MiR-384 inhibits human colorectal cancer metastasis by targeting KRAS and CDC42

Abstract

Colorectal cancer (CRC) is the third most common cancer worldwide. Metastatic progression is a primary factor contributing to lethality of CRC patients. However, the molecular mechanisms forming early local invasion and distant metastatic colonies are still unclear and the present therapeutic approaches for CRC are unsatisfactory. Therefore, novel therapies targeting metastatic invasion that could prevent tumor spreading and recurrence are urgently needed. Our study showed that the decrease of miR-384 was found in 83.0% (83/100) CRC patients. And low-leveled expression of miR-384 was closely correlated with the invasive depth, lymph node and distant metastasis of CRC. Overexpression of miR-384 could inhibit the invasive and migrating abilities of CRC cells in vitro and the metastatic potential in vivo. Luciferase assays showed that miR-384 repressed the expression of Kirsten Ras (KRAS) and Cell division cycle 42 (CDC42) by directly targeting their 3'-untranslated regions. There is functional and mechanistic relationship between miRNA-384 and KRAS, CDC42 in the invasion and metastasis of CRC. And our findings suggest that miR-384could be a potent therapeutic target for CRC. Restoration of miR-384 expression might provide novel therapeutic approach to the reduction of CRC metastasis.

Keywords: CDC42; KRAS; MiR-384; colorectal cancer; metastasis.

Figure 1. MiR-384 is down-regulated in CRC patients

A. Expression of miR-384 in 100 cases of fresh human CRC tissues and their matched adjacent normal tissues by real-time PCR analyses; miR-384 expression was normalized to U6 and expressed relative to the matched adjacent normal tissues (2^-ΔΔCт). B. Mean expression of miR-384 in 100 cases of fresh human CRC tissues and their matched adjacent normal tissues by real-time PCR (2^-ΔCт, n=100, ^**p<0.01). C. Mean expression of miR-384 by real-time PCR according to the T classification (2^-ΔCт, n=100, ^*p<0.05). D. Mean expression of miR-384 by real-time PCR according to the N classification (2^-ΔCт, n=100, ^**p<0.01). E. Mean expression of miR-384 by real-time PCR according to M classification (2^-ΔCт, n=100, ^**p<0.01). Boundaries of boxes represent bounding of the boxes and stand for the lower and upper quartile. Lines within the boxes and whiskers represent median and extremum (maximum and minimum).

Figure 2. Over-expression of miR-384 inhibits the invasive and metastatic abilities of CRC cells in vitro and in vivo

A. Expression of miR-384 in six CRC cell lines and one normal colorectal mucosa cell line FHC were detected by real-time PCR. B. Over-expression of miR-384 in SW480 and HCT116 CRC cells verified by real-time PCR. C. Wound-healing assay. Histograms represent the average migrated distances at the indicated times. Error bars represent mean±s.d. from three independent experiments. D-E. The migratory and invasive properties of SW480/Vector, SW480/miR-384 and HCT116/Vector, HCT116/miR-384 cells were analyzed using Boyden chambers or Matrigel-coated Boyden chambers. Error bars represent mean±s.d. from three independent experiments. F. Three-dimensional morphologies assay. Histograms represent the average number of filopodia formed by each cell sphere from three independent experiments. Error bars represent mean±s.d.. G. Representative images of gross specimens of liver metastatic lesions formed in mice injected intrasplenically with SW480/Vector and SW480/miR-384. H. The number of liver visible metastatic nodules was shown (^**p<0.01). (I) Overall survival of mice bearing liver metastases of SW480/Vector and SW480/miR-384 (log-rank test, n=8, ^** p<0.01).

Figure 3. Inhibition of endogenous miR-384 promotes the invasive and metastatic abilities of CRC cells in vitro and in vivo

A. Expression of miR-384 in LOVO and SW620 CRC cells transfected with inhibitor or their paired negative control lentiviral vector was detected by real-time PCR. B. Wound-healing assay. Histograms represent the average migrated distances at the indicated times. Error bars represent mean±s.d. from three independent experiments. C-D. The migratory and invasive properties of LOVO/NC, LOVO/miR-384-in and SW620/NC, SW620/miR-384-in cells were analyzed using Boyden chambers or Matrigel-coated Boyden chambers. Error bars represent mean±s.d. from three independent experiments. E. Three-dimensional morphologies assay. Histograms represent the average number of filopodia formed by each cell sphere from three independent experiments. Error bars represent mean±s.d.. F. Representative images of gross specimens a of liver metastatic lesions formed in mice injected intrasplenically with LOVO/NC and LOVO/miR-384-in. G. The number of liver visible metastatic nodules was shown(^**p <0.01). H. Overall survival of mice bearing liver metastases of LOVO/NC and LOVO/miR-384-in (log-rank test, n=8, ^** p<0.01).

Figure 4. KRAS and CDC42 are direct targets of miR-384

A. Predicted miR-384 target sequences in the 3’UTRs of KRAS and CDC42, and their mutants containing altered nucleotides in the 3’UTRs. B. Western blot analysis of KRAS and CDC42 in the indicated cells. C. Real-time PCR analysis of KRAS and CDC42 mRNA expression. D. Luciferase assay analyses of the indicated cells transfected with the indicated reporters with miR-384 (Error bars represent mean ± SD from three independent experiments; ^**p< 0.01). E-G. Luciferase assay analyses with the variable endogenous levels of mir-384 in FHC, HCT116 and LOVO cell lines (Error bars represent mean ± SD from three independent experiments; ^*p< 0.05, ^**p< 0.01).

Figure 5. Repression of KRAS and CDC42 plays an important role in miR-384-inhibited invasion and metastasis of CRC cells

A-C. KRAS and CDC42 over-expression in SW480 cells by real-time PCR analysis or Western blot. D. Wound-healing assay. Histograms represent the average migrated distances at the indicated times. Error bars represent mean±s.d. from three independent experiments. E-F. The migratory and invasive properties of SW480/Vector, SW480/miR-384 and SW480/miR-384+KRAS, SW480/miR-384+CDC42 cells were analyzed using Boyden chambers or Matrigel-coated Boyden chambers. Error bars represent mean±s.d. from three independent experiments. G. Three-dimensional morphologies assay. Histograms represent the average number of filopodia formed by each cell sphere from three independent experiments. Error bars represent mean±s.d.. H. Representative images of gross specimens of liver metastatic lesions formed in mice injected intrasplenically with SW480/Vector, SW480/miR-384, SW480/miR-384+KRAS and SW480/miR-384+CDC42. I. Number of visible metastatic nodules in the liver. ^**p<0.01. J. Overall survival of mice bearing liver metastases (log-rank test, n=8, ^*p <0.05).

Figure 6. Correlation between the expression of miR-384 and KRAS or CDC42 in CRC tissues

A. The expression of miR-384 and KRAS, CDC42 were detected by real-time PCR(ΔCт, n=10). B. Spearman correlation analyses of miR-384 expression and KRAS mRNA expression. C. Spearman correlation analyses of miR-384 expression and CDC42 mRNA expression.