MiR-124 Radiosensitizes human colorectal cancer cells by targeting PRRX1

Abstract

One of the challenges in the treatment of colorectal cancer patients is that these tumors show resistance to radiation. MicroRNAs (miRNAs) are involved in essential biological activities, including chemoresistance and radioresistance. Several research studies have indicated that miRNA played an important role in sensitizing cellular response to ionizing radiation (IR). In this study, we found that miR-124 was significantly down-regulated both in CRC-derived cell lines and clinical CRC samples compared with adjacent non-tumor colorectal tissues, MiR-124 could sensitize human colorectal cancer cells to IR in vitro and in vivo. We identified PRRX1, a new EMT inducer and stemness regulator as a novel direct target of miR-124 by using target prediction algorithms and luciferase assay. PRRX1 knockdown could sensitize CRC cells to IR similar to the effects caused by miR-124. Overexpression of PRRX1 in stably overexpressed-miR-124 cell lines could rescue the effects of radiosensitivity enhancement brought by miR-124. Taking these observations into consideration, we illustrated that miR-124 could increase the radiosensitivity of CRC cells by blocking the expression of PRRX1, which indicated miR-124 could act as a great therapeutic target for CRC patients.

Figure 1. miR-124 is down-regulated both in primary CRC tissues and cell lines.

(A) miR-124 was expressed at significantly lower levels in six CRC cell lines in comparison with normal colonic mucosa pooled from three healthy individuals. The figure is representative of three experiments with similar results.**P<0.01, *P<0.05. (B) The expression of miR-124 in CRC tissues and the matched normal tissues was detected by qRT-PCR and normalized to that of U6. Data are presented as individual samples (N = 24) with the line indicating the mean level.

Figure 2. miR-124 sensitizes colorectal cancer cells to irradiation treatment in vitro.

(A) LOVO and SW480 cells stably over-expression of miR-124 were treated with 0, 2, 4, 6, or 8Gy of IR. Survival fractions were calculated as described in Figure 2A. The results are presented as the means SD of values obtained in 3 independent experiments. The statistical significance of differences between the groups was calculated using Student t tests. *p<0.05. (B) Apoptosis assay showing induction of apoptosis after miR-124 over-expression, in particular combination with radiation in miR-124-overexpressed cell lines LOVO and SW480. *p<0.05. (C) Representative western blot for the effect of miR-124 over-expression or/and radiation(4Gy) on the expression of apoptosis and DNA damage related genes(Caspase-3, Bcl-2 andγ-H2AX).

Figure 3. PRRX1 is a direct target of miR-124.

(A) The predicted binding sequences for miR-124 within the human PRRX1 3′UTR. Seed sequences are highlighted and underlined. (B), (C) and(D) PRRX1 expression was determined in colorectal cancer cells stably overexpressed-miR-124 and cells transfected with miR-124 inhibitors, or antimiR-con by real-time PCR and Western blot analysis. ANOVA and Student t tests were used to determine the statistical significance of the differences between groups. *p<0.05. (E) Luciferase activity assays using a luciferase reporter with wild-type or mutant human PRRX1 3′UTRs were performed after co-transfection of miR-124 mimics or inhibitors into HEK293 cells. And mt 3′UTR has a significantly increase compared with wt 3′UTR.The bar graph showed the mean±SD in three independent transfection experiments. *p<0.05.

Figure 4. The effect of PRRX1 knockdown on radiosensitivity of CRC cells in vitro.

(A) Western blot analyzed PRRX1 interference efficiency in LOVO and SW480 cells (B) The quantification of the number formed from CRC cells that were stably infected with empty vector lentiviruses (control shRNA) and PRRX1-shRNA lentiviruses (PRRX1 shRNA) or without lentiviral infection (untreated). (C) The Annexin-V assay of apoptosis for the PRRX1 knockdown cells compared with the control cells.*p<0.05. (E) Representative western blot for the effect of PRRX1 knockdown or/and radiation(4Gy) on the expressions of apoptosis related genes(Caspase-3, Bcl-2 andγ-H2AX).

Figure 5. Restoration of PRRX1 expression in miR-124-overexpressed cells rescues the effects of miR-124 on radiosensitivity.

(A) PRRX1 expression was detected by western blot after transfecting pcDNA3.1-PRRX1 into miR-124-overexpressed cells. (B) Cells were followed by treatment as described in Figure 2A. Survival fractions were calculated for each group. The results are presented as the means ±SD of values obtained in 3 independent experiments. ANOVA or Student t tests were used to determine the statistical significance of the differences between groups. Statistical significance(*P<0.05) is indicated vs LV-con and miR-124 group. (C) Representative western blot for the effect of PRRX1 restoration or/and radiation(4Gy) on the expressions of apoptosis related genes(Caspase-3, Bcl-2 andγ-H2AX).

Figure 6. Detecting EMT and stemness-related genes expression by western blot.

Western blot analyzed the EMT-related genes like E-cadherin, ZO-1, Vimentin and N-caherin and stemness-related genes such as ABCG2, SOX2 and Oct4 in miR-124-tranfected cells and miR-124-PRRX1 co-transfected cells compared with control group. Data suggested over-expression of miR-124 could down-regulate Vimentin, N-cadherin, ABCG2, SOX2 and Oct4 expression and up-regulate E-cadherin, ZO-1 expression, while over-expression of PRRX1 could rescue this effect.

Figure 7. miR-124 sensitizes colorectal cancer cells SW480 to irradiation treatment in vivo.

(A) Over-expression of miR-124 sensitized SW480 tumor xenografts to IR in vivo. Tumor sizes were measured with caliper and irradiation was delivered to tumors area on the eleventh day(IR:10Gy, d11). (B)The volume curves of xenografts generated by LV-con, LV-miR-124, LV-con+Radiation, LV-miR-124+Radiation. (p<0.05).