYSUCC, AHSU and combined samples. (E) Kaplan-Meier estimates of progression-free survival time (upper panel) and overall survival time (lower panel) in CRC patients with different serum piR-54265 levels. HR: hazard ratio; CI: confidence interval. (F-H) Outcomes of 5-FU and oxaliplatin-based neoadjuvant chemotherapy in patients with CRC as a function of serum piR-54265 level. Shown are the results of SYSUCC2 sample (F), CHCAMS sample (G) and combined samples (H). P-value by Chi-square test. (I) Diagnostic performance of serum piR-54265 levels in predicting curative effect of 5-FU and oxaliplatin-based chemotherapy by receiver operating characteristic (ROC) curves analyses in the two patient sets and combined samples. AUC: area under ROC curves. See also Table 4.

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