The microRNA-200 family acts as an oncogene in colorectal cancer by inhibiting the tumor suppressor RASSF2

Abstract

This study aimed to determine whether manipulation of the microRNA-200 (miR-200) family could influence colon adenocarcinoma cell behavior. The miR-200 family has a significant role in tumor suppression and functions as an oncogene. In vitro studies on gain and loss of function with small interfering RNA demonstrated that the miR-200 family could regulate RASSF2 expression. Knockdown of the miR-200 family in the HT-29 colon cancer cell line increased KRAS expression but decreased signaling in the MAPK/ERK signaling pathway through reduced ERK phosphorylation. Increased expression of the miR-200 family in the CCD-841 colon epithelium cell line increased KRAS expression and led to increased signaling in the MAPK/ERK signaling pathway but increased ERK phosphorylation. Functionally, knockdown of the miR-200 family led to decreased cell proliferation in the HT-29 cells; therefore, increased miR-200 family expression could increase cell proliferation in the CCD-841 cell line. The present study included a large paired miR array dataset (n=632), in which the miR-200 family was significantly found to be increased in colon cancer when compared with normal adjacent colon epithelium. In a miR-seq dataset (n=199), the study found that miR-200 family expression was increased in localized colon cancer compared with metastatic disease. Decreased expression was associated with poorer overall survival. The miR-200 family directly targeted RASSF2 and was inversely correlated with RASSF2 expression (n=199, all P<0.001). Despite the well-defined role of the miR-200 family in tumor suppression, the present findings demonstrated a novel function of the miR-200 family in tumor proliferation.

Keywords: MAPK pathway; Ras association domain family member 2; colorectal cancer; microRNA-200 family; oncogene.

Figure 1.

Waterfall plot of the significantly dysregulated miRs in colon cancer cell lines HT-29 and T-84 as compared to the normal colon epithelial cell line (CCD-841). miR200 family is highlighted in yelow. miR, microRNA.

Figure 2.

Baseline miR-200 family expression and expression post transfection with miR-200 mimics and antagomirs. (A) Significant upregulation of all members of the miR-200 family in the HT-29 cell line vs. the CCD-841 cell line. (B) Significant upregulation of all of the miR-200 family compared to scramble following transfection with individual miR-200 family mimics in the CCD-841 cell line. (C) Significant downregulation of miR-200b and miR-200c following transfection with antagomirs as compared with scramble in the HT-29 cell line. ***P<0.0001 vs. CCD-841; ^††P<0.01 and ^†††P<0.001 vs. scramble. miR, microRNA.

Figure 3.

(A-C) miR-200a is increased in cancer tissue compared with normal adjacent epithelium (P<0.001). miR-200a expression is decreased in advanced stage (stage III or IV) compared with localized disease (stage I or II, P=0.006.) Low expression of miR-200a is associated with worse overall survival (log-rank=0.02). (D-F) miR-200b is increased in cancer tissue compared to normal adjacent epithelium (P<0.001). miR-200b expression is decreased in advanced stage (stage III or IV) compared to localized disease (stage I or II) (P=0.06). Low expression of miR-200b is associated with worse overall survival (log-rank=0.05). (G-I) miR-200c is increased in cancer tissue compared to normal adjacent epithelium (P<0.001). miR-200c expression is decreased in advanced stage (stage III or IV) compared to localized disease (stage I or II, P<0.001). Low expression of miR-200c is associated with worse overall survival (log-rank=0.03). (J-L) miR-141 is increased in cancer tissue compared to normal adjacent epithelium (P<0.001). miR-141 expression is decreased in advanced stage (stage III or IV) compared to localized disease (stage I or II, P=0.003). No difference in the overall survival between high and low miR-141 (log-rank=0.61). (M-O) miR-429 is increased in cancer tissue compared to normal adjacent epithelium (P<0.001). miR-429 expression is decreased in advanced stage (stage III or IV) compared to localized disease (stage I or II, P=0.08). No difference in the overall survival between high and low miR-429 (log-rank=0.30). miR, microRNA.

Figure 4.

Correlation of miR-200 family expression with RASSF2 expression in the colon adenocarcinoma TCGA dataset. (A-E) The miR-200 family is significantly inversely correlated with RASSF2 expression (n=199, P<0.001). miR, microRNA.

Figure 5.

Reduced expression of RASSF2 was associated a trend towards worse recurrence-free survival (log-rank=0.058).

Figure 6.

Expression of RASSF2 mRNA following transfection with miR-200 mimics and with miR-200 antagomirs. (A) Increased trend in the expression of RASSF2 in the HT-29 cell line following transfection with miR-200 antagomirs as compared with scramble (A-). *P=0.01. (B) Decreased trend in the expression of RASSF2 mRNA in the CCD-841 cell line following transfection with miR-200 mimics as compared with scramble (M-). *P=0.01 and **P<0.001 vs. scramble. miR, microRNA.

Figure 7.

Baseline RASSF2 protein expression and following transfection miR-200 mimics or antagomirs. (A) RASSF2 protein is expressed in the CCD-841 cell line at baseline. (B) There is no RASSF2 protein expression in the HT-29 cell line at baseline. (C) Following transfection with miR-200 mimics, there is reduced expression of RASSF2 protein compared to scramble with miR-200a, miR-200b, and miR-429 (*P<0.05 vs. scramble). Histograms are representative of n=5 experimental repeats for all transfections. Representative blots demonstrating significant decrease in RASSF2 expression following transfection with miR-429 mimics compared to scramble. (D) Following transfection with miR-200a and miR-200c antagomirs, there was an increased trend in RASSF2 protein expression. RASSF2 expression was significantly increased with miR-141 and miR-429 antagomir transfection (*P<0.05 vs. scramble). Histograms are representative of n=5 experimental repeats for all transfections. Representative blots demonstrating significant increase in RASSF2 expression following transfection with miR-429 antagomirs compared to scramble. miR, microRNA.

Figure 8.

Expression of KRAS protein following transfection of miR-200 mimics or antagomirs. (A) Increased KRAS protein expression in the CCD-841 cell line following transfection of miR-200a, miR-141, and miR-429 mimics compared to scramble (M-). (B) Increased expression of KRAS in the HT-29 following transfection of miR-200a and miR-200c antagomirs compared with scramble (A-). *P<0.05 vs. scramble. miR, microRNA.

Figure 9.

MAPK/ERK pathway activity and cell proliferation. (A) Significant upregulation of the ratio of phosphorylated to total ERK1/2 compared to negative control (M-) indicating increased MAPK pathway activation following transfection with all miR-200 family mimics in the CCD-841 CoN (normal colon epithelial) cell line. **P<0.002 vs. scramble. (B) Significant decrease in the ratio of ERK1/2 compared to negative control (A-) indicating decreased MAPK pathway activation following transfection with miR-200a, miR-200b, and miR-141 antagomirs in HT-29 (stage III colon cancer) cell line. *P<0.05 vs. scramble. (C) There is significantly increased proliferation in the CCD-841 CoN cell line following transfection with miR-200a, miR-200c, and miR-141 mimics. *P<0.05 vs. scramble. (D) There is significantly decreased cellular proliferation following transfection with antagomirs of all members of the miR-200 family. ***P<0.001 vs. scramble. miR, microRNA.

Figure 10.

Differences in miR-200 and RASSF2 expression in human colon cancer tissue compared with normal adjacent epithelial tissue from the same patients (n=5). (A) Sample H&E slides for tumor and control tissue used for reference during laser capture microdissection. (B) We observed downregulation in miR-200a, miR-200b, miR-141, and miR-429, and no change in miR-200c in colon cancer as compared with normal adjacent epithelial tissue using RT-qPCR and U6 for reference. (C) We observed downregulation of RASSF2 mRNA in colon cancer as compared with normal adjacent epithelial tissue; with an average fold change of −2.72 using RT-qPCR and 18S rRNA for reference. (D) A representative western blot showing RASSF2 protein downregulation using β-actin for reference. We observed a RASSF2: β-actin of 0.8 for CRC and 5.3 for normal adjacent for a −6.02 fold downregulation. miR, microRNA.