MicroRNA-340-5p inhibits colon cancer cell migration via targeting of RhoA

Abstract

Colon cancer is the third most common cancer and a significant cause of cancer-related deaths worldwide. Metastasis is the most insidious aspect of cancer progression. Convincing data suggest that microRNAs (miRs) play a key function in colon cancer biology. We examined the role of miR-340-5p in regulating RhoA expression as well as cell migration and invasion in colon cancer cells. Levels of miR-340-5p and RhoA mRNA varied inversely in serum-free and serum-grown HT-29 and AZ-97 colon cancer cells. It was found transfection with miR-340-5p not only decreased expression of RhoA mRNA and protein levels in HT-29 cells but also reduced colon cancer cell migration and invasion. Bioinformatics analysis predicted one putative binding sites at the 3'-UTR of RhoA mRNA. Targeting this binding site with a specific blocker reversed mimic miR-340-5p-induced inhibition of RhoA activation and colon cancer cell migration and invasion. These novel results suggest that miR-340-5p is an important regulator of colon cancer cell motility via targeting of RhoA and further experiments are warranted to evaluate the role of miR-340-5p in colon cancer metastasis.

Figure 1

Gene expression of miR340-5p and RhoA in HT-29 and AZ-97 colon cancer cells lines. Expression of (A) miR-340-5p and (B) RhoA mRNAs and the housekeeping gene U6 were determined using qRT-PCR in serum-free and serum-grown HT-29 cells. Relative expressions were demonstrated using qRT-PCR where U6 was used as a housekeeping gene for mir-340-5p and beta-actin was used as a housekeeping gene for RhoA mRNA and expressions were determined using 2–ΔΔCT method. Data represent mean ± SEM and n = 4.

Figure 2

Mir-340-5p regulates RhoA mRNA expression in HT-29 and AZ-97 colon cancer cells. Transfection with Mimic-Ctrl (50 nM) or miR-40-5p mimic (25 nM and 50 nM) (A) upregulates miR-340-5p and (B) downregulates RhoA mRNA expression in colon cancer cells. Relative expressions were demonstrated using qRT-PCR where U6 was used as a housekeeping gene for mir-340-5p and beta-actin was used as a housekeeping gene for RhoA mRNA and expressions were determined using 2^–ΔΔCT method. Data represents mean ± SEM and (n = 4).

Figure 3

RhoA is a direct target of miR-340-5p. (A) Predicted target site of miR-340-5p in RhoA mRNA 3′-UTR sequence containing an (AAUAUUUC) motif. The seeding region of miR-340-5p complementary to (UUAUAAAG) was blocked using TSB (red sequence). (B) TSB dose-dependently reversed the inhibitory effect of miR-340-5p on RhoA mRNA expression in HT-29 colon cancer cells. (C) TSB dose-dependently reversed the inhibitory effect of miR-340-5p on RhoA mRNA expression in AZ-97 colon cancer cells. Data represent mean ± SEM and (n = 4).

Figure 4

Mir-340-5p regulates colon cancer cell migration and RhoA activation. (A) RhoA-GTP activation was quantified using the G-LISA activation assay kit. (B) Total RhoA was measured by total RhoA assay kit. Cells were transfected with miR-340-5p mimic, mimic control, TSB control and TSB. Data represent mean ± SEM and n = 4.

Figure 5

(A) Migration of colon cancer cells were stimulated by use of 10% FBS. Cells were counted microscopically using high power fields in five different fields. (B) Invasion of colon cancer cells were stimulated by use of 10% FBS. Cells were transfected with miR-340-5p mimic, mimic control, TSB control and TSB. In one group, cells were pre-incubated with the Rho kinase inhibitor Y-27632 (50 µM) for 30 min before loading into the inserts. Cells were counted microscopically using high power fields in five different fields. Data represent mean ± SEM and n = 4.