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. 2021 Aug 11:11:699475.
doi: 10.3389/fonc.2021.699475. eCollection 2021.

Screening of MicroRNA Related to Irradiation Response and the Regulation Mechanism of miRNA-96-5p in Rectal Cancer Cells

Fengpeng Wu  1 , Bingyue Wu  2 , Xiaoxiao Zhang  1 , Congrong Yang  1 , Chaoxi Zhou  3 , Shuguang Ren  4 , Jun Wang  1 , Yafan Yang  3 , Guiying Wang  3   5
Affiliations

Screening of MicroRNA Related to Irradiation Response and the Regulation Mechanism of miRNA-96-5p in Rectal Cancer Cells

Fengpeng Wu et al. Front Oncol. .

Abstract

Neoadjuvant chemoradiotherapy has been widely used in the treatment of locally advanced rectal cancer due to the excellent advantages of irradiation in cancer therapy. Unfortunately, not every patient can benefit from this treatment, therefore, it is of great significance to explore biomarkers that can predict irradiation sensitivity. In this study, we screened microRNAs (miRNAs) which were positively correlated with irradiation resistance and found that miRNA-552 and miRNA-183 families were positively correlated with the irradiation resistance of rectal cancer, and found that high expression of miRNA-96-5p enhanced the irradiation resistance of rectal cancer cells through direct regulation of the GPC3 gene and abnormal activation of the canonical Wnt signal transduction pathway. Based on the radioreactivity results of patient-derived xenograft models, this is the first screening report for radio-resistant biomarkers in rectal cancer. Our results suggest that miRNA-96-5p expression is an important factor affecting the radiation response of colorectal cancer cells.

Keywords: GPC3; Wnt/β-catenin; irradiation resistance; miRNA-96-5p; rectal cancer.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Differential screening of irradiation reactivity to rectal cancer tissues and to rectal cancer cell lines. (A) Fresh specimen cut from the center of tumor; (B) Rectal cancer tissue was transplanted into nude mice to construct the first generation PDX model; (C) Second generation PDX model for screening irradiation reactivity; (D) Compensation tissue and irradiation field on the surface of xenograft tumor; (E) Irradiation caused liquefaction and necrosis of the tumor; (F) Measurement of xenograft tumor in vitro; (G) Sterilization with marked fibrosis (RCRG1); (H) Marked fibrosis but macroscopic disease present (RCRG2); (I) Little or no fibrosis, with abundant macroscopic disease (RCRG3); (J) Status of five rectal cancer cell lines exposed to irradiation, and survival fraction fitted to the linear quadratic equation; (K) Colony formation assays of five rectal cancer cell lines under irradiation interference.
Figure 2
Figure 2
A flowchart of each step in this study.
Figure 3
Figure 3
Differential miRNAs expression between RCRG1 and RCRG3. (A) MiRNAs microarray was used to detect the differential expression of miRNAs between RCRG1 and RCRG3, the scale bar indicates miRNAs expression level: red represented high expression, green represented low expression; (B) Differential expression miRNAs were confirmed by qRT-PCR.
Figure 4
Figure 4
Detecting the expression of miRNAs positively correlated with irradiation resistance in rectal cancer cell lines showed that the expression of miRNA-96-5p was positively correlated with the radiation resistance of the cells.
Figure 5
Figure 5
Changes in irradiation reactivity and biological behavior of HR-8348 cells before and after miRNA-96-5p knockdown. (A) MiRNA-96-5p was significantly down-regulated in HR-8348-IN cells; (B) MiRNA-96-5p low-expression in radio-resistant HR-8348 cells results in increased sensitivity to irradiation, and survival fraction fitted to the linear quadratic equation; (C) The effect of miRNA-96-5p on rectal cancer cells growth, as measured using the MTS assay; (D–G) The role of miRNA-96-5p in migration and invasion of rectal cancer cells was detected by wound healing test and Transwell assays *P < 0.05, **P < 0.01.
Figure 6
Figure 6
The expression profiles of seven target genes that may be regulated by miRNA-96-5p obtained from the GEPIA website in rectal cancer tissues and normal tissues. The median values of CAV1, DAB2, DDIT3, FOXO3, GPC3, MBD4 and PDCD4 in rectal cancer tissues and normal tissues were 17.16 and 180.87, 23.78 and 22.54, 27.78 and 33.00, 19.58 and 15.76, 1.24 and 4.3, 17.76 and 18.13, 92.51 vs.110.35, respectively. The red line represents the tumor, and the green line represents normal tissue.
Figure 7
Figure 7
Screening of target genes regulated by miRNA-96-5p and the effect of up-regulation of GPC3 expression on radiation reactivity of HR-8348 cells. (A–C) qRT-PCR assays were used to detect the expression levels of target genes in rectal cancer cells before and after miRNA-96-5p knockdown, and the mRNA expressions of FOXO3 and GPC3 were significantly up-regulated in HR-8348-IN cells; (D–G) Western blot assays were used to detect the expression of target genes in rectal cancer cells before and after miRNA-96-5p knockdown, and the protein expression of GPC3 was not significantly changed in HR-8348-NC cells, but was significantly up-regulated in HR-8348-IN cells; (H) Luciferase activity of the WT and mutant GPC3 3′UTR co-transfected with miRNA-NC and miRNA-96-5p mimics, the GPC3-WT group was inhibited, but not in the GPC3-MUT group; (I) GPC3 was significantly up-regulated in HR-8348-GPC3 cells; (J) GPC3 over-expression in HR-8348 cells results in increased sensitivity to irradiation, and survival fraction fitted to the linear quadratic equation. **P < 0.01.
Figure 8
Figure 8
Effect of miRNA-96-5p on the status of Wnt/β-catenin signal transduction pathway in rectal cancer cells. (A) Screening of enriched signal pathway indicated that Wnt signal pathway was one of the important factors affecting irradiation resistance of rectal cancer. (B, C) Down-regulation of miRNA-96-5p significantly reduced the expression of β-catenin in the cytoplasm and the phosphorylation level of GSK3β in rectal cancer cells. (D) After the down-regulation of miRNA-96-5p, the expression of CD44 and c-Myc was significantly reduced in rectal cancer cells. (E, F) GPC3 over-expression significantly reduced the expression of β-catenin in the cytoplasm and the phosphorylation level of GSK3β in rectal cancer cells. **P < 0.01.
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