MicroRNA-21 is involved in ionizing radiation-promoted liver carcinogenesis

Abstract

It has been known for decades that ionizing radiation (IR) promotes carcinogenesis and high-linear energy transfer (LET) IR has a higher risk than low-LET IR for carcinogenesis; however, the mechanism remains unclear. MicroRNAs (miRNAs) have a critical effect on carcinogenesis through post-transcriptional modification. In this study, our purpose is to explore whether miRNAs are involved in IR-(especially high-LET IR) promoted liver carcinogenesis. We showed here that among several hundred miRNAs, miR-21 was the only one that increased 6 folds in high-LET IR-promoted mouse liver tumors when compared with that in the non-irradiated liver tissues. We also showed that miR-21 was up-regulated in human or mouse hepatocytes after exposure to IR, as well as in liver tissues derived from whole body irradiated mice. The increased level of miR-21 was more significant in high-LET irradiated cells or liver tissues. After the non-irradiated, low-LET or high-LET irradiated human hepatocytes were over-expressed with miR-21, these cells became tumorigenesis in nude mice. The tumors derived from high-LET-irradiated-cells were largest, and accompanied by more significant changes in the miR-21-targets: PTEN and RECK. In addition, we showed that IR-induced up-regulation of miR-21 depended on the up-regulation/activation of AP-1 (at an earlier time, within 2 h) and the ErbB/Stat3 pathway (at a later time, more than 2 h), which was also IR dose dependent. Taken together, we conclude that IR-induced up-regulation of miR-21 plays an important role in IR (especially high-LET IR)-promoted liver carcinogenesis.

Keywords: carcinogenesis; ionizing radiation; miR-21; microRNA.

Figure 1

Over-expression of miR-21 in liver carcinoma from irradiated mice. (A) Histology of liver tissues or tumors (H&E, × 200) from non-irradiated or high-LET irradiated (1 Gy) mice. (B) The miR-21 expression level was derived from the microarray data (LC, Sciences, LLC). The mice were irradiated with 1 Gy high-LET IR (iron) and the mice were sacrificed two years after IR. The tumor data were mean + SE obtained from three mice. (C) Histology of liver tissues or tumors (H&E, × 200) from non-irradiated or high-LET irradiated (1 Gy) mice. (D) The miR-21 expression level was analyzed by qRT-PCR. The mice were irradiated with 0.5 Gy high-LET IR (neutron) and the mice were sacrificed two years after IR. The tumor data were mean +SE obtained from ten mice.

Figure 2

IR-stimulated miR-21 expression in human or mouse hepatocytes and liver tissue. (A) miR-21 expression in mouse or human hepatocytes survived 0.5 Gy low-LET or high-LET IR after more than 10 generations. The miR-21 level was derived from the microarray data (LC, Sciences, LLC). The analysis is based on the non-irradiated controls (as 1). (B) miR-21 expression in liver tissue from mice (6-8 month old) at 1 day or 20 days after low or high-LET IR (0.5 Gy). The miR-21 level was derived from the microarray data (LC, Sciences, LLC). Each group had 3 mice, the analysis is based on the non-irradiated controls (as 1) *, p < 0.05.

Figure 3

IR-stimulated miR-21 expression correlates with up-regulation/activation of AP-1 and ErbB/Stat3. (A) The levels of AP-1 (c-Fos and c-Jun) or miR-21 in LO2 (human hepatocytes) cells at different times after 1 Gy exposure. Actin was used as an internal loading control. L: the cells (LO2) that survived 0.5 Gy low-LET IR. H: the cells (LO2) that survived 0.5 Gy high-LET IR. Top panel: the image from Western blot. Bottom panel: analysis of the protein levels shown in the top panel by ImageQuant. The miR-21 level was measured by qRT-PCR. (B) The levels of Stat3, phosphorylated Stat3, and miR-21 in LO2 cells at different times after 1 Gy exposure. (C) The levels of ErbB (EGFR and ErbB2), phosphorylated ErbB and miR-21 in LO2 cells at different times after 1Gy exposure.

Figure 4

IR-stimulated miR-21 expression is via AP -1 at an earlier time and ErbB/Stat3 at a later time. (A) The effects of AP-1 siRNAs on the expression of miR-21 targets. LO2 cells were treated with c-Jun siRNA, c-Fos siRNA or both for 24 h and were then irradiated with 1 Gy. The cells were collected at 1 h after IR and the protein levels were measured by Western blot. Actin was used as an internal loading control. (B) The effects of AP-1 siRNA on the expression of miR-21. The cells were collected at 1 h after IR, RNA was prepared and the miR-21 levels were detected by qRT-PCR. The results were obtained from two separate experiments with 6 samples of each treatment, **, p< 0.01. (C) The effects of EGFR siRNA and ErbB2 siRNA on Stat3 activation and the expression of miR-21 targets. LO2 cells were treated with EGFR siRNA, ErbB2 siRNA, or both for 24 h and were irradiated with 1 Gy. The cells were then collected at 72 h after IR and the protein levels were measured by Western blot. (D) The effects of EGFR siRNA and ErbB2 siRNA on the miR-21 expression. The cells were treated and collected as described above, RNA was prepared and the miR-21 levels were detected by qRT-PCR. The results were obtained from two separate experiments using a total of 6 samples for each treatment, **, p< 0.01.

Figure 5

Up-regulation of miR-21 contributes to IR-promoted hepatocyte tumorigenesis. (A) The levels of miR-21 in NR (non-irradiated LO2 cells), L05 or H05 cells that were transfected with miR-21 or a control vector. (B) Imaging of the sizes of tumors that developed in the nude mice from the cells over-expressed with miR-21: NR-miR-21, the non-irradiated LO2 cells that were over-expressed with miR-21; L05-miR-21, the LO2 cells that survived 0.5 Gy low-LET IR and over-expressed with miR-21; H05-miR-21, the LO2 cells that survived 0.5 Gy high-LET IR and over-expressed with miR-21. Each mouse was injected subcutaneously with the same type cells at two sides of the body. Each group contained 6 mice and 12 injections. (C) Analysis of the tumor weight in the mice from different types of cell lines: NR-miR -21, L05-miR-21 or H05-miR-21: *, P< 0.05; **, P<0.01. (D) Histopathological image of tumors derived from NR-miR-21, L05-miR-21 or H05-miR-21 cells. The tumor tissues were prepared for histopathological slides with hematoxylin and easin (H&E, × 100) staining. The black arrows indicated the small blood vessels. (E) Compare the levels of miR21 targets (RECT and PTEN) in NR-miR-21, L05-miR-21 or H05-miR-21 cells versus that in NR-v, L05-v and H05-v cells. Western blot was performed and Actin was used as an internal loading control.

Figure 6

The effects of IR with different doses on AP-1, ErbB/Stat3 and miR-21 expression/activation. (A) The levels of AP-1 (c-Fos and c-Jun) or miR-21 in LO2 cells at 1 h after different doses of IR exposure. L and H are the same as described in Figure 3. Top panel: the image from Western blot. Bottom panel: analysis of the protein levels shown in the top panel by ImageQuant. The miR-21 level was measured by qRT-PCR. (B) The levels of EGFR, phosphorylated ErbB or Stat3 and miR-21 in LO2 cells at 72 h after different doses of IR exposure. L and H are the same as described above. Top panel: the image from Western blot. Bottom panel: analysis of the protein levels shown in the top panel by ImageQuant. The miR-21 level was measured by qRT-PCR. (C) A summary of the effects of IR-promoted liver carcinogenesis is as follows: IR stimulates miR-21 expression through the up-regulation or activation of AP-1 (at an earlier time) and ErbB/Stat3 (at a later time) pathways. AP-1 could also directly or indirectly activate the ErbB/Stat3 pathways. The over-expression of miR-21 promotes liver tumorigenesis and other IR-stimulated factors that may also facilitate the function of miR-21 and contribute to liver tumorigenesis.