Inhibition of colorectal cancer stem cell survival and invasive potential by hsa-miR-140-5p mediated suppression of Smad2 and autophagy

Abstract

Colorectal cancer (CRC) is the third highest mortality cancer in the United States and frequently metastasizes to liver and lung. Smad2 is a key element downstream of the TGF-β signaling pathway to regulate cancer metastasis by promoting epithelial to mesenchymal transition and maintaining the cancer stem cell (CSC) phenotype. In this study, we show that hsa-miR-140-5p directly targets Smad2 and overexpression of hsa-miR-140-5p in CRC cell lines decreases Smad2 expression levels, leading decreased cell invasion and proliferation, and increasing cell cycle arrest. Ectopic expression of hsa-miR-140-5p in colorectal CSCs inhibited CSC growth and sphere formation in vitro by disrupting autophagy. We have systematically identified targets of hsa-miR-140-5p involved in autophagy. Furthermore, overexpression of hsa-miR-140-5p in CSCs abolished tumor formation and metastasis in vivo. In addition, there is a progressive loss of hsa-miR-140-5p expression from normal colorectal mucosa to primary tumor tissues, with further reduction in liver metastatic tissues. Higher hsa-miR-140 expression is significantly correlated with better survival in stage III and IV colorectal cancer patients.The functional and clinical significance of hsa-miR-140-5p suggests that it is a key regulator in CRC progression and metastasis, and may have potential as a novel therapeutic molecule to treat CRC.

Keywords: Smad2; autophagy; colon cancer stem cell; hsa-miR-140-5p; metastasis.

Figure 1. Smad2 is the direct target of hsa-miR-140-5p

Four putative hsa-miR-140-5p binding sites exist in the 3′-UTR of Smad2 mRNA A. Ectopic expression of hsa-miR-140-5p (140) or siRNAs against Smad2 (siSmad2, Si) in HCT116, RKO and SW480 cells decreased Smad2 protein levels by Western blot analysis B. and mRNA level by real-time qRT-PCR C. Transfection of hsa-miR-140-5p inhibited firefly luciferase activity of pMIR-REPORT-3UTRSmad2 D. The negative miRNA (NEG) was used as the negative control in all experiments. The impact of hsa-miR-140-5p and siSmad2 on Smad2 expression was normalized and compared to those of negative miRNA (n = 3).

Figure 2. Hsa-miR-140-5p inhibits CRC cells invasion in vitro through Smad2

Ectopic expression of hsa-miR-140-5p in HCT116, RKO and SW480 cells decreased invasive activity measured by an ECM invasion assay. Knockdown of Smad2 by siRNAs has similar effects. (n = 3, p < 0.001).

Figure 3. Hsa-miR-140-5p inhibited colon cancer cell growth and induced cell cycle arrest partially through targeting Smad2

HCT116 A. RKO B. and SW480 cells C. were transfected with hsa-miR-140-5p or siSmad2, and cell numbers were measured with WST-1 assay at day 1, 3 and 5. Cell cycle analysis was performed to determine the impacts of hsa-miR-140-5p and siSmad2. The G1/S ratio was shown in D. (n = 3).

Figure 4. Hsa-miR-140-5p disrupts CSC growth through interrupting autophagy

A. HCT116 CSCs were transfected with hsa-miR-140-5p, and cell numbers were measured with WST-1 assay at day 1, 3 and 5 (n = 3). B. Representative bight field pictures of morphological changes were taken at 3 days after transfection (20×). C. The cell-death related genes were screened with PCRarray and gene changes over 2 folds are listed.

Figure 5. Hsa-miR-140-5p impacts CRC initiation and metastasis in vivo

Prior to injection, HCT116 CSCs were either transfected with hsa-miR-140-5p precursors or infected with lentivirus expressing hsa-miR-140-5p. Corresponding negative controls were used for both treatments. A. 10⁵ HCT116 CSCs were subcutaneously injected into NOD/SCID mice. The tumor sizes were measured for 8 weeks and the final volumes are shown (n = 5). Representative images of mice bearing HCT116 tumors at 8 weeks after injection are shown. B. 10⁵ HCT116 CSCs were injected into NOD/SCID mice through tail vein. Lungs and livers were collected at 8 weeks after injection, and were subjected to H&E staining (n = 5). Representative images are shown (upper panel 10×, lower panel 20×).

Figure 6. The expression level of hsa-miR-140-5p is significantly correlated with CRC patients survival

A. Hsa-miR-140-5p was progressively downregulated in primary and metastatic CRC tissue. Eighteen CRC patients with lymph node and/or liver metastasis were selected, and hsa-miR-140-5p levels were quantified by real-time qRT-PCR and normalized to the internal control. The hsa-miR-140-5p levels in primary and metastatic tumor tissues were compared to corresponding normal tissues. B. Kaplan-Meier survival curves of 174 stage III and IV CRC patients were generated based on different expression level of hsa-miR-140-5p. HR = 0.49 (0.27–0.91); Cutoff value: median, p = 0.02)

Figure 7. Proposed model of hsa-miR-140-5p function in CRC development and progression

Hsa-miR-140-5p acts as a master regulator of colorectal cancer survival, invasion and metastasis through the suppression of Smad2. Hsa-miR-140-5p also inhibits colorectal CSC survival by suppressing ATG12 and disrupting autophagy.